Table of Content
I.Project Summary
II.Workflow Checklist
III.NGS Sequencing Results
IV.Complete Report Download
V.Raw Sequence Data Download
VI.Analysis - DADA2 Read Processing
 Sample Meta Info
 Read Count by Sample
VII.Analysis - Read Taxonomy Assignment
 Taxonomy Barplots
VIII.Analysis - Alpha Diversity
IX.Analysis - Beta Diversity
X.Analysis - Differential Abundance
 ANCOM Result
 LEfSe Result
XI.Analysis - Heatmap Profile
XII.Analysis - Network Association

(Click to navigate)

FOMC Service Report

16S rRNA Gene V1V3 Amplicon Sequencing

Version V1.41

Version History

The Forsyth Institute, Cambridge, MA, USA
August 30, 2022

Project ID: F6620A

I. Project Summary

Project F6620A services include NGS sequencing of the V1V3 region of the 16S rRNA gene amplicons from the samples. First and foremost, please download this report, as well as the sequence raw data from the download links provided below. These links will expire after 60 days. We cannot guarantee the availability of your data after 60 days.

Full Bioinformatics analysis service was requested. We provide many analyses, starting from the raw sequence quality and noise filtering, pair reads merging, as well as chimera filtering for the sequences, using the DADA2 denosing algorithm and pipeline.

We also provide many downstream analyses such as taxonomy assignment, alpha and beta diversity analyses, and differential abundance analysis.

For taxonomy assignment, most informative would be the taxonomy barplots. We provide an interactive barplots to show the relative abundance of microbes at different taxonomy levels (from Phylum to species) that you can choose.

If you specify which groups of samples you want to compare for differential abundance, we provide both ANCOM and LEfSe differential abundance analysis.

 

II. Workflow Checklist

☑1.Sample Received
☑2.Sample Quality Evaluated
☑3.Sample Prepared for Sequencing
☑4.Next-Gen Sequencing
☑5.Sequence Quality Check
☑6.Absolute Abundance
☑7.Report and Raw Sequence Data Available for Download
☑8.Bioinformatics Analysis - Reads Processing (DADA2 Quality Trimming, Denoising, Paired Reads Merging)
☑9.Bioinformatics Analysis - Reads Taxonomy Assignment
☑10.Bioinformatics Analysis - Alpha Diversity Analysis
☑11.Bioinformatics Analysis - Beta Diversity Analysis
☑12.Bioinformatics Analysis - Differential Abundance Analysis
☑13.Bioinformatics Analysis - Heatmap Profile
☑14.Bioinformatics Analysis - Network Association
 

III. NGS Sequencing

The samples were processed and analyzed with the ZymoBIOMICS® Service: Targeted Metagenomic Sequencing (Zymo Research, Irvine, CA).

DNA Extraction: If DNA extraction was performed, one of three different DNA extraction kits was used depending on the sample type and sample volume and were used according to the manufacturer’s instructions, unless otherwise stated. The kit used in this project is marked below:

☐ZymoBIOMICS® DNA Miniprep Kit (Zymo Research, Irvine, CA)
☐ZymoBIOMICS® DNA Microprep Kit (Zymo Research, Irvine, CA)
☐ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research, Irvine, CA)
☑N/A (DNA Extraction Not Performed)
Elution Volume: 50µL
Additional Notes: NA

Targeted Library Preparation: The DNA samples were prepared for targeted sequencing with the Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA). These primers were custom designed by Zymo Research to provide the best coverage of the 16S gene while maintaining high sensitivity. The primer sets used in this project are marked below:

☐Quick-16S™ Primer Set V1-V2 (Zymo Research, Irvine, CA)
☑Quick-16S™ Primer Set V1-V3 (Zymo Research, Irvine, CA)
☐Quick-16S™ Primer Set V3-V4 (Zymo Research, Irvine, CA)
☐Quick-16S™ Primer Set V4 (Zymo Research, Irvine, CA)
☐Quick-16S™ Primer Set V6-V8 (Zymo Research, Irvine, CA)
☐Other: NA
Additional Notes: NA

The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned up with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research, Irvine, CA), then quantified with TapeStation® (Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific, Waltham, WA).

Control Samples: The ZymoBIOMICS® Microbial Community Standard (Zymo Research, Irvine, CA) was used as a positive control for each DNA extraction, if performed. The ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e. blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process.

Sequencing: The final library was sequenced on Illumina® MiSeq™ with a V3 reagent kit (600 cycles). The sequencing was performed with 10% PhiX spike-in.

 

IV. Complete Report Download

The complete report of your project, including all links in this report, can be downloaded by clicking the link provided below. The downloaded file is a compressed ZIP file and once unzipped, open the file “REPORT.html” (may only shown as "REPORT" in your computer) by double clicking it. Your default web browser will open it and you will see the exact content of this report.

Please download and save the file to your computer storage device. The download link will expire after 60 days upon your receiving of this report.

Complete report download link:

To view the report, please follow the following steps:
1.Download the .zip file from the report link above.
2.Extract all the contents of the downloaded .zip file to your desktop.
3.Open the extracted folder and find the "REPORT.html" (may shown as only "REPORT").
4.Open (double-clicking) the REPORT.html file. Your default browser will open the top age of the complete report. Within the report, there are links to view all the analyses performed for the project.

 

V. Raw Sequence Data Download

The raw NGS sequence data is available for download with the link provided below. The data is a compressed ZIP file and can be unzipped to individual sequence files. Since this is a pair-end sequencing, each of your samples is represented by two sequence files, one for READ 1, with the file extension “*_R1.fastq.gz”, another READ 2, with the file extension “*_R1.fastq.gz”. The files are in FASTQ format and are compressed. FASTQ format is a text-based data format for storing both a biological sequence and its corresponding quality scores. Most sequence analysis software will be able to open them. The Sample IDs associated with the R1 and R2 fastq files are listed in the table below:



Sample IDOriginal Sample IDRead 1 File NameRead 2 File Name


F6620.S100B3_39zr6620_100V1V3_R1.fastq.gzzr6620_100V1V3_R2.fastq.gz


F6620.S101L1_39zr6620_101V1V3_R1.fastq.gzzr6620_101V1V3_R2.fastq.gz


F6620.S102L2_39zr6620_102V1V3_R1.fastq.gzzr6620_102V1V3_R2.fastq.gz


F6620.S103L3_39zr6620_103V1V3_R1.fastq.gzzr6620_103V1V3_R2.fastq.gz


F6620.S104C1_39zr6620_104V1V3_R1.fastq.gzzr6620_104V1V3_R2.fastq.gz


F6620.S105C2_39zr6620_105V1V3_R1.fastq.gzzr6620_105V1V3_R2.fastq.gz


F6620.S106C3_39zr6620_106V1V3_R1.fastq.gzzr6620_106V1V3_R2.fastq.gz


F6620.S107FB1_46zr6620_107V1V3_R1.fastq.gzzr6620_107V1V3_R2.fastq.gz


F6620.S108FB2_46zr6620_108V1V3_R1.fastq.gzzr6620_108V1V3_R2.fastq.gz


F6620.S109FB3_46zr6620_109V1V3_R1.fastq.gzzr6620_109V1V3_R2.fastq.gz


F6620.S010FA1_4zr6620_10V1V3_R1.fastq.gzzr6620_10V1V3_R2.fastq.gz


F6620.S110FA1_46zr6620_110V1V3_R1.fastq.gzzr6620_110V1V3_R2.fastq.gz


F6620.S111FA2_46zr6620_111V1V3_R1.fastq.gzzr6620_111V1V3_R2.fastq.gz


F6620.S112FA3_46zr6620_112V1V3_R1.fastq.gzzr6620_112V1V3_R2.fastq.gz


F6620.S113B1_46zr6620_113V1V3_R1.fastq.gzzr6620_113V1V3_R2.fastq.gz


F6620.S114B2_46zr6620_114V1V3_R1.fastq.gzzr6620_114V1V3_R2.fastq.gz


F6620.S115B3_46zr6620_115V1V3_R1.fastq.gzzr6620_115V1V3_R2.fastq.gz


F6620.S116L1_46zr6620_116V1V3_R1.fastq.gzzr6620_116V1V3_R2.fastq.gz


F6620.S117L2_46zr6620_117V1V3_R1.fastq.gzzr6620_117V1V3_R2.fastq.gz


F6620.S118L3_46zr6620_118V1V3_R1.fastq.gzzr6620_118V1V3_R2.fastq.gz


F6620.S119C1_46zr6620_119V1V3_R1.fastq.gzzr6620_119V1V3_R2.fastq.gz


F6620.S011FA2_4zr6620_11V1V3_R1.fastq.gzzr6620_11V1V3_R2.fastq.gz


F6620.S120C2_46zr6620_120V1V3_R1.fastq.gzzr6620_120V1V3_R2.fastq.gz


F6620.S121C3_46zr6620_121V1V3_R1.fastq.gzzr6620_121V1V3_R2.fastq.gz


F6620.S122FB1_53zr6620_122V1V3_R1.fastq.gzzr6620_122V1V3_R2.fastq.gz


F6620.S123FB2_53zr6620_123V1V3_R1.fastq.gzzr6620_123V1V3_R2.fastq.gz


F6620.S124FB3_53zr6620_124V1V3_R1.fastq.gzzr6620_124V1V3_R2.fastq.gz


F6620.S125FA1_53zr6620_125V1V3_R1.fastq.gzzr6620_125V1V3_R2.fastq.gz


F6620.S126FA2_53zr6620_126V1V3_R1.fastq.gzzr6620_126V1V3_R2.fastq.gz


F6620.S127FA3_53zr6620_127V1V3_R1.fastq.gzzr6620_127V1V3_R2.fastq.gz


F6620.S128B1_53zr6620_128V1V3_R1.fastq.gzzr6620_128V1V3_R2.fastq.gz


F6620.S129B2_53zr6620_129V1V3_R1.fastq.gzzr6620_129V1V3_R2.fastq.gz


F6620.S012FA3_4zr6620_12V1V3_R1.fastq.gzzr6620_12V1V3_R2.fastq.gz


F6620.S130B3_53zr6620_130V1V3_R1.fastq.gzzr6620_130V1V3_R2.fastq.gz


F6620.S131L1_53zr6620_131V1V3_R1.fastq.gzzr6620_131V1V3_R2.fastq.gz


F6620.S132L2_53zr6620_132V1V3_R1.fastq.gzzr6620_132V1V3_R2.fastq.gz


F6620.S133L3_53zr6620_133V1V3_R1.fastq.gzzr6620_133V1V3_R2.fastq.gz


F6620.S134C1_53zr6620_134V1V3_R1.fastq.gzzr6620_134V1V3_R2.fastq.gz


F6620.S135C2_53zr6620_135V1V3_R1.fastq.gzzr6620_135V1V3_R2.fastq.gz


F6620.S136C3_53zr6620_136V1V3_R1.fastq.gzzr6620_136V1V3_R2.fastq.gz


F6620.S137CF1_53zr6620_137V1V3_R1.fastq.gzzr6620_137V1V3_R2.fastq.gz


F6620.S138CF2_53zr6620_138V1V3_R1.fastq.gzzr6620_138V1V3_R2.fastq.gz


F6620.S139CF3_53zr6620_139V1V3_R1.fastq.gzzr6620_139V1V3_R2.fastq.gz


F6620.S013B1_4zr6620_13V1V3_R1.fastq.gzzr6620_13V1V3_R2.fastq.gz


F6620.S014B2_4zr6620_14V1V3_R1.fastq.gzzr6620_14V1V3_R2.fastq.gz


F6620.S015B3_4zr6620_15V1V3_R1.fastq.gzzr6620_15V1V3_R2.fastq.gz


F6620.S016L1_4zr6620_16V1V3_R1.fastq.gzzr6620_16V1V3_R2.fastq.gz


F6620.S017L2_4zr6620_17V1V3_R1.fastq.gzzr6620_17V1V3_R2.fastq.gz


F6620.S018L3_4zr6620_18V1V3_R1.fastq.gzzr6620_18V1V3_R2.fastq.gz


F6620.S019C1_4zr6620_19V1V3_R1.fastq.gzzr6620_19V1V3_R2.fastq.gz


F6620.S001SupraC1zr6620_1V1V3_R1.fastq.gzzr6620_1V1V3_R2.fastq.gz


F6620.S020C2_4zr6620_20V1V3_R1.fastq.gzzr6620_20V1V3_R2.fastq.gz


F6620.S021C3_4zr6620_21V1V3_R1.fastq.gzzr6620_21V1V3_R2.fastq.gz


F6620.S022CC1_4zr6620_22V1V3_R1.fastq.gzzr6620_22V1V3_R2.fastq.gz


F6620.S023CC2_4zr6620_23V1V3_R1.fastq.gzzr6620_23V1V3_R2.fastq.gz


F6620.S024CC3_4zr6620_24V1V3_R1.fastq.gzzr6620_24V1V3_R2.fastq.gz


F6620.S025CC4_4zr6620_25V1V3_R1.fastq.gzzr6620_25V1V3_R2.fastq.gz


F6620.S026CC5_4zr6620_26V1V3_R1.fastq.gzzr6620_26V1V3_R2.fastq.gz


F6620.S027CC6_4zr6620_27V1V3_R1.fastq.gzzr6620_27V1V3_R2.fastq.gz


F6620.S028CC7_4zr6620_28V1V3_R1.fastq.gzzr6620_28V1V3_R2.fastq.gz


F6620.S029CC8_4zr6620_29V1V3_R1.fastq.gzzr6620_29V1V3_R2.fastq.gz


F6620.S002SupraC2zr6620_2V1V3_R1.fastq.gzzr6620_2V1V3_R2.fastq.gz


F6620.S030CC9_4zr6620_30V1V3_R1.fastq.gzzr6620_30V1V3_R2.fastq.gz


F6620.S031CC10_4zr6620_31V1V3_R1.fastq.gzzr6620_31V1V3_R2.fastq.gz


F6620.S032FB1_11zr6620_32V1V3_R1.fastq.gzzr6620_32V1V3_R2.fastq.gz


F6620.S033FB2_11zr6620_33V1V3_R1.fastq.gzzr6620_33V1V3_R2.fastq.gz


F6620.S034FB3_11zr6620_34V1V3_R1.fastq.gzzr6620_34V1V3_R2.fastq.gz


F6620.S035FA1_11zr6620_35V1V3_R1.fastq.gzzr6620_35V1V3_R2.fastq.gz


F6620.S036FA2_11zr6620_36V1V3_R1.fastq.gzzr6620_36V1V3_R2.fastq.gz


F6620.S037FA3_11zr6620_37V1V3_R1.fastq.gzzr6620_37V1V3_R2.fastq.gz


F6620.S038B1_11zr6620_38V1V3_R1.fastq.gzzr6620_38V1V3_R2.fastq.gz


F6620.S039B2_11zr6620_39V1V3_R1.fastq.gzzr6620_39V1V3_R2.fastq.gz


F6620.S003SupraC3zr6620_3V1V3_R1.fastq.gzzr6620_3V1V3_R2.fastq.gz


F6620.S040B3_11zr6620_40V1V3_R1.fastq.gzzr6620_40V1V3_R2.fastq.gz


F6620.S041L1_11zr6620_41V1V3_R1.fastq.gzzr6620_41V1V3_R2.fastq.gz


F6620.S042L2_11zr6620_42V1V3_R1.fastq.gzzr6620_42V1V3_R2.fastq.gz


F6620.S043L3_11zr6620_43V1V3_R1.fastq.gzzr6620_43V1V3_R2.fastq.gz


F6620.S044C1_11zr6620_44V1V3_R1.fastq.gzzr6620_44V1V3_R2.fastq.gz


F6620.S045C2_11zr6620_45V1V3_R1.fastq.gzzr6620_45V1V3_R2.fastq.gz


F6620.S046C3_11zr6620_46V1V3_R1.fastq.gzzr6620_46V1V3_R2.fastq.gz


F6620.S047FB1_18zr6620_47V1V3_R1.fastq.gzzr6620_47V1V3_R2.fastq.gz


F6620.S048FB2_18zr6620_48V1V3_R1.fastq.gzzr6620_48V1V3_R2.fastq.gz


F6620.S049FB3_18zr6620_49V1V3_R1.fastq.gzzr6620_49V1V3_R2.fastq.gz


F6620.S004SupraT1zr6620_4V1V3_R1.fastq.gzzr6620_4V1V3_R2.fastq.gz


F6620.S050FA1_18zr6620_50V1V3_R1.fastq.gzzr6620_50V1V3_R2.fastq.gz


F6620.S051FA2_18zr6620_51V1V3_R1.fastq.gzzr6620_51V1V3_R2.fastq.gz


F6620.S052FA3_18zr6620_52V1V3_R1.fastq.gzzr6620_52V1V3_R2.fastq.gz


F6620.S053B1_18zr6620_53V1V3_R1.fastq.gzzr6620_53V1V3_R2.fastq.gz


F6620.S054B2_18zr6620_54V1V3_R1.fastq.gzzr6620_54V1V3_R2.fastq.gz


F6620.S055B3_18zr6620_55V1V3_R1.fastq.gzzr6620_55V1V3_R2.fastq.gz


F6620.S056L1_18zr6620_56V1V3_R1.fastq.gzzr6620_56V1V3_R2.fastq.gz


F6620.S057L2_18zr6620_57V1V3_R1.fastq.gzzr6620_57V1V3_R2.fastq.gz


F6620.S058L3_18zr6620_58V1V3_R1.fastq.gzzr6620_58V1V3_R2.fastq.gz


F6620.S059C1_18zr6620_59V1V3_R1.fastq.gzzr6620_59V1V3_R2.fastq.gz


F6620.S005SupraT2zr6620_5V1V3_R1.fastq.gzzr6620_5V1V3_R2.fastq.gz


F6620.S060C2_18zr6620_60V1V3_R1.fastq.gzzr6620_60V1V3_R2.fastq.gz


F6620.S061C3_18zr6620_61V1V3_R1.fastq.gzzr6620_61V1V3_R2.fastq.gz


F6620.S062FB1_25zr6620_62V1V3_R1.fastq.gzzr6620_62V1V3_R2.fastq.gz


F6620.S063FB2_25zr6620_63V1V3_R1.fastq.gzzr6620_63V1V3_R2.fastq.gz


F6620.S064FB3_25zr6620_64V1V3_R1.fastq.gzzr6620_64V1V3_R2.fastq.gz


F6620.S065FA1_25zr6620_65V1V3_R1.fastq.gzzr6620_65V1V3_R2.fastq.gz


F6620.S066FA2_25zr6620_66V1V3_R1.fastq.gzzr6620_66V1V3_R2.fastq.gz


F6620.S067FA3_25zr6620_67V1V3_R1.fastq.gzzr6620_67V1V3_R2.fastq.gz


F6620.S068B1_25zr6620_68V1V3_R1.fastq.gzzr6620_68V1V3_R2.fastq.gz


F6620.S069B2_25zr6620_69V1V3_R1.fastq.gzzr6620_69V1V3_R2.fastq.gz


F6620.S006SupraT3zr6620_6V1V3_R1.fastq.gzzr6620_6V1V3_R2.fastq.gz


F6620.S070B3_25zr6620_70V1V3_R1.fastq.gzzr6620_70V1V3_R2.fastq.gz


F6620.S071L1_32zr6620_71V1V3_R1.fastq.gzzr6620_71V1V3_R2.fastq.gz


F6620.S072L2_25zr6620_72V1V3_R1.fastq.gzzr6620_72V1V3_R2.fastq.gz


F6620.S073L3_25zr6620_73V1V3_R1.fastq.gzzr6620_73V1V3_R2.fastq.gz


F6620.S074C1_25zr6620_74V1V3_R1.fastq.gzzr6620_74V1V3_R2.fastq.gz


F6620.S075C2_25zr6620_75V1V3_R1.fastq.gzzr6620_75V1V3_R2.fastq.gz


F6620.S076C3_25zr6620_76V1V3_R1.fastq.gzzr6620_76V1V3_R2.fastq.gz


F6620.S077FB1_32zr6620_77V1V3_R1.fastq.gzzr6620_77V1V3_R2.fastq.gz


F6620.S078FB2_32zr6620_78V1V3_R1.fastq.gzzr6620_78V1V3_R2.fastq.gz


F6620.S079FB3_32zr6620_79V1V3_R1.fastq.gzzr6620_79V1V3_R2.fastq.gz


F6620.S007FB1_4zr6620_7V1V3_R1.fastq.gzzr6620_7V1V3_R2.fastq.gz


F6620.S080FA1_32zr6620_80V1V3_R1.fastq.gzzr6620_80V1V3_R2.fastq.gz


F6620.S081FA2_32zr6620_81V1V3_R1.fastq.gzzr6620_81V1V3_R2.fastq.gz


F6620.S082FA3_32zr6620_82V1V3_R1.fastq.gzzr6620_82V1V3_R2.fastq.gz


F6620.S083B1_32zr6620_83V1V3_R1.fastq.gzzr6620_83V1V3_R2.fastq.gz


F6620.S084B2_32zr6620_84V1V3_R1.fastq.gzzr6620_84V1V3_R2.fastq.gz


F6620.S085B3_32zr6620_85V1V3_R1.fastq.gzzr6620_85V1V3_R2.fastq.gz


F6620.S086L1_25zr6620_86V1V3_R1.fastq.gzzr6620_86V1V3_R2.fastq.gz


F6620.S087L2_32zr6620_87V1V3_R1.fastq.gzzr6620_87V1V3_R2.fastq.gz


F6620.S088L3_32zr6620_88V1V3_R1.fastq.gzzr6620_88V1V3_R2.fastq.gz


F6620.S089C1_32zr6620_89V1V3_R1.fastq.gzzr6620_89V1V3_R2.fastq.gz


F6620.S008FB2_4zr6620_8V1V3_R1.fastq.gzzr6620_8V1V3_R2.fastq.gz


F6620.S090C2_32zr6620_90V1V3_R1.fastq.gzzr6620_90V1V3_R2.fastq.gz


F6620.S091C3_32zr6620_91V1V3_R1.fastq.gzzr6620_91V1V3_R2.fastq.gz


F6620.S092FB1_39zr6620_92V1V3_R1.fastq.gzzr6620_92V1V3_R2.fastq.gz


F6620.S093FB2_39zr6620_93V1V3_R1.fastq.gzzr6620_93V1V3_R2.fastq.gz


F6620.S094FB3_39zr6620_94V1V3_R1.fastq.gzzr6620_94V1V3_R2.fastq.gz


F6620.S095FA1_39zr6620_95V1V3_R1.fastq.gzzr6620_95V1V3_R2.fastq.gz


F6620.S096FA2_39zr6620_96V1V3_R1.fastq.gzzr6620_96V1V3_R2.fastq.gz


F6620.S097FA3_39zr6620_97V1V3_R1.fastq.gzzr6620_97V1V3_R2.fastq.gz


F6620.S098B1_39zr6620_98V1V3_R1.fastq.gzzr6620_98V1V3_R2.fastq.gz


F6620.S099B2_39zr6620_99V1V3_R1.fastq.gzzr6620_99V1V3_R2.fastq.gz


F6620.S009FB3_4zr6620_9V1V3_R1.fastq.gzzr6620_9V1V3_R2.fastq.gz

Please download and save the file to your computer storage device. The download link will expire after 60 days upon your receiving of this report.

Raw sequence data download link:

 

VI. Analysis - DADA2 Read Processing

What is DADA2?

DADA2 is a software package that models and corrects Illumina-sequenced amplicon errors. DADA2 infers sample sequences exactly, without coarse-graining into OTUs, and resolves differences of as little as one nucleotide. DADA2 identified more real variants and output fewer spurious sequences than other methods.

DADA2’s advantage is that it uses more of the data. The DADA2 error model incorporates quality information, which is ignored by all other methods after filtering. The DADA2 error model incorporates quantitative abundances, whereas most other methods use abundance ranks if they use abundance at all. The DADA2 error model identifies the differences between sequences, eg. A->C, whereas other methods merely count the mismatches. DADA2 can parameterize its error model from the data itself, rather than relying on previous datasets that may or may not reflect the PCR and sequencing protocols used in your study.

DADA2 Publication: Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016 Jul;13(7):581-3. doi: 10.1038/nmeth.3869. Epub 2016 May 23. PMID: 27214047; PMCID: PMC4927377.

DADA2 Software Package is available as an R package at : https://benjjneb.github.io/dada2/index.html

Analysis Procedures:

DADA2 pipeline includes several tools for read quality control, including quality filtering, trimming, denoising, pair merging and chimera filtering. Below are the major processing steps of DADA2:

Step 1. Read trimming based on sequence quality The quality of NGS Illumina sequences often decreases toward the end of the reads. DADA2 allows to trim off the poor quality read ends in order to improve the error model building and pair mergicing performance.

Step 2. Learn the Error Rates The DADA2 algorithm makes use of a parametric error model (err) and every amplicon dataset has a different set of error rates. The learnErrors method learns this error model from the data, by alternating estimation of the error rates and inference of sample composition until they converge on a jointly consistent solution. As in many machine-learning problems, the algorithm must begin with an initial guess, for which the maximum possible error rates in this data are used (the error rates if only the most abundant sequence is correct and all the rest are errors).

Step 3. Infer amplicon sequence variants (ASVs) based on the error model built in previous step. This step is also called sequence "denoising". The outcome of this step is a list of ASVs that are the equivalent of oligonucleotides.

Step 4. Merge paired reads. If the sequencing products are read pairs, DADA2 will merge the R1 and R2 ASVs into single sequences. Merging is performed by aligning the denoised forward reads with the reverse-complement of the corresponding denoised reverse reads, and then constructing the merged “contig” sequences. By default, merged sequences are only output if the forward and reverse reads overlap by at least 12 bases, and are identical to each other in the overlap region (but these conditions can be changed via function arguments).

Step 5. Remove chimera. The core dada method corrects substitution and indel errors, but chimeras remain. Fortunately, the accuracy of sequence variants after denoising makes identifying chimeric ASVs simpler than when dealing with fuzzy OTUs. Chimeric sequences are identified if they can be exactly reconstructed by combining a left-segment and a right-segment from two more abundant “parent” sequences. The frequency of chimeric sequences varies substantially from dataset to dataset, and depends on on factors including experimental procedures and sample complexity.

Results

1. Read Quality Plots NGS sequence analaysis starts with visualizing the quality of the sequencing. Below are the quality plots of the first sample for the R1 and R2 reads separately. In gray-scale is a heat map of the frequency of each quality score at each base position. The mean quality score at each position is shown by the green line, and the quartiles of the quality score distribution by the orange lines. The forward reads are usually of better quality. It is a common practice to trim the last few nucleotides to avoid less well-controlled errors that can arise there. The trimming affects the downstream steps including error model building, merging and chimera calling. FOMC uses an empirical approach to test many combinations of different trim length in order to achieve best final amplicon sequence variants (ASVs), see the next section “Optimal trim length for ASVs”.

Quality plots for all samples:

2. Optimal trim length for ASVs The final number of merged and chimera-filtered ASVs depends on the quality filtering (hence trimming) in the very beginning of the DADA2 pipeline. In order to achieve highest number of ASVs, an empirical approach was used -

  1. Create a random subset of each sample consisting of 5,000 R1 and 5,000 R2 (to reduce computation time)
  2. Trim 10 bases at a time from the ends of both R1 and R2 up to 50 bases
  3. For each combination of trimmed length (e.g., 300x300, 300x290, 290x290 etc), the trimmed reads are subject to the entire DADA2 pipeline for chimera-filtered merged ASVs
  4. The combination with highest percentage of the input reads becoming final ASVs is selected for the complete set of data

Below is the result of such operation, showing ASV percentages of total reads for all trimming combinations (1st Column = R1 lengths in bases; 1st Row = R2 lengths in bases):

R1/R2281271261251241231221211201
32126.04%47.96%56.00%60.47%60.32%34.52%28.11%19.18%8.92%
31126.16%49.63%58.20%62.28%35.37%29.15%21.77%11.17%4.37%
30126.44%49.98%57.84%35.61%28.53%20.15%11.00%4.19%3.64%
29126.24%49.84%33.74%28.22%19.88%10.86%4.10%3.44%0.02%
28129.91%29.90%27.85%19.88%10.69%4.16%3.53%0.02%0.02%
2717.19%23.89%18.82%10.03%4.08%3.50%0.02%0.02%0.00%
2613.90%15.69%9.32%3.80%3.52%0.02%0.02%0.00%0.00%
2512.73%8.32%3.54%3.36%0.02%0.02%0.00%0.00%0.00%
2410.53%3.69%3.50%0.02%0.02%0.00%0.00%0.00%0.00%

Based on the above result, the trim length combination of R1 = 311 bases and R2 = 251 bases (highlighted red above), was chosen for generating final ASVs for all sequences. This combination generated highest number of merged non-chimeric ASVs and was used for downstream analyses, if requested.

3. Error plots from learning the error rates After DADA2 building the error model for the set of data, it is always worthwhile, as a sanity check if nothing else, to visualize the estimated error rates. The error rates for each possible transition (A→C, A→G, …) are shown below. Points are the observed error rates for each consensus quality score. The black line shows the estimated error rates after convergence of the machine-learning algorithm. The red line shows the error rates expected under the nominal definition of the Q-score. The ideal result would be the estimated error rates (black line) are a good fit to the observed rates (points), and the error rates drop with increased quality as expected.

Forward Read R1 Error Plot


Reverse Read R2 Error Plot

The PDF version of these plots are available here:

 

4. DADA2 Result Summary The table below shows the summary of the DADA2 analysis, tracking paired read counts of each samples for all the steps during DADA2 denoising process - including end-trimming (filtered), denoising (denoisedF, denoisedF), pair merging (merged) and chimera removal (nonchim).

Sample IDF6620.S001F6620.S002F6620.S003F6620.S004F6620.S005F6620.S006F6620.S007F6620.S008F6620.S009F6620.S010F6620.S011F6620.S012F6620.S013F6620.S014F6620.S015F6620.S016F6620.S017F6620.S018F6620.S019F6620.S020F6620.S021F6620.S022F6620.S023F6620.S024F6620.S025F6620.S026F6620.S027F6620.S028F6620.S029F6620.S030F6620.S031F6620.S032F6620.S033F6620.S034F6620.S035F6620.S036F6620.S037F6620.S038F6620.S039F6620.S040F6620.S041F6620.S042F6620.S043F6620.S044F6620.S045F6620.S046F6620.S047F6620.S048F6620.S049F6620.S050F6620.S051F6620.S052F6620.S053F6620.S054F6620.S055F6620.S056F6620.S057F6620.S058F6620.S059F6620.S060F6620.S061F6620.S062F6620.S063F6620.S064F6620.S065F6620.S066F6620.S067F6620.S068F6620.S069F6620.S070F6620.S071F6620.S072F6620.S073F6620.S074F6620.S075F6620.S076F6620.S077F6620.S078F6620.S079F6620.S080F6620.S081F6620.S082F6620.S083F6620.S084F6620.S085F6620.S086F6620.S087F6620.S088F6620.S089F6620.S090F6620.S091F6620.S092F6620.S093F6620.S094F6620.S095F6620.S096F6620.S097F6620.S098F6620.S099F6620.S100F6620.S101F6620.S102F6620.S103F6620.S104F6620.S105F6620.S106F6620.S107F6620.S108F6620.S109F6620.S110F6620.S111F6620.S112F6620.S113F6620.S114F6620.S115F6620.S116F6620.S117F6620.S118F6620.S119F6620.S120F6620.S121F6620.S122F6620.S123F6620.S124F6620.S125F6620.S126F6620.S127F6620.S128F6620.S129F6620.S130F6620.S131F6620.S132F6620.S133F6620.S134F6620.S135F6620.S136F6620.S137F6620.S138F6620.S139Row SumPercentage
input30,65135,99527,86330,08535,90725,32419,47329,90431,95631,10930,75028,39333,81636,33533,00035,64531,85831,93135,23932,49033,84337,93631,75235,82943,43735,20035,58536,35534,80328,49825,17731,25138,27630,61030,95523,33827,36031,32935,99246,25833,19536,03329,20528,23139,44441,98031,44231,71027,74831,59227,09628,48542,18938,30329,69030,07139,13233,16642,44423,13529,35732,55833,86635,24631,78823,44027,88833,02626,13628,98229,47428,43122,61030,52232,56423,16628,28828,82531,83425,11734,24529,66919,08628,23536,39026,80029,98928,02737,28130,41425,31026,97428,20130,71423,89035,83533,77529,35733,03430,69827,54226,72928,82827,48034,72821,78132,04834,59934,51226,01229,60928,02736,84733,04727,70732,21327,27529,98323,06428,96731,43536,93031,81327,32822,98439,40920,51930,11941,05131,67727,84327,64332,02226,81032,62122,09728,04140,67829,7994,318,658100.00%
filtered30,64535,96827,84130,06135,89125,30019,46429,89031,92631,09330,72528,37033,79836,30732,98635,63031,82931,91335,21632,46233,81237,90131,73235,81643,40935,17635,56036,33934,77528,48425,16631,23938,25130,58430,94323,32127,35031,30335,96746,24333,17536,01329,18528,21439,41841,95131,41831,68627,72331,58127,08828,46842,16738,28229,67030,05439,09833,14842,41323,12429,33832,53533,84235,22631,77023,42427,86633,00926,12328,96929,46328,40922,59130,50632,54523,14428,27028,80231,82025,10434,21929,65219,07428,22436,36826,78629,98028,01637,26230,39825,29826,94528,18230,68923,86335,82733,74929,33733,01030,66627,49926,71228,81827,46134,70221,76232,02734,58534,50025,98229,59428,01136,82533,03127,69132,19327,25529,95823,05028,93631,40236,90331,79827,31422,98039,37420,51030,10941,01931,66027,82027,63031,99826,79932,60122,08728,02240,64529,7744,315,90099.94%
denoisedF28,76433,78025,92728,04733,76923,46018,26728,26429,96928,96628,66226,23331,44233,90331,03533,76029,93829,61233,60630,95231,84036,25330,27434,31741,39633,45133,88534,61333,04227,20123,92229,94936,36129,01129,09621,91425,56029,89833,98743,86731,29234,15627,53426,68337,33139,87030,12130,14426,55430,38325,82526,89040,73636,61628,16628,72037,46831,62940,82321,98627,87231,35732,46633,96830,45422,42626,49631,87125,05827,73227,85826,91921,52629,16631,04721,89226,79627,57430,54724,07432,59028,36118,26026,87934,92825,59228,85626,81035,56729,12024,17725,63627,00129,31922,60234,31532,41928,04731,46129,21426,40525,31527,37326,07733,05420,51830,77633,13333,25424,40728,06726,82735,46931,60026,48230,83826,03528,31022,00527,52329,79135,41430,49926,39821,97737,82419,60628,71239,24930,59426,54126,52330,72525,62931,13920,92026,62238,72728,2254,107,95695.12%
denoisedR27,85232,39825,10027,48132,77222,90417,93227,48329,65428,20328,33625,94031,38433,37830,19933,08529,49329,61833,04629,75130,77434,01629,82633,82640,47232,86433,42434,20232,58926,69623,67628,91135,99527,94128,32521,41424,95328,91733,49443,17531,00533,36527,39626,02836,68639,06229,30229,74925,11829,50424,99926,28039,48235,67627,72128,61736,88030,90439,92221,33526,79129,71631,73033,29929,86821,64526,28530,66124,02827,12927,38326,79120,02128,53129,22921,23726,29626,96829,64623,04231,91427,53217,30926,10533,80324,82828,20625,95135,20428,64623,56925,00325,71528,74822,10333,67531,56926,66430,77228,79426,15124,83726,85125,53132,37319,24229,89032,19832,33324,30127,86725,88333,23530,23625,73130,22125,08327,89721,22626,54329,31034,59129,45225,60021,17936,64319,20827,71638,45829,13926,00225,71429,39924,79630,19420,21725,71138,23527,1374,003,26692.70%
merged23,70327,74121,03922,66027,36217,98115,39823,66425,54123,12723,35021,13325,73227,44024,73527,80225,09424,82527,59924,68826,13729,29825,82028,20232,24027,89527,43628,80926,85423,26820,80225,26831,60825,27924,86118,16321,40026,17129,78537,59526,11628,44223,30722,13431,60432,83625,70426,25522,26325,48922,15923,46734,91531,76822,84725,43832,96127,99135,53119,17824,14427,49228,31929,82925,98219,56622,77628,98622,73424,35724,15723,13217,73425,47825,75017,89323,17623,98026,55820,25927,77523,46215,20423,01429,86122,06425,20123,28130,57025,15921,21122,40123,23225,46419,45629,29127,64923,74627,98226,07023,61521,98623,44022,38627,62717,09427,16029,08628,91020,77824,06423,13329,95726,84123,93726,62622,45024,96416,39522,55125,79331,40926,80623,53818,84831,94517,03725,35436,41727,74423,57823,20926,55721,67927,52518,24522,38533,02423,0753,499,47381.03%
nonchim14,21716,13012,87413,67816,45110,50712,28717,70819,42115,77615,63914,74617,60618,06816,50517,65716,55715,63919,83318,65418,71020,38017,13419,40622,37919,61619,81719,39715,99213,98314,62821,03224,34620,17720,11314,69816,95818,50620,71126,56418,86419,51916,42815,70023,00422,76819,53620,34018,15119,81517,66018,64325,18823,29218,32918,27823,49119,83825,50015,11318,81722,61422,38724,67721,27615,04816,96418,41914,28016,80017,85518,04513,46720,97319,93611,99819,53619,25323,06817,34821,21917,59712,78717,53121,84816,85720,12617,74523,42818,03015,75517,64418,29322,11417,18322,56022,94717,72519,31018,82818,21117,47117,78115,81419,57112,75621,37522,59725,44116,91919,36919,64122,25319,26017,10521,98918,60620,42913,03016,63719,39124,28220,40720,82415,37024,32613,68016,44423,41517,78619,33319,73420,79015,62518,89413,91816,25623,31016,8962,593,18160.05%

This table can be downloaded as an Excel table below:

 

5. DADA2 Amplicon Sequence Variants (ASVs). A total of 6177 unique merged and chimera-free ASV sequences were identified, and their corresponding read counts for each sample are available in the "ASV Read Count Table" with rows for the ASV sequences and columns for sample. This read count table can be used for microbial profile comparison among different samples and the sequences provided in the table can be used to taxonomy assignment.

 

The table can be downloaded from this link:

 
 

Sample Meta Information

#SampleIDSampleNameConditionDayWeekCombinationGroup
F6620.S001SupraC1InoculumCalgary00InoculumCalgary-0Week 0 InoculumCalgary
F6620.S002SupraC2InoculumCalgary00InoculumCalgary-0Week 0 InoculumCalgary
F6620.S003SupraC3InoculumCalgary00InoculumCalgary-0Week 0 InoculumCalgary
F6620.S004SupraT1Inoculum00Inoculum-0Week 0 Inoculum
F6620.S005SupraT2Inoculum00Inoculum-0Week 0 Inoculum
F6620.S006SupraT3Inoculum00Inoculum-0Week 0 Inoculum
F6620.S007FB1_4Foam_sqBefore41Foam_sqBefore-1Week 1 Foam_sqBefore
F6620.S008FB2_4Foam_sqBefore41Foam_sqBefore-1Week 1 Foam_sqBefore
F6620.S009FB3_4Foam_sqBefore41Foam_sqBefore-1Week 1 Foam_sqBefore
F6620.S010FA1_4Foam_sqAfter41Foam_sqAfter-1Week 1 Foam_sqAfter
F6620.S011FA2_4Foam_sqAfter41Foam_sqAfter-1Week 1 Foam_sqAfter
F6620.S012FA3_4Foam_sqAfter41Foam_sqAfter-1Week 1 Foam_sqAfter
F6620.S013B1_4Bead41Bead-1Week 1 Bead
F6620.S014B2_4Bead41Bead-1Week 1 Bead
F6620.S015B3_4Bead41Bead-1Week 1 Bead
F6620.S016L1_4Liquid41Liquid-1Week 1 Liquid
F6620.S017L2_4Liquid41Liquid-1Week 1 Liquid
F6620.S018L3_4Liquid41Liquid-1Week 1 Liquid
F6620.S019C1_4Calgary41Calgary-1Week 1 Calgary
F6620.S020C2_4Calgary41Calgary-1Week 1 Calgary
F6620.S021C3_4Calgary41Calgary-1Week 1 Calgary
F6620.S022CC1_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S023CC2_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S024CC3_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S025CC4_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S026CC5_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S027CC6_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S028CC7_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S029CC8_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S030CC9_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S031CC10_4Calgary_ctrlReps41Calgary_ctrlReps-1Week 1 Calgary_ctrlReps
F6620.S032FB1_11Foam_sqBefore112Foam_sqBefore-2Week 2 Foam_sqBefore
F6620.S033FB2_11Foam_sqBefore112Foam_sqBefore-2Week 2 Foam_sqBefore
F6620.S034FB3_11Foam_sqBefore112Foam_sqBefore-2Week 2 Foam_sqBefore
F6620.S035FA1_11Foam_sqAfter112Foam_sqAfter-2Week 2 Foam_sqAfter
F6620.S036FA2_11Foam_sqAfter112Foam_sqAfter-2Week 2 Foam_sqAfter
F6620.S037FA3_11Foam_sqAfter112Foam_sqAfter-2Week 2 Foam_sqAfter
F6620.S038B1_11Bead112Bead-2Week 2 Bead
F6620.S039B2_11Bead112Bead-2Week 2 Bead
F6620.S040B3_11Bead112Bead-2Week 2 Bead
F6620.S041L1_11Liquid112Liquid-2Week 2 Liquid
F6620.S042L2_11Liquid112Liquid-2Week 2 Liquid
F6620.S043L3_11Liquid112Liquid-2Week 2 Liquid
F6620.S044C1_11Calgary112Calgary-2Week 2 Calgary
F6620.S045C2_11Calgary112Calgary-2Week 2 Calgary
F6620.S046C3_11Calgary112Calgary-2Week 2 Calgary
F6620.S047FB1_18Foam_sqBefore183Foam_sqBefore-3Week 3 Foam_sqBefore
F6620.S048FB2_18Foam_sqBefore183Foam_sqBefore-3Week 3 Foam_sqBefore
F6620.S049FB3_18Foam_sqBefore183Foam_sqBefore-3Week 3 Foam_sqBefore
F6620.S050FA1_18Foam_sqAfter183Foam_sqAfter-3Week 3 Foam_sqAfter
F6620.S051FA2_18Foam_sqAfter183Foam_sqAfter-3Week 3 Foam_sqAfter
F6620.S052FA3_18Foam_sqAfter183Foam_sqAfter-3Week 3 Foam_sqAfter
F6620.S053B1_18Bead183Bead-3Week 3 Bead
F6620.S054B2_18Bead183Bead-3Week 3 Bead
F6620.S055B3_18Bead183Bead-3Week 3 Bead
F6620.S056L1_18Liquid183Liquid-3Week 3 Liquid
F6620.S057L2_18Liquid183Liquid-3Week 3 Liquid
F6620.S058L3_18Liquid183Liquid-3Week 3 Liquid
F6620.S059C1_18Calgary183Calgary-3Week 3 Calgary
F6620.S060C2_18Calgary183Calgary-3Week 3 Calgary
F6620.S061C3_18Calgary183Calgary-3Week 3 Calgary
F6620.S062FB1_25Foam_sqBefore254Foam_sqBefore-4Week 4 Foam_sqBefore
F6620.S063FB2_25Foam_sqBefore254Foam_sqBefore-4Week 4 Foam_sqBefore
F6620.S064FB3_25Foam_sqBefore254Foam_sqBefore-4Week 4 Foam_sqBefore
F6620.S065FA1_25Foam_sqAfter254Foam_sqAfter-4Week 4 Foam_sqAfter
F6620.S066FA2_25Foam_sqAfter254Foam_sqAfter-4Week 4 Foam_sqAfter
F6620.S067FA3_25Foam_sqAfter254Foam_sqAfter-4Week 4 Foam_sqAfter
F6620.S068B1_25Bead254Bead-4Week 4 Bead
F6620.S069B2_25Bead254Bead-4Week 4 Bead
F6620.S070B3_25Bead254Bead-4Week 4 Bead
F6620.S071L1_32Liquid325Liquid-5Week 5 Liquid
F6620.S072L2_25Liquid254Liquid-4Week 4 Liquid
F6620.S073L3_25Liquid254Liquid-4Week 4 Liquid
F6620.S074C1_25Calgary254Calgary-4Week 4 Calgary
F6620.S075C2_25Calgary254Calgary-4Week 4 Calgary
F6620.S076C3_25Calgary254Calgary-4Week 4 Calgary
F6620.S077FB1_32Foam_sqBefore325Foam_sqBefore-5Week 5 Foam_sqBefore
F6620.S078FB2_32Foam_sqBefore325Foam_sqBefore-5Week 5 Foam_sqBefore
F6620.S079FB3_32Foam_sqBefore325Foam_sqBefore-5Week 5 Foam_sqBefore
F6620.S080FA1_32Foam_sqAfter325Foam_sqAfter-5Week 5 Foam_sqAfter
F6620.S081FA2_32Foam_sqAfter325Foam_sqAfter-5Week 5 Foam_sqAfter
F6620.S082FA3_32Foam_sqAfter325Foam_sqAfter-5Week 5 Foam_sqAfter
F6620.S083B1_32Bead325Bead-5Week 5 Bead
F6620.S084B2_32Bead325Bead-5Week 5 Bead
F6620.S085B3_32Bead325Bead-5Week 5 Bead
F6620.S086L1_25Liquid254Liquid-4Week 4 Liquid
F6620.S087L2_32Liquid325Liquid-5Week 5 Liquid
F6620.S088L3_32Liquid325Liquid-5Week 5 Liquid
F6620.S089C1_32Calgary325Calgary-5Week 5 Calgary
F6620.S090C2_32Calgary325Calgary-5Week 5 Calgary
F6620.S091C3_32Calgary325Calgary-5Week 5 Calgary
F6620.S092FB1_39Foam_sqBefore396Foam_sqBefore-6Week 6 Foam_sqBefore
F6620.S093FB2_39Foam_sqBefore396Foam_sqBefore-6Week 6 Foam_sqBefore
F6620.S094FB3_39Foam_sqBefore396Foam_sqBefore-6Week 6 Foam_sqBefore
F6620.S095FA1_39Foam_sqAfter396Foam_sqAfter-6Week 6 Foam_sqAfter
F6620.S096FA2_39Foam_sqAfter396Foam_sqAfter-6Week 6 Foam_sqAfter
F6620.S097FA3_39Foam_sqAfter396Foam_sqAfter-6Week 6 Foam_sqAfter
F6620.S098B1_39Bead396Bead-6Week 6 Bead
F6620.S099B2_39Bead396Bead-6Week 6 Bead
F6620.S100B3_39Bead396Bead-6Week 6 Bead
F6620.S101L1_39Liquid396Liquid-6Week 6 Liquid
F6620.S102L2_39Liquid396Liquid-6Week 6 Liquid
F6620.S103L3_39Liquid396Liquid-6Week 6 Liquid
F6620.S104C1_39Calgary396Calgary-6Week 6 Calgary
F6620.S105C2_39Calgary396Calgary-6Week 6 Calgary
F6620.S106C3_39Calgary396Calgary-6Week 6 Calgary
F6620.S107FB1_46Foam_sqBefore467Foam_sqBefore-7Week 7 Foam_sqBefore
F6620.S108FB2_46Foam_sqBefore467Foam_sqBefore-7Week 7 Foam_sqBefore
F6620.S109FB3_46Foam_sqBefore467Foam_sqBefore-7Week 7 Foam_sqBefore
F6620.S110FA1_46Foam_sqAfter467Foam_sqAfter-7Week 7 Foam_sqAfter
F6620.S111FA2_46Foam_sqAfter467Foam_sqAfter-7Week 7 Foam_sqAfter
F6620.S112FA3_46Foam_sqAfter467Foam_sqAfter-7Week 7 Foam_sqAfter
F6620.S113B1_46Bead467Bead-7Week 7 Bead
F6620.S114B2_46Bead467Bead-7Week 7 Bead
F6620.S115B3_46Bead467Bead-7Week 7 Bead
F6620.S116L1_46Liquid467Liquid-7Week 7 Liquid
F6620.S117L2_46Liquid467Liquid-7Week 7 Liquid
F6620.S118L3_46Liquid467Liquid-7Week 7 Liquid
F6620.S119C1_46Calgary467Calgary-7Week 7 Calgary
F6620.S120C2_46Calgary467Calgary-7Week 7 Calgary
F6620.S121C3_46Calgary467Calgary-7Week 7 Calgary
F6620.S122FB1_53Foam_sqBefore538Foam_sqBefore-8Week 8 Foam_sqBefore
F6620.S123FB2_53Foam_sqBefore538Foam_sqBefore-8Week 8 Foam_sqBefore
F6620.S124FB3_53Foam_sqBefore538Foam_sqBefore-8Week 8 Foam_sqBefore
F6620.S125FA1_53Foam_sqAfter538Foam_sqAfter-8Week 8 Foam_sqAfter
F6620.S126FA2_53Foam_sqAfter538Foam_sqAfter-8Week 8 Foam_sqAfter
F6620.S127FA3_53Foam_sqAfter538Foam_sqAfter-8Week 8 Foam_sqAfter
F6620.S128B1_53Bead538Bead-8Week 8 Bead
F6620.S129B2_53Bead538Bead-8Week 8 Bead
F6620.S130B3_53Bead538Bead-8Week 8 Bead
F6620.S131L1_53Liquid538Liquid-8Week 8 Liquid
F6620.S132L2_53Liquid538Liquid-8Week 8 Liquid
F6620.S133L3_53Liquid538Liquid-8Week 8 Liquid
F6620.S134C1_53Calgary538Calgary-8Week 8 Calgary
F6620.S135C2_53Calgary538Calgary-8Week 8 Calgary
F6620.S136C3_53Calgary538Calgary-8Week 8 Calgary
F6620.S137CF1_53CalgaryFinaldislodged538CalgaryFinaldislodged-8Week 8 CalgaryFinaldislodged
F6620.S138CF2_53CalgaryFinaldislodged538CalgaryFinaldislodged-8Week 8 CalgaryFinaldislodged
F6620.S139CF3_53CalgaryFinaldislodged538CalgaryFinaldislodged-8Week 8 CalgaryFinaldislodged
 
 

ASV Read Counts by Samples

#Sample IDRead Count
F6620.S00610,507
F6620.S07611,998
F6620.S00712,287
F6620.S10612,756
F6620.S08312,787
F6620.S00312,874
F6620.S11913,030
F6620.S07313,467
F6620.S00413,678
F6620.S12713,680
F6620.S13613,918
F6620.S03013,983
F6620.S00114,217
F6620.S06914,280
F6620.S03114,628
F6620.S03614,698
F6620.S01214,746
F6620.S06615,048
F6620.S06015,113
F6620.S12515,370
F6620.S13415,625
F6620.S01815,639
F6620.S01115,639
F6620.S04415,700
F6620.S09115,755
F6620.S01015,776
F6620.S10415,814
F6620.S02915,992
F6620.S00216,130
F6620.S13716,256
F6620.S04316,428
F6620.S12816,444
F6620.S00516,451
F6620.S01516,505
F6620.S01716,557
F6620.S12016,637
F6620.S07016,800
F6620.S08616,857
F6620.S13916,896
F6620.S11016,919
F6620.S03716,958
F6620.S06716,964
F6620.S11517,105
F6620.S02317,134
F6620.S09517,183
F6620.S08017,348
F6620.S10217,471
F6620.S08417,531
F6620.S08217,597
F6620.S01317,606
F6620.S09217,644
F6620.S01617,657
F6620.S05117,660
F6620.S00817,708
F6620.S09817,725
F6620.S08817,745
F6620.S10317,781
F6620.S13017,786
F6620.S07117,855
F6620.S09018,030
F6620.S07218,045
F6620.S01418,068
F6620.S04918,151
F6620.S10118,211
F6620.S05618,278
F6620.S09318,293
F6620.S05518,329
F6620.S06818,419
F6620.S03818,506
F6620.S11718,606
F6620.S05218,643
F6620.S02018,654
F6620.S02118,710
F6620.S06118,817
F6620.S10018,828
F6620.S04118,864
F6620.S13518,894
F6620.S07819,253
F6620.S11419,260
F6620.S09919,310
F6620.S13119,333
F6620.S11119,369
F6620.S12119,391
F6620.S02819,397
F6620.S02419,406
F6620.S00919,421
F6620.S04219,519
F6620.S07719,536
F6620.S04719,536
F6620.S10519,571
F6620.S02619,616
F6620.S11219,641
F6620.S13219,734
F6620.S05019,815
F6620.S02719,817
F6620.S01919,833
F6620.S05819,838
F6620.S07519,936
F6620.S03520,113
F6620.S08720,126
F6620.S03420,177
F6620.S04820,340
F6620.S02220,380
F6620.S12320,407
F6620.S11820,429
F6620.S03920,711
F6620.S13320,790
F6620.S12420,824
F6620.S07420,973
F6620.S03221,032
F6620.S08121,219
F6620.S06521,276
F6620.S10721,375
F6620.S08521,848
F6620.S11621,989
F6620.S09422,114
F6620.S11322,253
F6620.S02522,379
F6620.S06322,387
F6620.S09622,560
F6620.S10822,597
F6620.S06222,614
F6620.S04622,768
F6620.S09722,947
F6620.S04523,004
F6620.S07923,068
F6620.S05423,292
F6620.S13823,310
F6620.S12923,415
F6620.S08923,428
F6620.S05723,491
F6620.S12224,282
F6620.S12624,326
F6620.S03324,346
F6620.S06424,677
F6620.S05325,188
F6620.S10925,441
F6620.S05925,500
F6620.S04026,564
 
 
 

VII. Analysis - Read Taxonomy Assignment

Read Taxonomy Assignment - Methods

 

The species-level, open-reference 16S rRNA NGS reads taxonomy assignment pipeline

Version 20210310
 

1. Raw sequences reads in FASTA format were BLASTN-searched against a combined set of 16S rRNA reference sequences. It consists of MOMD (version 0.1), the HOMD (version 15.2 http://www.homd.org/index.php?name=seqDownload&file&type=R ), HOMD 16S rRNA RefSeq Extended Version 1.1 (EXT), GreenGene Gold (GG) (http://greengenes.lbl.gov/Download/Sequence_Data/Fasta_data_files/gold_strains_gg16S_aligned.fasta.gz) , and the NCBI 16S rRNA reference sequence set (https://ftp.ncbi.nlm.nih.gov/blast/db/16S_ribosomal_RNA.tar.gz). These sequences were screened and combined to remove short sequences (<1000nt), chimera, duplicated and sub-sequences, as well as sequences with poor taxonomy annotation (e.g., without species information). This process resulted in 1,015 from HOMD V15.22, 495 from EXT, 3,940 from GG and 18,044 from NCBI, a total of 25,120 sequences. Altogether these sequence represent a total of 15,601 oral and non-oral microbial species.

The NCBI BLASTN version 2.7.1+ (Zhang et al, 2000) was used with the default parameters. Reads with ≥ 98% sequence identity to the matched reference and ≥ 90% alignment length (i.e., ≥ 90% of the read length that was aligned to the reference and was used to calculate the sequence percent identity) were classified based on the taxonomy of the reference sequence with highest sequence identity. If a read matched with reference sequences representing more than one species with equal percent identity and alignment length, it was subject to chimera checking with USEARCH program version v8.1.1861 (Edgar 2010). Non-chimeric reads with multi-species best hits were considered valid and were assigned with a unique species notation (e.g., spp) denoting unresolvable multiple species.

2. Unassigned reads (i.e., reads with < 98% identity or < 90% alignment length) were pooled together and reads < 200 bases were removed. The remaining reads were subject to the de novo operational taxonomy unit (OTU) calling and chimera checking using the USEARCH program version v8.1.1861 (Edgar 2010). The de novo OTU calling and chimera checking was done using 98% as the sequence identity cutoff, i.e., the species-level OTU. The output of this step produced species-level de novo clustered OTUs with 98% identity. Representative reads from each of the OTUs/species were then BLASTN-searched against the same reference sequence set again to determine the closest species for these potential novel species. These potential novel species were pooled together with the reads that were signed to specie-level in the previous step, for down-stream analyses.

Reference:
Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010 Oct 1;26(19):2460-1. doi: 10.1093/bioinformatics/btq461. Epub 2010 Aug 12. PubMed PMID: 20709691.

3. Designations used in the taxonomy:

	1) Taxonomy levels are indicated by these prefixes:
	
	   k__: domain/kingdom
	   p__: phylum
	   c__: class
	   o__: order
	   f__: family
	   g__: genus  
	   s__: species
	
	   Example: 
	
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Blautia;s__faecis
		
	2) Unique level identified – known species:
	   
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Roseburia;s__hominis
	
	   The above example shows some reads match to a single species (all levels are unique)
	
	3) Non-unique level identified – known species:

	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Roseburia;s__multispecies_spp123_3
	   
	   The above example “s__multispecies_spp123_3” indicates certain reads equally match to 3 species of the 
	   genus Roseburia; the “spp123” is a temporally assigned species ID.
	
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__multigenus;s__multispecies_spp234_5
	   
	   The above example indicates certain reads match equally to 5 different species, which belong to multiple genera.; 
	   the “spp234” is a temporally assigned species ID.
	
	4) Unique level identified – unknown species, potential novel species:
	   
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Roseburia;s__ hominis_nov_97%
	   
	   The above example indicates that some reads have no match to any of the reference sequences with 
	   sequence identity ≥ 98% and percent coverage (alignment length)  ≥ 98% as well. However this groups 
	   of reads (actually the representative read from a de novo  OTU) has 96% percent identity to 
	   Roseburia hominis, thus this is a potential novel species, closest to Roseburia hominis. 
	   (But they are not the same species).
	
	5) Multiple level identified – unknown species, potential novel species:
	   k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Roseburia;s__ multispecies_sppn123_3_nov_96%
	
	   The above example indicates that some reads have no match to any of the reference sequences 
	   with sequence identity ≥ 98% and percent coverage (alignment length)  ≥ 98% as well. 
	   However this groups of reads (actually the representative read from a de novo  OTU) 
	   has 96% percent identity equally to 3 species in Roseburia. Thus this is no single 
	   closest species, instead this group of reads match equally to multiple species at 96%. 
	   Since they have passed chimera check so they represent a novel species. “sppn123” is a 
	   temporary ID for this potential novel species.

 
4. The taxonomy assignment algorithm is illustrated in this flow char below:
 
 
 
 

Read Taxonomy Assignment - Result Summary *

CodeCategoryMPC=0% (>=1 read)MPC=0.01%(>=259 reads)
ATotal reads2,593,1812,593,181
BTotal assigned reads2,591,8582,591,858
CAssigned reads in species with read count < MPC09,395
DAssigned reads in samples with read count < 50000
ETotal samples139139
FSamples with reads >= 500139139
GSamples with reads < 50000
HTotal assigned reads used for analysis (B-C-D)2,591,8582,582,463
IReads assigned to single species2,493,5042,487,764
JReads assigned to multiple species90,91590,564
KReads assigned to novel species7,4394,135
LTotal number of species307144
MNumber of single species204129
NNumber of multi-species127
ONumber of novel species918
PTotal unassigned reads1,3231,323
QChimeric reads2323
RReads without BLASTN hits5151
SOthers: short, low quality, singletons, etc.1,2491,249
A=B+P=C+D+H+Q+R+S
E=F+G
B=C+D+H
H=I+J+K
L=M+N+O
P=Q+R+S
* MPC = Minimal percent (of all assigned reads) read count per species, species with read count < MPC were removed.
* Samples with reads < 500 were removed from downstream analyses.
* The assignment result from MPC=0.1% was used in the downstream analyses.
 
 
 

Read Taxonomy Assignment - ASV Species-Level Read Counts Table

This table shows the read counts for each sample (columns) and each species identified based on the ASV sequences. The downstream analyses were based on this table.
SPIDTaxonomyF6620.S001F6620.S002F6620.S003F6620.S004F6620.S005F6620.S006F6620.S007F6620.S008F6620.S009F6620.S010F6620.S011F6620.S012F6620.S013F6620.S014F6620.S015F6620.S016F6620.S017F6620.S018F6620.S019F6620.S020F6620.S021F6620.S022F6620.S023F6620.S024F6620.S025F6620.S026F6620.S027F6620.S028F6620.S029F6620.S030F6620.S031F6620.S032F6620.S033F6620.S034F6620.S035F6620.S036F6620.S037F6620.S038F6620.S039F6620.S040F6620.S041F6620.S042F6620.S043F6620.S044F6620.S045F6620.S046F6620.S047F6620.S048F6620.S049F6620.S050F6620.S051F6620.S052F6620.S053F6620.S054F6620.S055F6620.S056F6620.S057F6620.S058F6620.S059F6620.S060F6620.S061F6620.S062F6620.S063F6620.S064F6620.S065F6620.S066F6620.S067F6620.S068F6620.S069F6620.S070F6620.S071F6620.S072F6620.S073F6620.S074F6620.S075F6620.S076F6620.S077F6620.S078F6620.S079F6620.S080F6620.S081F6620.S082F6620.S083F6620.S084F6620.S085F6620.S086F6620.S087F6620.S088F6620.S089F6620.S090F6620.S091F6620.S092F6620.S093F6620.S094F6620.S095F6620.S096F6620.S097F6620.S098F6620.S099F6620.S100F6620.S101F6620.S102F6620.S103F6620.S104F6620.S105F6620.S106F6620.S107F6620.S108F6620.S109F6620.S110F6620.S111F6620.S112F6620.S113F6620.S114F6620.S115F6620.S116F6620.S117F6620.S118F6620.S119F6620.S120F6620.S121F6620.S122F6620.S123F6620.S124F6620.S125F6620.S126F6620.S127F6620.S128F6620.S129F6620.S130F6620.S131F6620.S132F6620.S133F6620.S134F6620.S135F6620.S136F6620.S137F6620.S138F6620.S139
SP1Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Stomatobaculum;longum935300005811103253353012082657001050000000000017700000151017600038237833800000380054000000000000520073038200000693006700002024160065000000000008800677000000272700000053100000008600126000491000000
SP10Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella;atypica000000015011914719419510896027023417800000000163000000075085000226303233000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP101Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Tannerella;sp. HMT28601131223502654838570850000250000000000000037044605388000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP102Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Eikenella;corrodens000000000000000000000005893545661550650000000000000024000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP103Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;oris23559001129400007100000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP104Bacteria;Firmicutes;Negativicutes;Veillonellales;Veillonellaceae;Veillonellaceae_[G-1];bacterium HMT12900000000000000000000000000000000000000000000000000000000000000121600336108301000000110004018160000018001000034800002734000000000000000000000000000000024011
SP105Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Fretibacterium;sp. HMT3600000000000000000000000000000000000000000000000000000046660000000000006100059000000000050000000490000012043710000095000000086747300000000000019829911900005900078
SP106Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;cristatus13012214910216517000012207186652931000021470000065060000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP107Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Solobacterium;moorei0000004042044008111413416075105233224268104180106172221238184012593000018022620236539087170454743828179157130122686649855146833730317080736538341245116713622022852425217337935823496178018512913712211219721345643710213793195256779215806361402902572510058238360140108110546055712891612107876185198168112566600190274118113400203113263021150209
SP108Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidales_[F-2];Bacteroidales_[G-2];sp._Oral_Taxon_2740001100759821799605538642101700000000000000831311396678924767961000131188194543682058485050000011750750221900000012513975000090000000000377879700015001200001187510873144064886283128000931061566635005803048560006211753504585
SP109Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;parasanguinis_I00000010352023214445312145393934231006222544153570000000300001030500000000007800000000000000000019000000000009000000000000000000000000000000000000000000000000000
SP11Bacteria;Firmicutes;Clostridia;Eubacteriales;Eubacteriales Family XIII. Incertae Sedis;Mogibacterium;vescum000014064222182289290242360359411262307254000000000017200991230209171183153191318414397225782532907849694969941621269225043108904937333834000021147185421613229500160015704165200450581220000000100115120021444617862000000159202124615220444115690800001281874200120211020650114
SP110Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;naeslundii1032011300129480000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP111Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Mogibacterium;timidum0000000006400000000000000000000960015000000000063617064515011035000000262919009390000000017010026367650000000000001000000000918038260180000000012250000000000000000
SP112Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-1];bacterium HMT9570000017000000301825000000000000000000000000360032622869000000000000013000000000014231700000000000000000000000000000000000000000000000000000000000000001115
SP113Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT21503849000447602161251491781882612042192561038000036590030000015006100028940000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP114Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;flueggei0000000000000000000000000000000000000000000000000000000000000000000000000000000054000000000083470038000000000097650022000000000058000490000000000000
SP115Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._Oral_Taxon_718321413818820816333633201284280000042733333149646351553151648765827422348600000000000000114000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP116Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-4];bacterium HMT36900000017514217717313784169181126070000000000000000485003134610105127024006255557053550620000000033300000432800000003525005347386600000000000005962000000006852046079620006200590220000585100010200210410
SP117Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;gingivalis95001171247900000000000000000000016000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP119Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-7];bacterium HMT0810000000000000000000000000000000000000001120005400000000257271950000000000000009000000000000000000000000000000000130000000117000000000000000000000028000
SP12Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;peroris4194353933914292990005962130000224000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP120Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;parasanguinis_II00000000000000000000000000000000000000010000000000001140000000000000010917990000500000000000860000000000000000000000000000000000000000000000000000
SP121Bacteria;Spirochaetes;Spirochaetia;Spirochaetales;Treponemataceae;Treponema;socranskii000001100462823000000000000000000000626135273500000000000000000000000000000000000000000000000000000400000000000000000000000000048014000000000000000000
SP122Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;mucosa015000000000000000000000000000000000000033600021100000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP123Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT1717310954711108100000000042000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP128Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Mogibacterium;neglectum00000040001431571784414311302800000000000174000000490000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP13Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-4];bacterium HMT10300000024032012700039000000000000000000000000000000000000003600000000000000000000000000000000000001921000300000000003100700000000420000000014000000000000
SP130Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;noxia00869077440000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP132Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp. HMT06475387356671896846700616410152784760000147157233195192133332181325252157124121000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP133Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT498170070560570000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP134Bacteria;Actinobacteria;Actinomycetia;Actinomycetales;Actinomycetaceae;Actinomyces;dentalis0017429620900000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP136Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT22194117991672141920000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP138Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;loescheii000170090000000005500000000000000000000000001191221410000000000000000000000000000000000000000000000000000000000000000000000000003600000000000000000000
SP14Bacteria;Firmicutes;Negativicutes;Veillonellales;Veillonellaceae;Veillonellaceae_[G-1];bacterium HMT15500000000000000000000000000000000000000000000001500000000000000214306216420185383213904400001461667608793016218500000601321401643406912312493000861580611821890000717800076000801690017625900001159187980102
SP140Bacteria;Firmicutes;Erysipelotrichia;Erysipelotrichales;Erysipelotrichaceae;Erysipelotrichaceae_[G-1];bacterium HMT9040000000000000000000000000000000000000000000000000000000000000053000020000000002132003238000000000600031260251100000000000000000000000000024320000000000
SP146Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;leadbetteri73680080450000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP148Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;wadei21735725966115176000000000090450000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP149Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Micrococcaceae;Rothia;dentocariosa1971799601351140000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP15Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;massiliensis3893672392871921650000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP151Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Eubacterium_[XI][G-7];yurii000000000000650035000000000000000000000104116160020000000000069000068000000000000000000000000095930000000000008369730000000000000000000000000000550000000000
SP153Bacteria;Spirochaetes;Spirochaetia;Spirochaetales;Spirochaetaceae;Treponema;maltophilum000000802100011000000000000000000376256141817001100001100000000000001800000000000170000000000000000022543001806700010000242115020001318000000272714012001500000001900000
SP154Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;Lautropia;mirabilis1301051241311781580000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP156Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Atopobiaceae;Olsenella;uli0000000000000000000000000000000000000000000000000000000000000000000000000000000004100000000000000000000007400000000000000111000000000000000010100
SP159Bacteria;Absconditabacteria_(SR1);Absconditabacteria_(SR1)_[C-1];Absconditabacteria_(SR1)_[O-1];Absconditabacteria_(SR1)_[F-1];Absconditabacteria_(SR1)_[G-1];bacterium HMT345000000027441900152600000000000000000255746121322000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP16Bacteria;Firmicutes;Clostridia;Eubacteriales;Eubacteriaceae;Pseudoramibacter;alactolyticus00000000000000000000000000000000501200000000000139156134292185188146944720825800050840040238124526101371153280023812401922274143825155652674055571598801180011314510818525119520621229208819100088725619424317715381100165016800121049551133194111118515912118520702800046
SP162Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;viscosus10301421811621630000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP163Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT17517726816916492420000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP164Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Stomatobaculum;sp. HMT09700000000075115645910869003300000000006000045004548000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP17Bacteria;Firmicutes;Negativicutes;Veillonellales;Veillonellaceae;Anaeroglobus;geminatus13000280130521331329944003952813130000000000000621107816113517907564330749535002881024638531336613792655008674161083435133700443014000022553563521328023049304053314112152645110214385487981419445729560673275104148754610290535570433612275233249263498542113538127744198510961092777819636236207165614841643015701791396967043879208262239
SP172Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._Oral_Taxon_B66112122115120111830000230048370000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP175Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;shahii011775606800000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP177Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;artemidis6344708469490000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP18Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Oribacterium;sinus000000002500000048580000000000000000000000445300064850000002506100032487700000000000000000000000000000000000000000000000000000000000000210000000000000510
SP182Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;tannerae78102358392590000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP184Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT41400196665800000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP19Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp. HMT2230000000000000000000000000000000011000000000000012401145100000000110031600322488299000550000040605164995314880000006300111115582021701620000490841906114283631631506500004000533694006122140000013203600005
SP2Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcaceae_[G-2];bacterium HMT08500000013728117022131232174166948128238633800000000001400037642564839000000170001600230230000000000001000000000000000090006900000000000000161000000000000001710000000000000086000000000
SP20Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XIII];Parvimonas;sp._Oral_Taxon_1100000000000009788262470073664459864142515241792394119910851520871105282131645454487184463094419642541199124220673150821164398054721441116516032225270917732340367629366645441023198111108159610178785945214901968172618652159198398900196422342560288513261392276234512619236531651997206600018032835296698712372643029563208201824012447811284271860273925348629816823702271223391296146427592381452881982358717641972893403259263519909585141382182042002151
SP22Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum9202843911779355370848132442923500072778137453024847464532711574361649144140457437032995236612535307224643756045920006684683101475213217382242720018819144064192420389368270158014420501240004953324063925520268113903144000019457000801706432421110841340013800690057262246421101700130011400108678803873796326170921319000001235948036215627810331546
SP24Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._str._2136FAA581271311031266302501631002425132825000000000000000001812000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP25Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;anginosus6459092104825344660999323762289437342701244924751891218118837005974123912553749084035733621922332451152411545101701066161667541205222952788522549994747265030822863115181217795131130610187103228240694798077862107258381153446410104361512015466184971372099241062814679684829838864656233108966333261410709122931307292158400799249725610595250059976769010718635158079913898516482884810510110102276193934241375075567784321648633583128169537182338557826992312627419349091075490988366190928666237133087214142616911107124822737230618549642107787810302520475182377099483745
SP28Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Oribacterium;sp. HMT102000000000004323752100350000000000000000001120990934432350360000430139921286906016580460059001130005318811259900650114104002467920016673344610009761000117495911816519420312656878780000570153157037815812015080147052005000002200635602120550000
SP29Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;Phocaeicola;abscessus00000000542312017000000000000000000171825552584576401479854770125419000002634146621402572463233291930000011812417222098120100212133000000340340480684975630000018938830417554919119515269920000014720454124491821538981118495997900000298299424261626559838690577519600008460044301930
SP3Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;parainfluenzae6276044554837932550000000000016142004583994820405246401207390274000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP30Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;adiacens813408514600000071050972131252651237670461031873221431821431202167200000000013422029312013319400000000017114195450850000000000320799819700000000055541998900000000000036646000000000000012000000000000006600000000
SP31Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;paradiacens43421653845177000000064015913721869610936211891163137124277604500000000019116723621614811400000000068900000000000000000100100171000000000317121000000000000000258123000000000000980000000000000097027000000
SP32Bacteria;Firmicutes;Clostridia;Eubacteriales;Peptostreptococcaceae;Peptoanaerobacter;[Eubacterium] yurii000000000001772971070272800000000000002452028314221013161615214612900001629180047110153025420000000007862310320014000000000366000050140000003878196000000043000000810230015000000000580000001400
SP33Bacteria;Actinobacteria;Actinomycetia;Propionibacteriales;Propionibacteriaceae;Propionibacterium;acidifaciens0000000000000000000000000000000000000000000000000000000000000000000000019413200000000000000011075819000084003100006459827350054711221098800000914117213891430117250404196000085015135991468215229371643
SP34Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium;periodonticum000000000000000000000016103621010023100000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP35Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;oralis0000001952182619787822051369272008733675756346827996225414093845552639354374556223618323316911521032538829651052364420331561202192088776410287847645676372932615115011282743321029401493133517097765037301188976824102611502632444184084673910198834951140124079093293382590470722101488149650011318846876363611645905417879758591972917580260379432598513343569630300650604801511381246221341200250274271465519337543435477443407475
SP36Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;oralis761791852874104362253466097781890867828552093230340382476269219101469190831813115241325252969215133731596146929044597357886708096461590239462692718307018482505287345613543015595505207724074322262897297939332942583330000670000075001706350000000063293600245000000000000424005600000000002792600116022300000000030211701071011464170
SP37Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;odontolyticus06710501090000625439000000000008851480034500000000000002100000000000012618615200000000000000000000000000096000000000000000320000000000000000000000000000000000000
SP38Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Staphylococcus;epidermidis0000000000000000000002482262700000770000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP39Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Atopobiaceae;Lancefieldella;rimae07000100068568271832640228000086148000000000000003232983742251721693500217344173143407141395402328268206147871020050563781201716804811550915000203173761990020718213015817726510088460013101801822191466889116130213185150151092509311717016014595127552171854862127159022912617006703705349650931040534873689082
SP4Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_ss_polymorphum16218303934512134853436042252191000347529314914464644043085149333924649387945423467287321861029274617327215149402000423247681275186316541331200002020001340018617200103001590460000005592436000001190000179055305940000000000007550000000000000871724068750000000000117882908117861000
SP40Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT3150000000000140000000000000000000000000000030434200000000000000000000000000000000000000000000002500000500000000020000000000000054361700000000000003515000
SP41Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;elongata058000370000000000000000000000000000000560182000117000000000000007064741580000000001301460000000000000000000000000000000000000000000000000000000000000000000
SP42Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Tannerella;sp. HMT808000000000000000000000000000000006700000000000000101100159131854845000000029362400286119000000000170001562745400000150002081851462382121000003510271011915023619823818874000022949420415312416622218309611100033178427249111
SP43Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;sp. HMT13600000000000000000000000000000000000000000004703600000000000000000000000000013014000000000001500000000000001817014000000000035025000000000000260000003720
SP44Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp. HMT0660000000000000000000000000000000000000000000000000000001000440000000000008123009400000000110015700900000000500100120000000000000000000000000000000000
SP45Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._str._C30013111425111013191625102535773815212719309839381391041731492071791571113441652792251105189000001500004801919000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP46Bacteria;Actinobacteria;Actinobacteria;Bifidobacteriales;Bifidobacteriaceae;Bifidobacterium;dentium00000000000000000000000000000000000000000000008284003600000000009201104838400002960000096653413651215270410250116183001760001334224848315512705181516774841700108619900933172970016342835209653780231100988112001696250682122532851226142448950000561107000487058
SP47Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIVa];Catonella;morbi000000831771031731851711711401553263622828117311132251513200045022233305169221190271118111362534646549134194217300010187360636215229617526717621513811371348852016103824725723548655114915220033821430433115827825239536744835424715821535720234144053426321614102932961252561661871783051721762242631871185073413321931972811175210220231316911214353469504154149219150222321267
SP48Bacteria;Firmicutes;Negativicutes;Selenomonadales;Selenomonadaceae;Selenomonas;sputigena7399287900000001504400000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP49Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sanguinis7021013605575684413144556794850102188713241402602601445077464444270170035111721361690005962440000040014800082440000001600460000000000026451520000050000001115020000000000000001419600001100000003912110000000460
SP5Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella;parvula309037492850250530471940591123493223832399187116291246233625262157233812351161128414741217816792874102012771192135915465778395011205109211435005972335149819741634154313361676250023013618700012131171114832110329200000520005935692241302137625021417292000000446180518801294162152000002000001032109094800000000000115914241619563432020000000006097446450012810610180
SP50Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;showae0320405300027120008057000000000350016300274166762600906915200061111109362602401534344300015812687000000000108520000000000000000094500000000000004661420000000000007858000000000000003538488555
SP51Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT44855923573101970000002906500000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP52Bacteria;Actinobacteria;Actinomycetia;Propionibacteriales;Propionibacteriaceae;Arachnia;propionica366201066010100180000000000000000000000000000000000000000000000000000000000000000000000000000000007800000000000011343000000000000000000000000000000098207161
SP53Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;mitis6433715240679101400032002222210000023753700006100000000000133000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP54Bacteria;Firmicutes;Erysipelotrichia;Erysipelotrichales;Erysipelotrichaceae;Erysipelotrichaceae_[G-1];bacterium HMT9050000000018011008160000000000000000789252494537220520000001000202629000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP55Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;concisus433204246354179110786578000131154279721779118831491551561626093480021000000011717610645576400000000000000000000000000000280000000000007000000000000011447000000000000081540000000000000051000000
SP56Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;Enterobacter;sp._str._HCB0000000000000000000000000000000000000126513180000063500000001155126500000394000000019381700988015900000000005486737300000000000001445130711780000000000001966141611380012807030062100001648239720630084001592000290
SP57Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcus;stomatis0000001252164612891895200215591452159917562192353840005380019123835831530735117712679971169109811101181130520896435934320283149169619921867168017641736193517071643302242502414634304774791733053703461861022262233250000683764456397594260023400001422811285385202224183711473351406280018247514747849633239641121547601820005387212551352891451651951135830388000132382112329301468
SP58Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Coriobacteriaceae;Atopobium;rimae000540016117916277791440005364000000000000001168010159149200074862710910927103115431145789420000099507100092047000408301240061746201015509112068870985124467100013582212600630945851187202624030651021502132250000000000064039060410
SP59Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;constellatus0000000020035019024000000000000000000702228401000093140214200611075599601001814420019901042737085022959437836000002431510637103117016036300097429161851035800330413212357269000418138501778366726914524744142916520979002641247721531220421431552262829517317237800433518241967281715322490
SP6Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._Oral_Taxon_18026010511631817924635119101163167102110193131132146000000000000000004900000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP60Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;sp. HMT338000000014121373511001013500480000170203422000002900330470004362978117104000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP61Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Dialister;invisus220035000003727048713600000000000000000002201805462000000567762989479757393441900000001004028000000000271565698895000000000423707810394000000320040450878067000000000430090178670000000901000
SP62Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Fretibacterium;fastidiosum00000000240013000000000000000000002653051320676800000000320005154000016696680000000000000000000000000000000000000000002400000016000000028710360001800000000109109112121226190
SP63Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;salivae035059001843862473012181605412510240542118284183000018002690893100500360230000430000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP64Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIVa];Shuttleworthia;satelles0000000000000017000000000000000000000000039875700000001300000059000008000008003700011300003200590000000000000003500740000002400000000000000000000000000000
SP65Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;gracilis290373803302600000000000000000000000013000000000000000000000000000001927014000000030006040554463591902500003705243965821323050330047330003565201501514102809300065544782847600062042000000
SP66Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-1];[Eubacterium]_infirmum0000003617643557457387231677234921874303343540532000062601560000566534341825707778463565184913781378892184344376394418263503495500805640882372615868482227343332262250270210288362175457319519241600298644324295493490712629362438670173456559455362255260389313393420355455372378036469621442130746535840953845940365131327240044578834924537523128321228733921837526214523629665624172217145236168
SP67Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;dentalis0000000180007281614000000000000000000000140020000000000000220000000000000000000000000000033000000000000004945000000000000000000000000010002800000500000
SP68Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-3];bacterium HMT3510000004567065705311710117543825900000000000000000029002042220000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP7Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;mutans00000000000000000000000000000000000000000000000000000000000000000000000005700053140000001300000089000000000000001344000000000000004000000000000000
SP71Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;vestibularis02200000000059000020690000182188049001952980000200000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP73Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;saccharolytica000000148265320136148202282262286131149902623372532701732702722282090141141084126119080481441132461251501360143249001923170000000360540000000007210100000010613211900000000891000100012217400000001181211110929398126012200000749811202757706106700000964210748880
SP76Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;sp. HMT317111115001520000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000044370000000000000
SP77Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;intermedius1581124810016711024562276577632532769111981030013191620161715267140210102517300282122170300001100000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP78Bacteria;Firmicutes;Clostridia;Clostridiales;Peptoniphilaceae;Parvimonas;micra07473888574015970177134113213275007600000003000000008300000000000001460244000000000000759195510144000000191000094962850902550000000012108031169796000000582001451280858986712011300000009900162285386504200000000000582132878
SP79Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;israelii01360254310005500000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000001400
SP8Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterobacteriaceae;Klebsiella;aerogenes0000000000000232000001311000000000000000005844597900000214800000005366492000000148900000009824817836140902000000000018512038301600002020000000486163514485000065100000006709694953700010810235300306500008438128219506003212068930001205
SP80Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIVa];Oribacterium;sp._Oral_Taxon_78000030000011588850600470340000000000000230048416800000000000035000000000000000009713415703201120000490596084021407294393413000000143197180005475870002654112691250020050521006100000431276900050078022484257
SP82Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Corynebacteriaceae;Corynebacterium;matruchotii030903672182760000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP83Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;oralis_subsp._tigurinus_clade_07025237737151745631310912091192224167215232194551632151060102613611623179418742364111613281896150915141835001217171900000000210001600000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP84Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Coriobacteriaceae;Slackia;exigua0000002793390000271361001031610000000000000029800001860826001400401001670000000000017100000000036804480003580650023500000000023200000000023600000020200001162000000065600000001060000187053458287600
SP85Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-2];bacterium HMT091000000004104756645949000000000000000003400003007602800000003100510590000029000000000000000000032000180000000024203100000000000000000000029290015000250000000000000
SP86Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-1];[Eubacterium]_sulci0000000000000001000252387712001221491833310150761170000000000005002500000000000000880000000016000000000000000000000000000000000000000000000000000000000000000000000
SP87Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;oris10814501181531861300056022621631700000000000000000000000062000000000000000000000000000000000710000034000000003800000000003000000004379000003466000000037000000000000000
SP9Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_ss_animalis000000000000610321472113517065100717155412766053815890607247745124411396774585104123312502705911037962926955905129934533045331218529601441365505363943442418082752212522132461424700916685649679007344886424983760225000181391756807647591497544378601747622322002710694570473497525649218492420484194025938410955424154285841447152766348470148036485722542623227567352
SP90Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Megasphaera;micronuciformis000017003100320395006761000000000002400000000000000261212117000000000000000000000000780000000000000000002400000000000003080000000000000000000000000000000000
SP91Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;oulorum420203891297471100216998610044400415600000000000000080000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP93Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_subsp._vincentii00000960193106700004112583000000000000029243011769000025044000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SP94Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;buccae00000000430051607004500006522522091632260020084191000000000000780000000002123000650380000000082000000000000000730000000000000000000000000000000000670000000009400000000
SP95Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;gordonii3572712862413731833155743596057237166897625991944495151190151839041922328919729922634031715036115126035030824827545902872982453676151629058571901600102207405806050000000076752366039800183308000000158342206340390347000000005610058501221590000000018500885002270000000000018400081416158
SP96Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;melaninogenica1792811361351811530000004465995257940000000024025000000000000061000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SPN11Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;anginosus_nov_95.749%000000036351402123720019016000000000000023437512000313804930253701101900001100380000000000000000000000000000170000000000000018000000000000002000000000000002900000000
SPN15Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;viscosus_nov_96.023%56144610148103000092000000000000000000000190000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SPN26Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp. HMT175 nov_97.822%146948718258430000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SPN3Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._str._2136FAA_nov_97.227%00000000520006065048000002245260000001605001501400000072817100046360000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SPN37Bacteria;Saccharibacteria_(TM7);Saccharibacteria_(TM7)_[C-1];Saccharibacteria_(TM7)_[O-1];Saccharibacteria_(TM7)_[F-1];Saccharibacteria_(TM7)_[G-1];bacterium HMT957 nov_97.639%000044000000029363513023000000000000000000013039110214233140000000000002802300000000001719000000000000000000000000000000000000000000000000000000000000000080
SPN47Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;anginosus_nov_97.597%0000000000000000000000000000000000000000000000000000000000000013000120006733390000070036000000450000002100000000000020001400870000000300000000001400280
SPN59Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;massiliensis_nov_96.357%93062936700000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
SPN69Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;constellatus_nov_97.393%000000000000000000000000000000000000000000000000000000000000002300000001921000000000000000000240000080713000000002600070000000000002300002100280004724
SPP10Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacterales;Enterobacteriaceae;Raoultella;multispecies_spp10_2000000000000010500000000000000000000000809892000003590000000713684000002490000000126398450300000000000016924748000000000000061981363400002400000009508276660000339003720000102617121161004060882000164
SPP12Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Oribacterium;multispecies_spp12_20000000000007062000000000000000000000000010067011623054000000575100018000982190008125320820700002019504300008531718207195510000000018023919105800000000002351411110286600000000088112004384000000
SPP2Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;multifamily;multigenus;multispecies_spp2_24105200700120558071014612011521673120205112001298297200108602278310800000000120000001960000000026000000000000008200000410000000000010500000000000000000000000000000107000000000000000000000
SPP6Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;multispecies_spp6_30000000000000004742870006041540540003455331611421700003362434242680000000001321000240000000005000000009400000000428201100000000000025000000000000002300000272600000000004300
SPP7Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;multispecies_spp7_200000000000007184999874121700037885513989210001316046516116554432864784614197333633705556366709824323535314961904333717919430861476370341566308163400411248230181101062383783010892644108167046463674570290766019994210928208916547798168436206988858863950515330397964819646734989757310459391549318037005427146486607966576603426248852022010312322428510559437441383435
SPP8Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;multispecies_spp8_260840520390443111394510462511638000000000000000000000000000000000000000000000000000001600000000000000000000000000000000000000000000000000000000000000000000
SPP9Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;multispecies_spp9_24280081590003482000001871585017216816415101709110849119216895000000000939915388501280000000000730000000000000620045137770000000002770960000000000000168000000000000025089000000000000000000000
 
 
Download OTU Tables at Different Taxonomy Levels
PhylumCount*: Relative**: CLR***:
ClassCount*: Relative**: CLR***:
OrderCount*: Relative**: CLR***:
FamilyCount*: Relative**: CLR***:
GenusCount*: Relative**: CLR***:
SpeciesCount*: Relative**: CLR***:
* Read count
** Relative abundance (count/total sample count)
*** Centered log ratio transformed abundance
;
 
The species listed in the table has full taxonomy and a dynamically assigned species ID specific to this report. When some reads match with the reference sequences of more than one species equally (i.e., same percent identiy and alignmnet coverage), they can't be assigned to a particular species. Instead, they are assigned to multiple species with the species notaton "s__multispecies_spp2_2". In this notation, spp2 is the dynamic ID assigned to these reads that hit multiple sequences and the "_2" at the end of the notation means there are two species in the spp2.

You can look up which species are included in the multi-species assignment, in this table below:
 
 
 
 
Another type of notation is "s__multispecies_sppn2_2", in which the "n" in the sppn2 means it's a potential novel species because all the reads in this species have < 98% idenity to any of the reference sequences. They were grouped together based on de novo OTU clustering at 98% identity cutoff. And then a representative sequence was chosed to BLASTN search against the reference database to find the closest match (but will still be < 98%). This representative sequence also matched equally to more than one species, hence the "spp" was given in the label.
 
 

Taxonomy Bar Plots for All Samples

 
 

Taxonomy Bar Plots for Individual Comparison Groups

 
 
Comparison No.Comparison NameFamiliesGeneraSpecies
Comparison 1Week 0 InoculumCalgary vs Week 0 InoculumPDFSVGPDFSVGPDFSVG
Comparison 2Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlRepsPDFSVGPDFSVGPDFSVG
Comparison 3Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 4Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 5Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 6Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 7Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 8Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 9Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodgedPDFSVGPDFSVGPDFSVG
 
 

VIII. Analysis - Alpha Diversity

 

In ecology, alpha diversity (α-diversity) is the mean species diversity in sites or habitats at a local scale. The term was introduced by R. H. Whittaker[1][2] together with the terms beta diversity (β-diversity) and gamma diversity (γ-diversity). Whittaker's idea was that the total species diversity in a landscape (gamma diversity) is determined by two different things, the mean species diversity in sites or habitats at a more local scale (alpha diversity) and the differentiation among those habitats (beta diversity).


References:
Whittaker, R. H. (1960) Vegetation of the Siskiyou Mountains, Oregon and California. Ecological Monographs, 30, 279–338. doi:10.2307/1943563
Whittaker, R. H. (1972). Evolution and Measurement of Species Diversity. Taxon, 21, 213-251. doi:10.2307/1218190

 

Alpha Diversity Analysis by Rarefaction

Diversity measures are affected by the sampling depth. Rarefaction is a technique to assess species richness from the results of sampling. Rarefaction allows the calculation of species richness for a given number of individual samples, based on the construction of so-called rarefaction curves. This curve is a plot of the number of species as a function of the number of samples. Rarefaction curves generally grow rapidly at first, as the most common species are found, but the curves plateau as only the rarest species remain to be sampled.


References:
Willis AD. Rarefaction, Alpha Diversity, and Statistics. Front Microbiol. 2019 Oct 23;10:2407. doi: 10.3389/fmicb.2019.02407. PMID: 31708888; PMCID: PMC6819366.

 
 
 

Boxplot of Alpha-diversity Indices

The two main factors taken into account when measuring diversity are richness and evenness. Richness is a measure of the number of different kinds of organisms present in a particular area. Evenness compares the similarity of the population size of each of the species present. There are many different ways to measure the richness and evenness. These measurements are called "estimators" or "indices". Below is a diversity of 3 commonly used indices showing the values for all the samples (dots) and in groups (boxes).

 
Alpha Diversity Box Plots for All Groups
 
 
 
 
 
 
 
Alpha Diversity Box Plots for Individual Comparisons
 
Comparison 1Week 0 InoculumCalgary vs Week 0 InoculumView in PDFView in SVG
Comparison 2Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlRepsView in PDFView in SVG
Comparison 3Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 CalgaryView in PDFView in SVG
Comparison 4Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 CalgaryView in PDFView in SVG
Comparison 5Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 CalgaryView in PDFView in SVG
Comparison 6Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 CalgaryView in PDFView in SVG
Comparison 7Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 CalgaryView in PDFView in SVG
Comparison 8Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 CalgaryView in PDFView in SVG
Comparison 9Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodgedView in PDFView in SVG
 
 
 

Group Significance of Alpha-diversity Indices

To test whether the alpha diversity among different comparison groups are different statisticall, we use the Kruskal Wallis H test provided the "alpha-group-significance" fucntion in the QIIME 2 diversity package. Kruskal Wallis H test is the non parametric alternative to the One Way ANOVA. Non parametric means that the test doesn’t assume your data comes from a particular distribution. The H test is used when the assumptions for ANOVA aren’t met (like the assumption of normality). It is sometimes called the one-way ANOVA on ranks, as the ranks of the data values are used in the test rather than the actual data points. The test determines whether the medians of two or more groups are different.

Below are the Kruskal Wallis H test results for each comparison based on three different alpha diversity measures: 1) Observed species (features), 2) Shannon index, and 3) Simpson index.

 
 
Comparison 1.Week 0 InoculumCalgary vs Week 0 InoculumObserved FeaturesShannon IndexSimpson Index
Comparison 2.Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlRepsObserved FeaturesShannon IndexSimpson Index
Comparison 3.Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 CalgaryObserved FeaturesShannon IndexSimpson Index
Comparison 4.Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 CalgaryObserved FeaturesShannon IndexSimpson Index
Comparison 5.Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 CalgaryObserved FeaturesShannon IndexSimpson Index
Comparison 6.Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 CalgaryObserved FeaturesShannon IndexSimpson Index
Comparison 7.Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 CalgaryObserved FeaturesShannon IndexSimpson Index
Comparison 8.Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 CalgaryObserved FeaturesShannon IndexSimpson Index
Comparison 9.Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodgedObserved FeaturesShannon IndexSimpson Index
 
 

IX. Analysis - Beta Diversity

 

NMDS and PCoA Plots

Beta diversity compares the similarity (or dissimilarity) of microbial profiles between different groups of samples. There are many different similarity/dissimilarity metrics. In general, they can be quantitative (using sequence abundance, e.g., Bray-Curtis or weighted UniFrac) or binary (considering only presence-absence of sequences, e.g., binary Jaccard or unweighted UniFrac). They can be even based on phylogeny (e.g., UniFrac metrics) or not (non-UniFrac metrics, such as Bray-Curtis, etc.).

For microbiome studies, species profiles of samples can be compared with the Bray-Curtis dissimilarity, which is based on the count data type. The pair-wise Bray-Curtis dissimilarity matrix of all samples can then be subject to either multi-dimensional scaling (MDS, also known as PCoA) or non-metric MDS (NMDS).

MDS/PCoA is a scaling or ordination method that starts with a matrix of similarities or dissimilarities between a set of samples and aims to produce a low-dimensional graphical plot of the data in such a way that distances between points in the plot are close to original dissimilarities.

NMDS is similar to MDS, however it does not use the dissimilarities data, instead it converts them into the ranks and use these ranks in the calculation.

In our beta diversity analysis, Bray-Curtis dissimilarity matrix was first calculated and then plotted by the PCoA and NMDS separately. Below are beta diveristy results for all groups together:

 
 
NMDS and PCoA Plots for All Groups
 
 
 
 
 

The above PCoA and NMDS plots are based on count data. The count data can also be transformed into centered log ratio (CLR) for each species. The CLR data is no longer count data and cannot be used in Bray-Curtis dissimilarity calculation. Instead CLR can be compared with Euclidean distances. When CLR data are compared by Euclidean distance, the distance is also called Aitchison distance.

Below are the NMDS and PCoA plots of the Aitchison distances of the samples:

 
 
 
 
 
 
 
NMDS and PCoA Plots for Individual Comparisons
 
 
Comparison No.Comparison NameNMDAPCoA
Bray-CurtisCLR EuclideanBray-CurtisCLR Euclidean
Comparison 1Week 0 InoculumCalgary vs Week 0 InoculumPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 2Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlRepsPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 3Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 CalgaryPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 4Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 CalgaryPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 5Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 CalgaryPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 6Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 CalgaryPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 7Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 CalgaryPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 8Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 CalgaryPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 9Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodgedPDFSVGPDFSVGPDFSVGPDFSVG
 
 
 
 
 

Interactive 3D PCoA Plots - Bray-Curtis Dissimilarity

 
 
 

Interactive 3D PCoA Plots - Euclidean Distance

 
 
 

Interactive 3D PCoA Plots - Correlation Coefficients

 
 
 

Group Significance of Beta-diversity Indices

To test whether the between-group dissimilarities are significantly greater than the within-group dissimilarities, the "beta-group-significance" function provided in the QIIME 2 "diversity" package was used with PERMANOVA (permutational multivariate analysis of variance) chosen s the group significan testing method.

Three beta diversity matrics were used: 1) Bray–Curtis dissimilarity 2) Correlation coefficient matrix , and 3) Aitchison distance (Euclidean distance between clr-transformed compositions).

 
 
Comparison 1.Week 0 InoculumCalgary vs Week 0 InoculumBray–CurtisCorrelationAitchison
Comparison 2.Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlRepsBray–CurtisCorrelationAitchison
Comparison 3.Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 CalgaryBray–CurtisCorrelationAitchison
Comparison 4.Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 CalgaryBray–CurtisCorrelationAitchison
Comparison 5.Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 CalgaryBray–CurtisCorrelationAitchison
Comparison 6.Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 CalgaryBray–CurtisCorrelationAitchison
Comparison 7.Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 CalgaryBray–CurtisCorrelationAitchison
Comparison 8.Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 CalgaryBray–CurtisCorrelationAitchison
Comparison 9.Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodgedBray–CurtisCorrelationAitchison
 
 
 

X. Analysis - Differential Abundance

16S rRNA next generation sequencing (NGS) generates a fixed number of reads that reflect the proportion of different species in a sample, i.e., the relative abundance of species, instead of the absolute abundance. In Mathematics, measurements involving probabilities, proportions, percentages, and ppm can all be thought of as compositional data. This makes the microbiome read count data “compositional” (Gloor et al, 2017). In general, compositional data represent parts of a whole which only carry relative information (http://www.compositionaldata.com/).

The problem of microbiome data being compositional arises when comparing two groups of samples for identifying “differentially abundant” species. A species with the same absolute abundance between two conditions, its relative abundances in the two conditions (e.g., percent abundance) can become different if the relative abundance of other species change greatly. This problem can lead to incorrect conclusion in terms of differential abundance for microbial species in the samples.

When studying differential abundance (DA), the current better approach is to transform the read count data into log ratio data. The ratios are calculated between read counts of all species in a sample to a “reference” count (e.g., mean read count of the sample). The log ratio data allow the detection of DA species without being affected by percentage bias mentioned above

In this report, a compositional DA analysis tool “ANCOM” (analysis of composition of microbiomes) was used. ANCOM transforms the count data into log-ratios and thus is more suitable for comparing the composition of microbiomes in two or more populations. "ANCOM" generates a table of features with W-statistics and whether the null hypothesis is rejected. The “W” is the W-statistic, or number of features that a single feature is tested to be significantly different against. Hence the higher the "W" the more statistical sifgnificane that a feature/species is differentially abundant.


References:

Gloor GB, Macklaim JM, Pawlowsky-Glahn V, Egozcue JJ. Microbiome Datasets Are Compositional: And This Is Not Optional. Front Microbiol. 2017 Nov 15;8:2224. doi: 10.3389/fmicb.2017.02224. PMID: 29187837; PMCID: PMC5695134.

Mandal S, Van Treuren W, White RA, Eggesbø M, Knight R, Peddada SD. Analysis of composition of microbiomes: a novel method for studying microbial composition. Microb Ecol Health Dis. 2015 May 29;26:27663. doi: 10.3402/mehd.v26.27663. PMID: 26028277; PMCID: PMC4450248.

Lin H, Peddada SD. Analysis of compositions of microbiomes with bias correction. Nat Commun. 2020 Jul 14;11(1):3514. doi: 10.1038/s41467-020-17041-7. PMID: 32665548; PMCID: PMC7360769.

 
 

ANCOM Differential Abundance Analysis

 
ANCOM Results for Individual Comparisons
Comparison No.Comparison Name
Comparison 1.Week 0 InoculumCalgary vs Week 0 Inoculum
Comparison 2.Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlReps
Comparison 3.Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 Calgary
Comparison 4.Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 Calgary
Comparison 5.Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 Calgary
Comparison 6.Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 Calgary
Comparison 7.Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 Calgary
Comparison 8.Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 Calgary
Comparison 9.Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodged
 
 

ANCOM-BC Differential Abundance Analysis

 

Starting with version V1.2, we also include the results of ANCOM-BC (Analysis of Compositions of Microbiomes with Bias Correction) (Lin and Peddada 2020). ANCOM-BC is an updated version of "ANCOM" that: (a) provides statistically valid test with appropriate p-values, (b) provides confidence intervals for differential abundance of each taxon, (c) controls the False Discovery Rate (FDR), (d) maintains adequate power, and (e) is computationally simple to implement. The bias correction (BC) addresses a challenging problem of the bias introduced by differences in the sampling fractions across samples. This bias has been a major hurdle in performing DA analysis of microbiome data. ANCOM-BC estimates the unknown sampling fractions and corrects the bias induced by their differences among samples. The absolute abundance data are modeled using a linear regression framework.

References:

Lin H, Peddada SD. Analysis of compositions of microbiomes with bias correction. Nat Commun. 2020 Jul 14;11(1):3514. doi: 10.1038/s41467-020-17041-7. PMID: 32665548; PMCID: PMC7360769.

 
 
ANCOM-BC Results for Individual Comparisons
 
Comparison No.Comparison Name
Comparison 1.Week 0 InoculumCalgary vs Week 0 Inoculum
Comparison 2.Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlReps
Comparison 3.Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 Calgary
Comparison 4.Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 Calgary
Comparison 5.Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 Calgary
Comparison 6.Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 Calgary
Comparison 7.Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 Calgary
Comparison 8.Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 Calgary
Comparison 9.Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodged
 
 
 

LEfSe - Linear Discriminant Analysis Effect Size

LEfSe (Linear Discriminant Analysis Effect Size) is an alternative method to find "organisms, genes, or pathways that consistently explain the differences between two or more microbial communities" (Segata et al., 2011). Specifically, LEfSe uses rank-based Kruskal-Wallis (KW) sum-rank test to detect features with significant differential (relative) abundance with respect to the class of interest. Since it is rank-based, instead of proportional based, the differential species identified among the comparison groups is less biased (than percent abundance based).

Reference:

Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C. Metagenomic biomarker discovery and explanation. Genome Biol. 2011 Jun 24;12(6):R60. doi: 10.1186/gb-2011-12-6-r60. PMID: 21702898; PMCID: PMC3218848.

 
Week 0 InoculumCalgary vs Week 0 Inoculum
 
 
 
 
 
 
 
LEfSe Results for All Comparisons
 
Comparison No.Comparison Name
Comparison 1.Week 0 InoculumCalgary vs Week 0 Inoculum
Comparison 2.Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlReps
Comparison 3.Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 Calgary
Comparison 4.Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 Calgary
Comparison 5.Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 Calgary
Comparison 6.Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 Calgary
Comparison 7.Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 Calgary
Comparison 8.Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 Calgary
Comparison 9.Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodged
 
 

XI. Analysis - Heatmap Profile

 

Species vs Sample Abundance Heatmap for All Samples

 
 
 

Heatmaps for Individual Comparisons

 
A) Two-way clustering - clustered on both columns (Samples) and rows (organism)
Comparison No.Comparison NameFamily LevelGenus LevelSpecies Level
Comparison 1Week 0 InoculumCalgary vs Week 0 InoculumPDFSVGPDFSVGPDFSVG
Comparison 2Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlRepsPDFSVGPDFSVGPDFSVG
Comparison 3Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 4Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 5Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 6Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 7Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 8Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 9Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodgedPDFSVGPDFSVGPDFSVG
 
 
B) One-way clustering - clustered on rows (organism) only
Comparison No.Comparison NameFamily LevelGenus LevelSpecies Level
Comparison 1Week 0 InoculumCalgary vs Week 0 InoculumPDFSVGPDFSVGPDFSVG
Comparison 2Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlRepsPDFSVGPDFSVGPDFSVG
Comparison 3Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 4Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 5Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 6Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 7Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 8Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 9Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodgedPDFSVGPDFSVGPDFSVG
 
 
C) No clustering
Comparison No.Comparison NameFamily LevelGenus LevelSpecies Level
Comparison 1Week 0 InoculumCalgary vs Week 0 InoculumPDFSVGPDFSVGPDFSVG
Comparison 2Week 1 Foam_sqBefore vs Week 1 Foam_sqAfter vs Week 1 Bead vs Week 1 Liquid vs Week 1 Calgary vs Week 1 Calgary_ctrlRepsPDFSVGPDFSVGPDFSVG
Comparison 3Week 2 Foam_sqBefore vs Week 2 Foam_sqAfter vs Week 2 Bead vs Week 2 Liquid vs Week 2 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 4Week 3 Foam_sqBefore vs Week 3 Foam_sqAfter vs Week 3 Bead vs Week 3 Liquid vs Week 3 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 5Week 4 Foam_sqBefore vs Week 4 Foam_sqAfter vs Week 4 Bead vs Week 4 Liquid vs Week 4 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 6Week 5 Foam_sqBefore vs Week 5 Foam_sqAfter vs Week 5 Bead vs Week 5 Liquid vs Week 5 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 7Week 6 Foam_sqBefore vs Week 6 Foam_sqAfter vs Week 6 Bead vs Week 6 Liquid vs Week 6 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 8Week 7 Foam_sqBefore vs Week 7 Foam_sqAfter vs Week 7 Bead vs Week 7 Liquid vs Week 7 CalgaryPDFSVGPDFSVGPDFSVG
Comparison 9Week 8 Foam_sqBefore vs Week 8 Foam_sqAfter vs Week 8 Bead vs Week 8 Liquid vs Week 8 Calgary vs Week 8 CalgaryFinaldislodgedPDFSVGPDFSVGPDFSVG
 
 

XII. Analysis - Network Association

To analyze the co-occurrence or co-exclusion between microbial species among different samples, network correlation analysis tools are usually used for this purpose. However, microbiome count data are compositional. If count data are normalized to the total number of counts in the sample, the data become not independent and traditional statistical metrics (e.g., correlation) for the detection of specie-species relationships can lead to spurious results. In addition, sequencing-based studies typically measure hundreds of OTUs (species) on few samples; thus, inference of OTU-OTU association networks is severely under-powered. Here we use SPIEC-EASI (SParse InversE Covariance Estimation for Ecological Association Inference), a statistical method for the inference of microbial ecological networks from amplicon sequencing datasets that addresses both of these issues (Kurtz et al., 2015). SPIEC-EASI combines data transformations developed for compositional data analysis with a graphical model inference framework that assumes the underlying ecological association network is sparse. SPIEC-EASI provides two algorithms for network inferencing – 1) Meinshausen-Bühlmann's neighborhood selection (MB method) and inverse covariance selection (GLASSO method, i.e., graphical least absolute shrinkage and selection operator). This is fundamentally distinct from SparCC, which essentially estimate pairwise correlations. In addition to these two methods, we provide the results of a third method - SparCC (Sparse Correlations for Compositional Data)(Friedman & Alm 2012), which is also a method for inferring correlations from compositional data. SparCC estimates the linear Pearson correlations between the log-transformed components.


References:

Kurtz ZD, Müller CL, Miraldi ER, Littman DR, Blaser MJ, Bonneau RA. Sparse and compositionally robust inference of microbial ecological networks. PLoS Comput Biol. 2015 May 7;11(5):e1004226. doi: 10.1371/journal.pcbi.1004226. PMID: 25950956; PMCID: PMC4423992.

Friedman J, Alm EJ. Inferring correlation networks from genomic survey data. PLoS Comput Biol. 2012;8(9):e1002687. doi: 10.1371/journal.pcbi.1002687. Epub 2012 Sep 20. PMID: 23028285; PMCID: PMC3447976.

 

SPIEC-EASI Network Inference by Neighborhood Selection (MB Method)

 
 

 

Association Network Inference by SparCC

 
 

 
 
Copyright FOMC 2022