FOMC Service Report

16S rRNA Gene V1V3 Amplicon Sequencing

Version V1.41 fork

Version History

The Forsyth Institute, Cambridge, MA, USA
October 10, 2023

Project ID: 20231006_PIQ30_2


I. Project Summary

Project 20231006_PIQ30_2 services do not include NGS sequencing of the V1V3 region of the 16S rRNA gene amplicons from the samples. First and foremost, please download this report. 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 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

Not available
 

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

Not available
 

VI. Analysis - DADA2 Read Processing

Not available
 

Sample Meta Information

#SampleIDGroup
IM1Patient_PI
IM2Patient_PI
IM3Patient_PI
IM4Patient_PI
IM5Patient_PI
IM6Patient_PI
IM7Patient_PI
IM8Patient_PI
IM9Patient_PI
IM10Patient_PI
IM11Patient_PI
IM12Patient_PI
IM13Patient_PI
IM14Patient_PI
IM15Patient_PI
IM16Patient_PI
IM17Patient_PI
IM18Patient_PI
IM19Patient_PI
IM20Patient_PI
IM21Patient_PI
IM22Patient_PI
IM24Healthy_Control
IM25Healthy_Control
IM26Healthy_Control
IM27Healthy_Control
IM28Healthy_Control
IM29Healthy_Control
IM30Healthy_Control
IM31Healthy_Control
IM32Healthy_Control
IM33Healthy_Control
IM34Healthy_Control
IM36Healthy_Control
IM37Healthy_Control
IM38Patient_no_PI
IM39Patient_no_PI
IM40Patient_no_PI
IM41Patient_no_PI
IM42Patient_no_PI
IM43Patient_no_PI
IM44Patient_no_PI
IM45Patient_no_PI
IM46Patient_no_PI
IM47Patient_no_PI
IM48Patient_no_PI
IM49Patient_no_PI
IM50Patient_no_PI
IM51Patient_no_PI
IM52Patient_no_PI
 
 

ASV Read Counts by Samples

#Sample IDRead Count
IM3640
IM26107
IM20120
IM44159
IM52163
IM33230
IM32296
IM34313
IM46346
IM17503
IM42514
IM27568
IM48677
IM43683
IM28690
IM47766
IM41786
IM16820
IM7879
IM15974
IM14986
IM91,018
IM291,145
IM181,233
IM511,243
IM371,254
IM301,329
IM381,490
IM131,573
IM11,593
IM241,636
IM61,740
IM221,749
IM51,774
IM311,792
IM252,023
IM112,859
IM392,977
IM403,120
IM213,320
IM43,661
IM104,021
IM454,066
IM194,746
IM496,455
IM88,055
IM38,590
IM129,131
IM5012,287
IM214,119
 
 
 

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%(>=10 reads)
ATotal reads120,619120,619
BTotal assigned reads105,212105,212
CAssigned reads in species with read count < MPC01,313
DAssigned reads in samples with read count < 100
ETotal samples5050
FSamples with reads >= 15050
GSamples with reads < 100
HTotal assigned reads used for analysis (B-C-D)105,212103,899
IReads assigned to single species103,416102,603
JReads assigned to multiple species255215
KReads assigned to novel species1,5411,081
LTotal number of species674264
MNumber of single species487232
NNumber of multi-species154
ONumber of novel species17228
PTotal unassigned reads15,40715,407
QChimeric reads7979
RReads without BLASTN hits3131
SOthers: short, low quality, singletons, etc.15,29715,297
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.
SPIDTaxonomyIM1IM10IM11IM12IM13IM14IM15IM16IM17IM18IM19IM2IM20IM21IM22IM24IM25IM26IM27IM28IM29IM3IM30IM31IM32IM33IM34IM36IM37IM38IM39IM4IM40IM41IM42IM43IM44IM45IM46IM47IM48IM49IM5IM50IM51IM52IM6IM7IM8IM9
SP1Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;timonensis1761004200060166008005061453191318078612512171702542411910015622
SP10Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Methylobacteriaceae;Methylobacterium;goesingense000000000000022000000000000100000000000000000400000
SP100Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;johnsonii0520121302204000000000010002102131020001210011220001100
SP101Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;Lautropia;mirabilis00100000002600550000000012000012000000000000000000000
SP102Bacteria;Proteobacteria;Alphabacteria;Rhizobiales;Methylobacteriaceae;Methylobacterium;rhodesianum0000000000000558100000000000111000000000010000500000
SP103Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;tigurinus022029000023120100000000000100000000200670000702901000
SP104Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Oribacterium;sp._oral_taxon_078020202302000000001000000000000109001001020214000020
SP105Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_175001300150200000010010021014100002100001302120100001012004
SP106Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Propionibacteriaceae;Propionibacterium;granulosum000000000000000000000001000024000000000000000000000
SP108Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;salivarius000000000010011000000000100000000000000010001300000
SP109Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Fretibacterium;sp._oral_taxon_3590001000000000000000000117000000000010000200000000000
SP11Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Mogibacterium;timidum065116123826825610125110463511113721051241591332154700500126266302320228243464801781041122680310
SP111Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Micrococcaceae;Rothia;aeria11500020104390192001000100012006100381130100010000004000
SP112Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._oral_taxon_056800010000001000000000000000001402000000000000000000
SP113Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Lachnospiraceae_[G-7];sp._oral_taxon_08610000400000700003000060000000004013100000022100100
SP114Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;australis38026070000980069000000000602014005000202750120000000000
SP116Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;oris0003000050110100000000034000000000000000001015001000
SP117Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;sputorum000000000000036000000000000000000000000000000000000
SP119Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;pharyngis000000000060148000000000000014000000000000000100000
SP12Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Atopobiaceae;Olsenella;uli2378727460421371421559730036002003219539030012041966511210122372480133663112843829580
SP120Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;infantis2030000001214102530000010032000000000000142020002000000
SP122Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Coriobacteriaceae;Slackia;exigua0021100300200001300003022500000010020004000061000115
SP123Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Fretibacterium;sp._oral_taxon_36000000100208100000000001100200001000000010010000001
SP124Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Coriobacteriaceae;Atopobium;parvulum0500420001708300000001017000000310132015031005310003100
SP125Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_896200000040040100020001040000101061011000100300190000000
SP126Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_1714000000300116001100000023320000113320151101003001003000
SP128Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;gracilis000000000023000001000000100000000000000000000000000
SP13Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;israelii4343410233662270169180302613623701833867718181062842513467912782011358221065300726372
SP130Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonellaceae_[G-1];sp._oral_taxon_129000000000010000000000401000100220000000000000000000
SP131Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Filifactor;alocis400201000034400070050012411000001001900000000306000108
SP132Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonellaceae_[G-1];sp._oral_taxon_14800000210003000000100020000000000000000000000000010
SP133Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonellaceae_[G-1];sp._oral_taxon_14500000120003200020000100000000000000001000500000400
SP134Bacteria;Bacteroidetes;Bacteroidetes_[C-1];Bacteroidetes_[O-1];Bacteroidetes_[F-1];Bacteroidetes_[G-5];sp._oral_taxon_51121004001006000101000000000000060000000000000000000
SP135Bacteria;Spirochaetes;Spirochaetia;Spirochaetales;Spirochaetaceae;Treponema;sp._oral_taxon_23700020000000006000300000600000000000000000001000001
SP136Bacteria;Firmicutes;Bacilli;Bacillales;Gemellaceae;Gemella;haemolysans10120000004610100000000127000090000000064040000101000
SP137Bacteria;Actinobacteria;Actinobacteria;Propionibacteriales;Propionibacteriaceae;Propionibacterium;acidifaciens0100001800050000000000000000000001430010001081102000300
SP139Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonella;parvula_group0300000006241190100000000010000321236032018143213300000
SP14Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_44801001000000000005100133900191000010000244000200020411001
SP140Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;dentisani03001000004016200000000011000011901000000101100500001
SP141Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Selenomonas;sp._oral_taxon_44200000000000000000000000800000000010000000510000000
SP142Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;constellatus010900008049500009000400400000010000007011050113100000
SP143Bacteria;Actinobacteria;Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium;sp._Oral_Taxon_A16000000000100000000000000000300001000000110000000000
SP144Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_subsp._nucleatum000000000430000000000000000000000500001100000000025
SP146Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;buccalis000000000097000000000000000000000000000000000000000
SP15Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Atopobiaceae;Olsenella;sp._oral_taxon_80711143016664114012142015670000496350111073171242193103471163851620131800
SP150Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Selenomonas;sp._oral_taxon_1340010001000115000100000308100000000010000002400000101
SP154Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._oral_taxon_066302600000263905200000000000000000000000650160001200000
SP156Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;intermedius5000200000000004000002000000031000002000220198000000
SP157Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._oral_taxon_43190000000000001200000000000001000000000026000000000000
SP158Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae;Sphingobium;japonicum0000000000000015100000000000000000000000000000000000
SP159Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._oral_taxon_06120000000040005000000000000000000000006000000000000
SP160Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;flavescens|subflava00000000003101119000000000200003000000000000001100000
SP161Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae;Sphingobium;limneticum000000000000003700000000000000000000000000000000000
SP162Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;lingnae_[NVP]0100000001200000000000000000000000000030160000000000
SP165Bacteria;Spirochaetes;Spirochaetia;Spirochaetales;Spirochaetaceae;Treponema;maltophilum000010000016301000110000000000000000000100000000003
SP166Bacteria;Spirochaetes;Spirochaetia;Spirochaetales;Spirochaetaceae;Treponema;lecithinolyticum000000000001001000490000000000000000000000000000005
SP167Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-3];sp._oral_taxon_49500062000000080004000001100000006600000000001113000126
SP168Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Geobacillus;stearothermophilus000000000000002400000000300000000000000000002000000
SP17Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Micrococcaceae;Rothia;mucilaginosa01250100110931225500001000190020000160004121350006300001
SP171Bacteria;Spirochaetes;Spirochaetia;Spirochaetales;Spirochaetaceae;Treponema;socranskii000512004023402010130003000000001020000000301000013
SP174Bacteria;Spirochaetes;Spirochaetia;Spirochaetales;Spirochaetaceae;Treponema;sp._oral_taxon_23100000000008000000010000000000000000100000001000000
SP177Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Catonella;morbi00000000009000000000000800000010000000000000000000
SP179Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae;Agrobacterium;tumefaciens00000000000000010001000001100001000100010400000000
SP18Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-1];[Eubacterium]_infirmum06250821101700000160000311700007022010202000293474000300
SP180Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Selenomonas;dianae00000000001000000000002800000010020000000001000001
SP182Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;massiliensis2000000002030001200061112200017121200000020000008000
SP184Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Johnsonella;ignava000000000016400000000010000000001000000000000000000
SP186Bacteria;Saccharibacteria_(TM7);TM7_[C-1];TM7_[O-1];TM7_[F-1];TM7_[G-1];sp._oral_taxon_348100080000030000000000000000000000000000000100000000
SP187Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._oral_taxon_05890000000014300582000000002000000000001002010000000000
SP188Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;periodonticum000000000229009000000002000000000000000000000000000
SP189Bacteria;Actinobacteria;Coriobacteriia;Eggerthellales;Eggerthellaceae;Cryptobacterium;curtum060010000000000000000002000000201000040106020000000
SP19Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Mogibacterium;diversum04100230110404000120000332401000592100027101102360000101
SP190Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-7];[Eubacterium]_yurii_subsp._yurii_&_margaretiae400000000019000000000000000000000000000000000000000
SP191Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Staphylococcus;epidermidis0000000000100140000000000000012100000000000000000000
SP195Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Aggregatibacter;sp._oral_taxon_458000000000050080000000000000014000000000000000000001
SP196Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;gingivalis000100000006650101000000111400000000034002100000000019
SP197Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;Bacteroides;heparinolyticus00060000000100000100000000000000000000000002000001
SP198Bacteria;Actinobacteria;Actinobacteria;Bifidobacteriales;Bifidobacteriaceae;Scardovia;wiggsiae0000000000000000000000000100000010000000018900000100
SP2Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Tannerella;forsythia40820742023371540601922334154045502500058812290004510700005416121
SP20Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonellaceae_[G-1];sp._oral_taxon_13220123300000000003200031601501000015204442000208902000401
SP200Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp._oral_taxon_219000000000043000000000000000000000000000000000000000
SP201Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._oral_taxon_0700000000000083000000000027000000004000050000261700100
SP202Bacteria;Actinobacteria;Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium;durum20000000010000000000022000700001001000001000000001
SP203Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;Klebsiella;pneumoniae000000000000010000000000000020000000000000000500000
SP204Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonellaceae_[G-1];sp._oral_taxon_13500000000002000000000030000000010050000000010100000
SP208Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;sp._oral_taxon_326000000000038000000000000000000000000000000000000000
SP209Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;sp._oral_taxon_279005000000900047000000000000000000000008030002500000
SP21Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_17000020003105300017500500178111137204002321210000104200027005241961
SP210Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;intermedia000000000014200000200000000000000000000010000000010
SP211Bacteria;Bacteroidetes;Bacteroidetes_[C-1];Bacteroidetes_[O-1];Bacteroidetes_[F-1];Bacteroidetes_[G-3];sp._oral_taxon_36500000700010000000120010000000110000000000000000010
SP214Bacteria;Firmicutes;Bacilli;Bacillales;Listeriaceae;Brochothrix;thermosphacta00003000000000000006000015300010000000030100600000
SP215Bacteria;Firmicutes;Erysipelotrichia;Erysipelotrichales;Erysipelotrichaceae;Bulleidia;extructa30001010020300000000000000000000000001000040600000
SP216Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp._oral_taxon_392000000000017000000000000000008000000000000000000000
SP218Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Stomatobaculum;sp._oral_taxon_373000000000000000200000010000000100000000000000000100
SP219Bacteria;Saccharibacteria_(TM7);TM7_[C-1];TM7_[O-1];TM7_[F-1];TM7_[G-1];sp._oral_taxon_86900000000000000042000000000000030042000000000000006
SP22Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;odontolyticus21150295039013300017000000105141010009010001301214911062300000
SP220Bacteria;Firmicutes;Bacilli;Bacillales;Gemellaceae;Gemella;sanguinis000000000000013000000001000000000000001010000000000
SP221Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Micrococcaceae;Kocuria;sp._Oral_Taxon_B5600000000001006000000000000000000000000000100300000
SP223Bacteria;Firmicutes;Clostridia;Clostridiales;Peptoniphilaceae;Finegoldia;magna000000000000001800000000000000000000000000000100000
SP225Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-5];[Eubacterium]_saphenum0003300000000000000000000000000200400000100030000041
SP227Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Selenomonas;artemidis000000000013000000000000000000000000000000000000000
SP229Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp._oral_taxon_498000000000017000000000000000000000000000000000000000
SP23Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae;Abiotrophia;defectiva4113800500102721416071000020013424061102454004803553210070302000
SP230Bacteria;Proteobacteria;Alphabacteria;Rhizobiales;Methylobacteriaceae;Methylobacterium;sp._Oral_Taxon_B84000000000000006900000000000200000000000000000200000
SP234Bacteria;Saccharibacteria_(TM7);TM7_[C-1];TM7_[O-1];TM7_[F-1];TM7_[G-2];sp._oral_taxon_350000010002000000000000003000000010000000000000000000
SP235Bacteria;Actinobacteria;Actinobacteria;Bifidobacteriales;Bifidobacteriaceae;Bifidobacterium;dentium091000000000200000000100010000111600001001840000200
SP24Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;gerencseriae070600001100001450370000029005016310106000142210122300000
SP241Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;trevisanii000000000021005000000000000000000000000000000000000
SP242Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._Oral_Taxon_171000000000002000000000000000000000000000000005003000
SP244Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_877000000000000100010001000000018020000000002000000000
SP246Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Lachnospiraceae_[G-8];sp._oral_taxon_50000020000003000020100000000000010080000000000000000
SP247Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;nanceiensis000000000000013000000000000000000000000000001000000
SP248Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-1];sp._oral_taxon_383010001000102000000000335000200045000001001000001100
SP249Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;Cardiobacteriaceae;Cardiobacterium;hominis00000000007001000000000001000104000000010000500000
SP25Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Coriobacteriaceae;Atopobium;sp._oral_taxon_1993120412400018010242018000001300000006450010000119015988010086560
SP251Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Dietziaceae;Dietzia;sp._Oral_Taxon_D12000000000000001200000000000000000000000000000000000
SP255Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-4];sp._oral_taxon_3690300000000325000000000030003000000000000002101300003
SP256Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_172020350000701000010000001000000000001016010020302000
SP258Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-4];sp._oral_taxon_10300002030000200000000111200000002000000100000000232
SP26Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;mitis8000000000297020639000000315021020000000001020012101001
SP264Bacteria;Firmicutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;Mycoplasma;faucium00010000000000000100007000000000010000000002000001
SP265Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_17800001300005000001000200702000900001000010100101000
SP267Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Bradyrhizobiaceae;Bradyrhizobium;elkanii0000000000000415000000000000017000000000000000310000
SP27Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_897057911660000057000018010032100000022110305626007202400000
SP271Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Tannerella;sp._oral_taxon_28600000000007003001000000000000100000000000000000000
SP274Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;Noviherbaspirillum;suwonense10002002000000030000011641210001101020130810001000
SP277Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-6];[Eubacterium]_minutum010010140100100400100020000000040010011003000000250
SP278Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcaceae_[G-1];sp._oral_taxon_07500000000006005000000000000000000000000000000000000
SP28Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;gordonii1130020000003180013100100001200400120050110186001011100100
SP280Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;cristatus000000000110010000000000000000001000000000000000000
SP281Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Eikenella;corrodens000000000010100000000000000000000000000000000000000
SP283Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;sp._oral_taxon_036000000000000037000000000200000000000000000000000000
SP286Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;oralis000000000032100000000000000000000000000000000000000
SP287Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;catoniae100000000010044000000000000000100000000000000000000
SP289Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;sp._Oral_Taxon_G43000000000000000000100000000000000000000000000000000
SP29Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sanguinis1702712000032039132351000011491000641250000469211010901000
SP294Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae;Rhizobium;etli000000000000001600000000000000000000000000000000000
SP295Bacteria;Actinobacteria;Actinobacteria;Micrococcales;Microbacteriaceae;Microbacterium;oleivorans000000000000000000000000000018000000000000000000000
SP296Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Staphylococcus;gallinarum000000000000001500000000000000000000000000000000000
SP3Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_5251339547755261450612810862015510401019403110120215171296146112024873256346594144000
SP30Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Dialister;invisus0200000002000003000010000000008000010000018027000110
SP301Bacteria;Actinobacteria;Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium;accolens000000000000001100000000000000000000000000000000000
SP304Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Geodermatophilaceae;Blastococcus;saxobsidens000000000000000000000000001010000000000000000000000
SP31Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;elegans000000000010062000000000000000000000000000000000000
SP317Bacteria;Actinobacteria;Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium;variabile00000000000000000009000011000000000000000000000000
SP32Bacteria;Saccharibacteria_(TM7);TM7_[C-1];TM7_[O-1];TM7_[F-2];TM7_[G-5];sp._oral_taxon_356012021105620000311830016002023270150001121115000260301758113002250
SP323Bacteria;Firmicutes;Clostridia;Clostridiales;Eubacteriaceae_[XV];Pseudoramibacter;alactolyticus00051000000800000001000000000000000000000310000000
SP324Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Acidovorax;defluvii001210020100000000010001000000000010150000000000000
SP327Plantae;Angiosperms;Magnoliids;Laurales;Calycanthaceae;Calycanthus;floridus_Oral_Taxon_D07000000000000002600000000000000000000000010000000000
SP328Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Geobacillus;sp._str._WCH70000000000000002400000000000009000000000000001050000
SP33Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;oralis1041000003100000000000107000000000000020070003000000
SP333Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Selenomonas;noxia000100000014000010000000100000000000000001000000000
SP334Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus;anthracis000000000000001000000000000004000000000000000000000
SP339Bacteria;Actinobacteria;Actinobacteria;Propionibacteriales;Propionibacteriaceae;Propionibacterium;sp._oral_taxon_915000000000012000000000000000000000000000000000000000
SP34Bacteria;Spirochaetes;Spirochaetia;Spirochaetales;Spirochaetaceae;Treponema;denticola100800100026060101000010000000100100000000000020019
SP344Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;sp._oral_taxon_2250000000000100017000000000000000000000000000000700000
SP347Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;sp._Oral_Taxon_B43000000000000000000000000000014000000000000000000000
SP348Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;sp._oral_taxon_336000000000012000000000000000000000000000000000000000
SP349Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;histicola000000000000002300000000000000000000000000000100000
SP35Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiales_[F];Clostridiales_[G];sp._Oral_Taxon_G7400093001200007000006000000000000700010000201021000570
SP350Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinobaculum;sp._oral_taxon_84800000000013000000000020000000220000100000000100000
SP352Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridium;butyricum000000000000002700000000000000000000000000000000000
SP36Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Tetragenococcus;halophilus0000000000000036000000006001016000000000000000120000
SP37Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;tannerae000000000004000001000001000000000000000100111000000
SP374Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Anoxybacillus;pushchinoensis_Oral_Taxon_B7200000000000000000000000000007000000000000000500000
SP38Bacteria;Firmicutes;Clostridia;Clostridiales;Peptoniphilaceae;Peptoniphilaceae_[G-1];sp._oral_taxon_113030282500000210134900487002010600200000030501012231110011110
SP381Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;Enterobacter;cloacae000100000000000000000000000011000000000000000000000
SP39Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_1801112464570010370150810020012017690010318141323283912625027911081812000
SP4Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Coriobacteriaceae;Atopobium;rimae66467703434013021001000042669021050950111180381014437313982017120
SP40Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae;Rhizobium;rhizogenes_Oral_Taxon_D340000000000230010637000020011600003000000000000002000000
SP41Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oricola1054013084102001010100231281510014000302142500068703035003815109
SP419Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;sputigena00000000009301000000000000000000000001000000000000
SP42Bacteria;Actinobacteria;Actinobacteria;Propionibacteriales;Propionibacteriaceae;Propionibacterium;sp._oral_taxon_194410000000004007040000000000000000000000000000000000
SP421Bacteria;Actinobacteria;Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium;singulare00000000000006000000000000000000000000000000500000
SP426Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Aggregatibacter;sp._oral_taxon_51300000000000005000000000000006000000000000000000000
SP43Bacteria;Saccharibacteria_(TM7);TM7_[C-1];TM7_[O-1];TM7_[F-1];TM7_[G-1];sp._oral_taxon_347000000000020002200000000000000000000000000000000000
SP44Bacteria;Saccharibacteria_(TM7);TM7_[C-1];TM7_[O-1];TM7_[F-1];TM7_[G-1];sp._oral_taxon_95285001000300270000000000010000017000200819120130000000
SP45Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._oral_taxon_423403050000003427113900000010681011301012110065312020303000
SP46Bacteria;Saccharibacteria_(TM7);TM7_[C-1];TM7_[O-1];TM7_[F-1];TM7_[G-1];sp._oral_taxon_34632902140140020132113191018145100025162503001359020714220354116417450041132
SP47Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sicca000000000000034000000000100000000000000000000000000
SP48Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Anaeroglobus;geminatus01000000000000000000000400000000000101000004700000
SP49Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;mutans00034000020254000000000000000000017010000009104000600
SP5Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;naeslundii400013090180168240073001131113234025177044103000322481000180000
SP50Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae_[XIV];Lachnospiraceae_[G-3];sp._oral_taxon_1000100110000019000000000000000000200000100000000000000
SP51Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Selenomonas;sputigena012600400143302041300046160100000010011020030753301002
SP52Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;cardiffensis006000002202107000060401100000000000090009510020100114063
SP53Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;downei000000000000000000010000010000000000000101260000041000
SP54Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;dentalis07110145107400512230200300085713411511113273626218143681004001161
SP55Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Micrococcaceae;Rothia;dentocariosa73086111001048410110040303000660101100090000207830630028000
SP56Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_169154622101001300000040015010510131229000000106414825508321041005
SP57Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcus;stomatis14412170401918400000000000200000010000412300016891000311
SP58Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;meyeri914002058002350100000000032600000000002910005200000299000
SP59Bacteria;Spirochaetes;Spirochaetia;Spirochaetales;Spirochaetaceae;Treponema;sp._oral_taxon_26900050000005000000000000000000000000000000000000000
SP6Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;Caulobacteraceae;Brevundimonas;diminuta80215003012421401443035481019191133851524003851521373139356932044612401225
SP60Bacteria;Actinobacteria;Actinobacteria;Propionibacteriales;Propionibacteriaceae;Propionibacterium;propionicum213901128200147531441011007890307385012500131272500917001501120120500111
SP61Bacteria;Chloroflexi;Anaerolineae;Anaerolineales;Anaerolineaceae;Anaerolineae_[G-1];sp._oral_taxon_439121268122002100011005001280000001722501300620010000101281
SP62Bacteria;Saccharibacteria_(TM7);TM7_[C-1];TM7_[O-1];TM7_[F-1];TM7_[G-1];sp._oral_taxon_3494101210922006002418200010416132510311310761202940000816057440501111990
SP64Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;parainfluenzae00100000005003010000000000000010000100000000006000000
SP65Bacteria;Firmicutes;Erysipelotrichia;Erysipelotrichales;Erysipelotrichaceae;Solobacterium;moorei071112810033117000000010317000003303723013001130122004020
SP66Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-6];[Eubacterium]_nodatum0000032001643200010020000000000008001010000000000005
SP67Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_subsp._animalis000000000080000000010000600000000000000000005000000
SP68Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;georgiae00215160002201001080010103100000000000000600000117001000
SP69Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-9];[Eubacterium]_brachy5105310010105320108502091100010016204010000055315020035
SP7Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_414000010001429020000000000000100000220000000100000000000
SP70Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidales_[F-2];Bacteroidales_[G-2];sp._oral_taxon_2740035413201004100001040900013638100000737160300003000031000150
SP71Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfobulbaceae;Desulfobulbus;sp._oral_taxon_041000200000100200006200300000030011147000002000000000207
SP72Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonellaceae_[G-1];sp._oral_taxon_150004000000117400011000100200200000150100000000100040
SP75Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_subsp._vincentii371178000012456811805122520110500003002026002600010673301077
SP76Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Fretibacterium;fastidiosum23050050002000031301021716001000020800000000116000233
SP77Bacteria;Actinobacteria;Actinobacteria;Pseudonocardiales;Pseudonocardiaceae;Pseudonocardia;carboxydivorans000000000000032100000000000000000000000000000000000
SP78Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;viscosus00200000010000000000200110000000000202000000006000
SP79Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;sp._oral_taxon_0649000000000120632000000017000007000000007003003100000
SP8Bacteria;Firmicutes;Clostridia;Clostridiales;Peptoniphilaceae;Parvimonas;micra97227229216026101701372112218033361368321233498901260371539451481399261157711021763684810359811242587
SP80Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Staphylococcus;warneri000000000000112000000000000000000000000000000430000
SP81Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;showae000000000011003000000000000000000000000000000000000
SP82Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Desemzia;incerta00000001000002000001000201300010000000010000000000
SP83Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Atopobiaceae;Olsenella;profusa000046000000000000000010000100000040000000100000610
SP85Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonellaceae_[G-1];sp._oral_taxon_1550000000000060160020000100000000000000000002900200010
SP86Bacteria;Actinobacteria;Actinobacteria;Propionibacteriales;Propionibacteriaceae;Propionibacterium;acnes00000200002101167410000003212101223560000001100100044400202
SP87Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;sp._oral_taxon_20300000000002800000000007960000000000000000000000000000
SP88Bacteria;Firmicutes;Clostridia;Clostridiales;Peptoniphilaceae;Parvimonas;sp._oral_taxon_1100001004940000000000480000000100000000000710000254000
SP89Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Tannerella;sp._oral_taxon_80885100100000100000000020000000000000001000201000000
SP9Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;anginosus01037012500202670200000001037000034001000029100500700000
SP90Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;parasanguinis_II011270000016015075000000004000000008000050802011500000
SP91Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Carnobacterium;divergens1511014001821106011100279611010301012948260103427590370165682910
SP92Bacteria;Firmicutes;Bacilli;Bacillales;Gemellaceae;Gemella;morbillorum3820100320731000000100407000004100011042000020000200
SP93Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;leadbetteri000000000019000000000000700000000000000000000100000
SP94Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;endodontalis33101106002139151300175070344030030035326000040010222111300002
SP95Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinobaculum;sp._oral_taxon_183050024000021001417911000600000020400000003200320000000
SP96Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;nucleatum_subsp._polymorphum0000000002930050000100017000010000000002000000100000
SP97Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;adiacens404010070030937617601200005127104005009113126245400131104000
SP99Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oris0028221025000123000001022102710000300100210302111001531011000
SPN110Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae;Porphyrobacter;tepidarius_nov_95.662%000000000000003000000000000000000000000000000000000
SPN121Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_897_nov_97.988%000000000000000000000230000000040000000000020000000
SPN133Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Enterococcus;saccharolyticus_nov_88.867%000000000000001600000000100006000000000000000100000
SPN145Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;oricola_nov_97.421%000010000000000000000060000000000000000000000001151
SPN154Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_175_nov_97.967%001000001000000000000000000000000000007000000000000
SPN155Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_171_nov_96.450%0000120000000100000000000010030035004200600000010300
SPN156Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Coriobacteriaceae;Slackia;exigua_nov_89.339%00100231000000060000000000000000000002010000000000
SPN157Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Atopobiaceae;Olsenella;uli_nov_97.872%000000000000000000000000000000000000000000010000120
SPN158Bacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Atopobiaceae;Olsenella;sp._oral_taxon_807_nov_97.872%03000000000000000000060000000020000000000000000020
SPN160Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Hymenobacter;roseosalivarius_nov_91.176%00000000010000000021010020100000002002000001000000
SPN161Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;israelii_nov_94.922%00000000000000500000401200000000000000000000000000
SPN162Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;sp._oral_taxon_018_nov_97.959%000000000012000000000000000000000000000000000000000
SPN163Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Bradyrhizobiaceae;Afipia;broomeae_nov_92.466%000000000000000000000001200000000000000000000000000
SPN164Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;israelii_nov_97.804%00000000000000000000040000000350000000000000000000
SPN165Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_178_nov_89.400%000000000010000000000000000000000100000000000000000
SPN166Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_170_nov_97.843%00000000000000000000012000000006000000000000000100
SPN167Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Peptostreptococcaceae_[XI][G-3];sp._oral_taxon_950_nov_96.473%000900000000000000000000000000000000000100000000000
SPN19Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae;Rhizobium;loti_nov_90.930%0000000000000135400000000000000000000000000000200000
SPN30Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;timonensis_nov_93.927%0000000000000201000000000000000001100000100000090040
SPN42Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Tannerella;forsythia_nov_97.737%050053000023001000000000000000000100000000000000000
SPN43Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Mogibacterium;timidum_nov_95.851%000000000005600000000000000000000000000000000000060
SPN55Bacteria;Saccharibacteria_(TM7);TM7_[C-1];TM7_[O-1];TM7_[F-1];TM7_[G-1];sp._oral_taxon_349_nov_97.788%000000000000000000000055000000000000000000000000000
SPN67Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae;Acinetobacter;sp._oral_taxon_408_nov_92.480%20000002011010020101002123200031260011210120220100
SPN82Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;israelii_nov_96.857%0410112021017500001000000401011670100103021001000100
SPN87Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;sp._oral_taxon_525_nov_95.257%003600000000000000000000000000000000000000000000000
SPN93Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;dentalis_nov_97.804%0500010100050001000006000000001112000000000000000000
SPN99Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;Lautropia;mirabilis_nov_95.902%000000000060028000000000000000000000000000000000000
SPP1Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;multispecies_spp1_20000000000110140000000002000013000000002010000000000
SPP10Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;NA;Reyranella;multispecies_spp10_20000000000000360000000000000025000000000000000000000
SPP5Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;multispecies_spp5_2302000000400020000000000000000000001042090000000000
SPP9Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;multispecies_spp9_22001000000140000000100000000000100000001000000000100
SPPN1Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae_[XI];Mogibacterium;multispecies_sppn1_2_nov_91.393%000000000700000000000000000000000000000020000000000
 
 
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 1Healthy_Control vs Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVG
Comparison 2Healthy_Control vs Patient_PIPDFSVGPDFSVGPDFSVG
Comparison 3Healthy_Control vs Patient_no_PIPDFSVGPDFSVGPDFSVG
Comparison 4Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVG
 
 

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 1Healthy_Control vs Patient_PI vs Patient_no_PIView in PDFView in SVG
Comparison 2Healthy_Control vs Patient_PIView in PDFView in SVG
Comparison 3Healthy_Control vs Patient_no_PIView in PDFView in SVG
Comparison 4Patient_PI vs Patient_no_PIView 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.Healthy_Control vs Patient_PI vs Patient_no_PIObserved FeaturesShannon IndexSimpson Index
Comparison 2.Healthy_Control vs Patient_PIObserved FeaturesShannon IndexSimpson Index
Comparison 3.Healthy_Control vs Patient_no_PIObserved FeaturesShannon IndexSimpson Index
Comparison 4.Patient_PI vs Patient_no_PIObserved 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 1Healthy_Control vs Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 2Healthy_Control vs Patient_PIPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 3Healthy_Control vs Patient_no_PIPDFSVGPDFSVGPDFSVGPDFSVG
Comparison 4Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVGPDFSVG
 
 
 
 
 

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.Healthy_Control vs Patient_PI vs Patient_no_PIBray–CurtisCorrelationAitchison
Comparison 2.Healthy_Control vs Patient_PIBray–CurtisCorrelationAitchison
Comparison 3.Healthy_Control vs Patient_no_PIBray–CurtisCorrelationAitchison
Comparison 4.Patient_PI vs Patient_no_PIBray–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.Healthy_Control vs Patient_PI vs Patient_no_PI
Comparison 2.Healthy_Control vs Patient_PI
Comparison 3.Healthy_Control vs Patient_no_PI
Comparison 4.Patient_PI vs Patient_no_PI
 
 

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.Healthy_Control vs Patient_PI vs Patient_no_PI
Comparison 2.Healthy_Control vs Patient_PI
Comparison 3.Healthy_Control vs Patient_no_PI
Comparison 4.Patient_PI vs Patient_no_PI
 
 
 

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.

 
Healthy_Control vs Patient_PI vs Patient_no_PI
 
 
 
 
 
 
 
LEfSe Results for All Comparisons
 
Comparison No.Comparison Name
Comparison 1.Healthy_Control vs Patient_PI vs Patient_no_PI
Comparison 2.Healthy_Control vs Patient_PI
Comparison 3.Healthy_Control vs Patient_no_PI
Comparison 4.Patient_PI vs Patient_no_PI
 
 

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 1Healthy_Control vs Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVG
Comparison 2Healthy_Control vs Patient_PIPDFSVGPDFSVGPDFSVG
Comparison 3Healthy_Control vs Patient_no_PIPDFSVGPDFSVGPDFSVG
Comparison 4Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVG
 
 
B) One-way clustering - clustered on rows (organism) only
Comparison No.Comparison NameFamily LevelGenus LevelSpecies Level
Comparison 1Healthy_Control vs Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVG
Comparison 2Healthy_Control vs Patient_PIPDFSVGPDFSVGPDFSVG
Comparison 3Healthy_Control vs Patient_no_PIPDFSVGPDFSVGPDFSVG
Comparison 4Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVG
 
 
C) No clustering
Comparison No.Comparison NameFamily LevelGenus LevelSpecies Level
Comparison 1Healthy_Control vs Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVG
Comparison 2Healthy_Control vs Patient_PIPDFSVGPDFSVGPDFSVG
Comparison 3Healthy_Control vs Patient_no_PIPDFSVGPDFSVGPDFSVG
Comparison 4Patient_PI vs Patient_no_PIPDFSVGPDFSVGPDFSVG
 
 

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

 

 

 
 

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