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DNA Isolation and Next Generation Sequencing.
ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research, Irvine, CA) was used to extract DNA from samples according to the manufacturer’s instructions.
DNA samples were prepared for targeted sequencing with the Quick-16S Plus NGS Library Prep Kit (V1-V3) (Cat No. D6440-PS1, Zymo Research, Irvine, CA) targeting V1-V3 region of 16S rRNA sequences.
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).
The final library was sequenced on Illumina® NextSeq 2000™ with a p1 (Illumina, Sand Diego, CA) reagent kit (600 cycles). The sequencing was performed with 25% PhiX spike-in.
Bioinformatics Analysis of Sequence Data. Raw 16S V1V3 amplicon sequences obtained from Zymo Research were quality-filtered, denoised,
pair-end merged, and chimera removed with the DADA2 tool (version 1.12.1) [1]. Amplicon sequence variants (ASVs) generated by DADA2
were subjected to species level taxonomy assignment based on the approach developed by Al-Hebshi et. al. [2] against the FOMC Reference Sequence Set
(https://microbiome.forsyth.org/ftp/refseq) that consists of 1,015 full-length 16S rRNA sequences
from HOMD V15.22, 356 from MOMD V5.1, and 22,126 from NCBI, a total of 23,497 sequences. Species-level taxonomy assignment algorithm is available at
https://microbiome.forsyth.org/algorithm.php. Altogether these sequences represent a total of
17,035 oral and non-oral microbial species. Specie-level read count tables were imported using R’s “phyloseq” package (version 1.28.0) [3] for downstream
analyses. Alpha diversity was calculated in 3 measurements: 1) number of species (observed), 2) Shannon index, and 3) Simpson index using “phyloseq” package’s “plot_richness”
function (3). Alpha diversity significance tests were evaluated with QIIME2 (version 2020.11) “alpha-group-significance” function [4]. Beta diversity NMDS
plots were generated with the “ordinate” function in “phyloseq” and beta diversity significance tests were evaluated with QIIME2 “beta-group-significance” function
[4]. ANCOMB-BC2 (Analysis of Compositions of Microbiomes with Bias Correction) (version 2.0.2) was used to test the significance of differential abundance
between two test groups [5]. Linear discriminant analysis LDA Effect Size (LEfSe) (version 1.0.0) was used to plot the effect size of differentially abundant
features [6]. The FOMC analysis pipepline software version information is available at https://microbiome.forsyth.org/software.php.
References:
1. 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.
2. Al-Hebshi NN, Nasher AT, Idris AM, Chen T. Robust species taxonomy assignment algorithm for 16S rRNA NGS reads: application to oral carcinoma samples. J Oral Microbiol. 2015 Sep 29;7:28934. doi: 10.3402/jom.v7.28934. PMID: 26426306; PMCID: PMC4590409.
3. McMurdie PJ, Holmes S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One. 2013 Apr 22;8(4):e61217. doi: 10.1371/journal.pone.0061217. PMID: 23630581; PMCID: PMC3632530.
4. Bolyen et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019 Aug;37(8):852-857. doi: 10.1038/s41587-019-0209-9. Erratum in: Nat Biotechnol. 2019 Sep;37(9):1091. doi: 10.1038/s41587-019-0252-6. PMID: 31341288; PMCID: PMC7015180.
5. 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.
6. 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.
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