Efficient nucleic acid extraction and 16s rRNA gene sequencing for bacterial community characterization

Journal of Visualized Experiments

Volumn 2016, Issue 110, 2016, Pages

  Anahtar, Melis N ^a   Bowman, Brittany A ^a   Kwon, Douglas S ^a  

^a MASSACHUSETTS GENERAL HOSPITAL   (United States)

Author keywords

16S; Bacteria; DNA extraction; High throughput sequencing; Human sequencing; Issue 110; Metagenomics; Microbiome; Molecular biology; Next generation sequencing; RNA extraction; Stool; Swab; Vaginal

Indexed keywords

RNA 16S;

BACTERIUM; DNA SEQUENCE; FECES; GENETICS; HIGH THROUGHPUT SEQUENCING; HUMAN; METAGENOMICS;

BACTERIA; FECES; HIGH-THROUGHPUT NUCLEOTIDE SEQUENCING; HUMANS; METAGENOMICS; RNA, RIBOSOMAL, 16S; SEQUENCE ANALYSIS, DNA;

EID: 84964808197     PISSN: 1940087X     EISSN: None     Source Type: Journal    
DOI: 10.3791/53939     Document Type: Article

References (33)

1. Huttenhower, C. Structure, function and diversity of the healthy human microbiome. Nature. 486, 207-214 (2012).
2. O'Hanlon, D. E., Moench, T. R., & Cone, R. A. Vaginal pH and microbicidal lactic acid when lactobacilli dominate the microbiota. PloS one. 8, e80074 (2013).
3. Aldunate, M. et al. Vaginal concentrations of lactic acid potently inactivate HIV. The Journal of antimicrobial chemotherapy 68, 2015-2025 (2013).
4. Anahtar, M. N. et al. Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract. Immunity 42, 965-976 (2015).
5. Reyes, A., Wu, M., McNulty, N. P., Rohwer, F. L., & Gordon, J. I. Gnotobiotic mouse model of phage-bacterial host dynamics in the human gut. Proceedings of the National Academy of Sciences of the United States of America. 110, 20236-20241 (2013).
6. Chomczynski, P., & Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical biochemistry. 162, 156-159 (1987).
7. Srinivasan, S. et al. Temporal variability of human vaginal bacteria and relationship with bacterial vaginosis. PloS one 5, e10197 (2010).
8. Dols, J. A. et al. Microarray-based identification of clinically relevant vaginal bacteria in relation to bacterial vaginosis. American journal of obstetrics and gynecology 204, 305 e301-307 (2011).
9. Segata, N. et al. Metagenomic microbial community profiling using unique clade-specific marker genes. Nature methods 9, 811-814 (2012).
10. Caporaso, J. G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME journal 6, 1621-1624 (2012).
11. Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences of the United States of America 108 Suppl 1, 4516-4522 (2011).
12. Earthmicrobiome Project. 16S rRNA Amplification Protocol. (2015).
13. Ravel, J. et al. Vaginal microbiome of reproductive-age women. Proceedings of the National Academy of Sciences of the United States of America 108 Suppl 1, 4680-4687 (2011).
14. Srinivasan, S. et al. Bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria. PloS one 7, e37818 (2012).
15. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nature methods 7, 335-336 (2010).
16. Werner, J. MacQIIME. Source: http://www.wernerlab.org/software/macqiime (2015).
17. Schloss, P. D. et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and environmental microbiology 75, 7537-7541 (2009).
18. Edgar, R. C. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature methods. 10, 996-998 (2013).
19. Thermo Fisher Scientific. NanoDrop 2000/2000c Spectrophotometer, V1.0 User Manual. Source: http://www.thermoscientific.com/content/dam/tfs/ATG/CAD/CADDocuments/ProductManuals&Specifications/MolecularSpectroscopy/UVVisibleSpectrophotometers/Spectrophotometer Systems/NanoDrop/NanoDrop-2000-User-Manual-EN.pdf (2009).
20. Qiagen. AllPrep DNA/RNA Mini Kit. Source: https://http://www.qiagen.com/us/shop/sample-technologies/rna-sample-technologies/dna-rna-protein/allprep-dnarna-mini-kit/-orderinginformation (2015).
21. Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H. Agarose gel electrophoresis for the separation of DNA fragments. Journal of visualized experiments: JoVE. (2012).
22. MoBio. UltraClean PCR Clean-Up Kit Instruction Manual. Source: http://www.mobio.com/images/custom/file/12500(1).pdf (2013).
23. Invitrogen. Quant-iT PicoGreen dsDNA Reagent. Source: https://tools.thermofisher.com/content/sfs/manuals/mp07581.pdf (2008).
24. Illumina. Quality Scores for Next-Generation Sequencing. Source: (2011).
25. QIIME. split_libraries_fastq.py. Source: http://qiime.org/scripts/split_libraries_fastq.html (2015).
26. DeSantis, T. Z. et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Applied and environmental microbiology 72, 5069-5072 (2006).
27. QIIME. pick_open_reference:otus.py. Source: http://qiime.org/scripts/pick_open_reference:otus.html (2015).
28. QIIME. summarize_taxa.py. Source: http://qiime.org/scripts/summarize_taxa.html (2015).
29. Vazquez-Baeza, Y., Pirrung, M., Gonzalez, A., & Knight, R. EMPeror: a tool for visualizing high-throughput microbial community data. GigaScience. 2, 16 (2013).
30. QIIME. Comparing categories. Source: http://qiime.org/tutorials/category_comparison.html (2015).
31. Eren, A. M. et al. Exploring the diversity of Gardnerella vaginalis in the genitourinary tract microbiota of monogamous couples through subtle nucleotide variation. PloS one 6, e26732 (2011).
32. Illumina. Diagnosing and preventing flow cell overclustering on the MiSeq system. Source: http://support.illumina.com/content/dam/illumina-marketing/documents/products/other/miseq-overclustering-primer-770-2014-038.pdf (2015).
33. Langille, M. G. et al. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nature biotechnology 31, 814-821 (2013).