16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles
Tóm tắt
Sample storage conditions, extraction methods, PCR primers, and parameters are major factors that affect metagenomics analysis based on microbial 16S rRNA gene sequencing. Most published studies were limited to the comparison of only one or two types of these factors. Systematic multi-factor explorations are needed to evaluate the conditions that may impact validity of a microbiome analysis. This study was aimed to improve methodological options to facilitate the best technical approaches in the design of a microbiome study. Three readily available mock bacterial community materials and two commercial extraction techniques, Qiagen DNeasy and MO BIO PowerSoil DNA purification methods, were used to assess procedures for 16S ribosomal DNA amplification and pyrosequencing-based analysis. Primers were chosen for 16S rDNA quantitative PCR and amplification of region V3 to V1. Swabs spiked with mock bacterial community cells and clinical oropharyngeal swabs were incubated at respective temperatures of -80°C, -20°C, 4°C, and 37°C for 4 weeks, then extracted with the two methods, and subjected to pyrosequencing and taxonomic and statistical analyses to investigate microbiome profile stability. The bacterial compositions for the mock community DNA samples determined in this study were consistent with the projected levels and agreed with the literature. The quantitation accuracy of abundances for several genera was improved with changes made to the standard Human Microbiome Project (HMP) procedure. The data for the samples purified with DNeasy and PowerSoil methods were statistically distinct; however, both results were reproducible and in good agreement with each other. The temperature effect on storage stability was investigated by using mock community cells and showed that the microbial community profiles were altered with the increase in incubation temperature. However, this phenomenon was not detected when clinical oropharyngeal swabs were used in the experiment. Mock community materials originated from the HMP study are valuable controls in developing 16S metagenomics analysis procedures. Long-term exposure to a high temperature may introduce variation into analysis for oropharyngeal swabs, suggestive of storage at 4°C or lower. The observed variations due to sample storage temperature are in a similar range as the intrapersonal variability among different clinical oropharyngeal swab samples.
Tài liệu tham khảo
Blaser M, Bork P, Fraser C, Knight R, Wang J: The microbiome explored: recent insights and future challenges. Nat Rev Microbiol. 2013, 11 (3): 213-217. 10.1038/nrmicro2973.
Caporaso JG, Lauber CL, Costello EK, Berg-Lyons D, Gonzalez A, Stombaugh J, Knights D, Gajer P, Ravel J, Fierer N, Gordon JI, Knight R: Moving pictures of the human microbiome. Genome Biol. 2011, 12 (5): R50-10.1186/gb-2011-12-5-r50.
Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R: Bacterial community variation in human body habitats across space and time. Science. 2009, 326 (5960): 1694-1697. 10.1126/science.1177486.
Zhou Y, Gao H, Mihindukulasuriya KA, La Rosa PS, Wylie KM, Vishnivetskaya T, Podar M, Warner B, Tarr PI, Nelson DE, Fortenberry JD, Holland MJ, Burr SE, Shannon WD, Sodergren E, Weinstock GM: Biogeography of the ecosystems of the healthy human body. Genome Biol. 2013, 14 (1): R1-10.1186/gb-2013-14-1-r1.
Human Microbiome Project C: A framework for human microbiome research. Nature. 2012, 486 (7402): 215-221. 10.1038/nature11209.
Human Microbiome Project C: Structure, function and diversity of the healthy human microbiome. Nature. 2012, 486 (7402): 207-214. 10.1038/nature11234.
Nelson KE, Weinstock GM, Highlander SK, Worley KC, Creasy HH, Wortman JR, Rusch DB, Mitreva M, Sodergren E, Chinwalla AT, Feldgarden M, Gevers D, Haas BJ, Madupu R, Ward DV, Birren BW, Gibbs RA, Methe B, Petrosino JF, Strausberg RL, Sutton GG, White OR, Wilson RK, Durkin S, Giglio MG, Gujja S, Howarth C, Kodira CD, Kyrpides N, Human Microbiome Jumpstart Reference Strains Consortium: A catalog of reference genomes from the human microbiome. Science. 2010, 328 (5981): 994-999.
Pflughoeft KJ, Versalovic J: Human microbiome in health and disease. Annu Rev Pathol. 2012, 7: 99-122. 10.1146/annurev-pathol-011811-132421.
Ding T, Schloss PD: Dynamics and associations of microbial community types across the human body. Nature. 2014, 509 (7500): 357-360. 10.1038/nature13178.
Ren T, Glatt DU, Nguyen TN, Allen EK, Early SV, Sale M, Winther B, Wu M: 16S rRNA survey revealed complex bacterial communities and evidence of bacterial interference on human adenoids. Environ Microbiol. 2013, 15 (2): 535-547. 10.1111/1462-2920.12000.
Marchesi JR, Dutilh BE, Hall N, Peters WH, Roelofs R, Boleij A, Tjalsma H: Towards the human colorectal cancer microbiome. PloS one. 2011, 6 (5): e20447-10.1371/journal.pone.0020447.
Ahn J, Sinha R, Pei Z, Dominianni C, Wu J, Shi J, Goedert JJ, Hayes RB, Yang L: Human gut microbiome and risk for colorectal cancer. J Natl Canc Inst. 2013, 105 (24): 1907-1911. 10.1093/jnci/djt300.
Zackular JP, Baxter NT, Iverson KD, Sadler WD, Petrosino JF, Chen GY, Schloss PD: The gut microbiome modulates colon tumorigenesis. mBio. 2013, 4 (6): e00692-00613.
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI: An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006, 444 (7122): 1027-1031. 10.1038/nature05414.
Finucane MM, Sharpton TJ, Laurent TJ, Pollard KS: A taxonomic signature of obesity in the microbiome? Getting to the guts of the matter. PloS one. 2014, 9 (1): e84689-10.1371/journal.pone.0084689.
Naseer MI, Bibi F, Alqahtani MH, Chaudhary AG, Azhar EI, Kamal MA, Yasir M: Role of gut microbiota in obesity, type 2 diabetes and Alzheimer's disease. CNS Neurol Disord Drug Targets. 2014, 13 (2): 305-311. 10.2174/18715273113126660147.
Larsen N, Vogensen FK, van den Berg FW, Nielsen DS, Andreasen AS, Pedersen BK, Al-Soud WA, Sorensen SJ, Hansen LH, Jakobsen M: Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. PloS one. 2010, 5 (2): e9085-10.1371/journal.pone.0009085.
Kuczynski J, Lauber CL, Walters WA, Parfrey LW, Clemente JC, Gevers D, Knight R: Experimental and analytical tools for studying the human microbiome. Nat Rev Genet. 2012, 13 (1): 47-58.
Fodor AA, DeSantis TZ, Wylie KM, Badger JH, Ye Y, Hepburn T, Hu P, Sodergren E, Liolios K, Huot-Creasy H, Birren BW, Earl AM: The “most wanted” taxa from the human microbiome for whole genome sequencing. PloS one. 2012, 7 (7): e41294-10.1371/journal.pone.0041294.
Group NHW, Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA, Bonazzi V, McEwen JE, Wetterstrand KA, Deal C, Baker CC, Di Francesco V, Howcroft TK, Karp RW, Lunsford RD, Wellington CR, Belachew T, Wright M, Giblin C, David H, Mills M, Salomon R, Mullins C, Akolkar B, Begg L, Davis C, Grandison L, Humble M, Khalsa J: The NIH Human Microbiome Project. Genome Res. 2009, 19 (12): 2317-2323.
Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky N, Chen F, Ochman H, Hugenholtz P: Experimental factors affecting PCR-based estimates of microbial species richness and evenness. ISME J. 2010, 4 (5): 642-647. 10.1038/ismej.2009.153.
Hamady M, Knight R: Microbial community profiling for human microbiome projects: tools, techniques, and challenges. Genome Res. 2009, 19 (7): 1141-1152. 10.1101/gr.085464.108.
Lauber CL, Zhou N, Gordon JI, Knight R, Fierer N: Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples. FEMS Microbiol Lett. 2010, 307 (1): 80-86. 10.1111/j.1574-6968.2010.01965.x.
Biesbroek G, Sanders EA, Roeselers G, Wang X, Caspers MP, Trzcinski K, Bogaert D, Keijser BJ: Deep sequencing analyses of low density microbial communities: working at the boundary of accurate microbiota detection. PloS one. 2012, 7 (3): e32942-10.1371/journal.pone.0032942.
Lozupone C, Stombaugh J, Gonzalez A, Ackermann G, Wendel D, Vazquez-Baeza Y, Jansson JK, Gordon JI, Knight R: Meta-analyses of studies of the human microbiota. Genome Res. 2013, 23 (10): 1704-1714. 10.1101/gr.151803.112.
Wu GD, Lewis JD, Hoffmann C, Chen YY, Knight R, Bittinger K, Hwang J, Chen J, Berkowsky R, Nessel L, Li H, Bushman FD: Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags. BMC Microbiol. 2010, 10: 206-10.1186/1471-2180-10-206.
Jumpstart Consortium Human Microbiome Project Data Generation Working G: Evaluation of 16S rDNA-based community profiling for human microbiome research. PloS one. 2012, 7 (6): e39315-10.1371/journal.pone.0039315.
Schloss PD, Gevers D, Westcott SL: Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PloS one. 2011, 6 (12): e27310-10.1371/journal.pone.0027310.
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG: Primer3—new capabilities and interfaces. Nucleic Acids Res. 2012, 40 (15): e115-10.1093/nar/gks596.
Sacchi CT, Whitney AM, Mayer LW, Morey R, Steigerwalt A, Boras A, Weyant RS, Popovic T: Sequencing of 16S rRNA gene: a rapid tool for identification of Bacillus anthracis. Emerg Infect Dis. 2002, 8 (10): 1117-1123. 10.3201/eid0810.020391.
Wang Y, Qian PY: Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PloS one. 2009, 4 (10): e7401-10.1371/journal.pone.0007401.
Nossa CW, Oberdorf WE, Yang L, Aas JA, Paster BJ, Desantis TZ, Brodie EL, Malamud D, Poles MA, Pei Z: Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome. World J Gastroenterol. 2010, 16 (33): 4135-4144. 10.3748/wjg.v16.i33.4135.
Amplification primers. [http://vamps.mbl.edu/resources/primers.php]
Wang Q, Garrity GM, Tiedje JM, Cole JR: Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007, 73 (16): 5261-5267. 10.1128/AEM.00062-07.
Group HDGW: 16S 454 Default Protocol Version 4.2—Pilot Study P.1 (15). [http://www.hmpdacc.org/doc/HMP_MDG_454_16S_Protocol.pdf]
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R: QIIME allows analysis of high-throughput community sequencing data. Br J Pharmacol. 2010, 7 (5): 335-336.
Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R: UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011, 27 (16): 2194-2200. 10.1093/bioinformatics/btr381.
Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R: UniFrac: an effective distance metric for microbial community comparison. ISME J. 2011, 5 (2): 169-172. 10.1038/ismej.2010.133.
HMP: hypothesis testing and power calculations for comparing metagenomic samples from HMP. R-package. [http://cran.r-project.org/web/packages/HMP/index.html]
La Rosa PS, Brooks JP, Deych E, Boone EL, Edwards DJ, Wang Q, Sodergren E, Weinstock G, Shannon WD: Hypothesis testing and power calculations for taxonomic-based human microbiome data. PloS one. 2012, 7 (12): e52078-10.1371/journal.pone.0052078.
metagenomeSeq: statistical analysis for sparse high-throughput sequencing. [http://www.bioconductor.org/packages/devel/bioc/vignettes/metagenomeSeq/inst/doc/metagenomeSeq.pdf]
Paulson JN, Stine OC, Bravo HC, Pop M: Differential abundance analysis for microbial marker-gene surveys. Nat Methods. 2013, 10 (12): 1200-1202. 10.1038/nmeth.2658.
Cabrera-Rubio R, Garcia-Nunez M, Seto L, Anto JM, Moya A, Monso E, Mira A: Microbiome diversity in the bronchial tracts of patients with chronic obstructive pulmonary disease. J Clin Microbiol. 2012, 50 (11): 3562-3568. 10.1128/JCM.00767-12.
Pinto AJ, Raskin L: PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets. PloS one. 2012, 7 (8): e43093-10.1371/journal.pone.0043093.
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF: Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009, 75 (23): 7537-7541. 10.1128/AEM.01541-09.
Quince C, Lanzen A, Davenport RJ, Turnbaugh PJ: Removing noise from pyrosequenced amplicons. BMC Bioinformatics. 2011, 12: 38-10.1186/1471-2105-12-38.
Willner D, Daly J, Whiley D, Grimwood K, Wainwright CE, Hugenholtz P: Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples. PloS one. 2012, 7 (4): e34605-10.1371/journal.pone.0034605.
Zhao J, Li J, Schloss PD, Kalikin LM, Raymond TA, Petrosino JF, Young VB, LiPuma JJ: Effect of sample storage conditions on culture-independent bacterial community measures in cystic fibrosis sputum specimens. J Clin Microbiol. 2011, 49 (10): 3717-3718. 10.1128/JCM.01189-11.
Cardona S, Eck A, Cassellas M, Gallart M, Alastrue C, Dore J, Azpiroz F, Roca J, Guarner F, Manichanh C: Storage conditions of intestinal microbiota matter in metagenomic analysis. BMC Microbiol. 2012, 12: 158-10.1186/1471-2180-12-158.
Rubin BE, Gibbons SM, Kennedy S, Hampton-Marcell J, Owens S, Gilbert JA: Investigating the impact of storage conditions on microbial community composition in soil samples. PloS one. 2013, 8 (7): e70460-10.1371/journal.pone.0070460.
Carroll IM, Ringel-Kulka T, Siddle JP, Klaenhammer TR, Ringel Y: Characterization of the fecal microbiota using high-throughput sequencing reveals a stable microbial community during storage. PloS one. 2012, 7 (10): e46953-10.1371/journal.pone.0046953.
Charlson ES, Bittinger K, Chen J, Diamond JM, Li H, Collman RG, Bushman FD: Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts. PloS one. 2012, 7 (9): e42786-10.1371/journal.pone.0042786.
Charlson ES, Bittinger K, Haas AR, Fitzgerald AS, Frank I, Yadav A, Bushman FD, Collman RG: Topographical continuity of bacterial populations in the healthy human respiratory tract. Am J Respir Crit Care Med. 2011, 184 (8): 957-963. 10.1164/rccm.201104-0655OC.
Lemon KP, Klepac-Ceraj V, Schiffer HK, Brodie EL, Lynch SV, Kolter R: Comparative analyses of the bacterial microbiota of the human nostril and oropharynx. mBio. 2010, 1 (3): e00129-10-