An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform

Microbiome - Tập 2 - Trang 1-7 - 2014
Douglas W Fadrosh1, Bing Ma1, Pawel Gajer1, Naomi Sengamalay1, Sandra Ott1, Rebecca M Brotman2, Jacques Ravel1
1Institute for Genome Sciences, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, USA
2Institute for Genome Sciences, Department of Epidemiology and Public Health, University of Maryland School of Medicine, Baltimore, USA

Tóm tắt

To take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development of improved methods to overcome technical limitations inherent to the sequencing platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on the Illumina MiSeq platform and often generates sequences of suboptimal quality. Here we present an improved dual-indexing amplification and sequencing approach to assess the composition of microbial communities from clinical samples using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform. We introduced a 0 to 7 bp “heterogeneity spacer” to the index sequence that allows an equal proportion of samples to be sequenced out of phase. Our approach yields high quality sequence data from 16S rRNA gene amplicons using both 250 bp and 300 bp paired-end MiSeq protocols and provides a flexible and cost-effective sequencing option.

Tài liệu tham khảo

Staley JT, Konopka A: Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Ann Rev Microbiol. 1985, 39: 321-346. 10.1146/annurev.mi.39.100185.001541. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R: Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012, 6: 1621-1624. 10.1038/ismej.2012.8. Illumina: High-speed multiplexed 16S microbial sequencing on the MiSeq System. Application Note: DNA Sequencing. http://res.illumina.com/documents/products/appnotes/appnote_miseq_16s.pdf Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD: Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol. 2013, 79: 5112-5120. 10.1128/AEM.01043-13. Human Microbiome Project Consortium: Structure, function and diversity of the healthy human microbiome. Nature. 2012, 486: 207-214. 10.1038/nature11234. Ewing B, Hillier L, Wendl MC, Green P: Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998, 8: 175-185. 10.1101/gr.8.3.175. Ewing B, Green P: Base-calling of automated sequencer traces using phred II. Error probabilities. Genome Res. 1998, 8: 186-194. Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009, 25: 1754-1760. 10.1093/bioinformatics/btp324. Seatk: Toolkit for processing sequences in FASTA/Q formats. [https://github.com/lh3/seqtk] 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. Nature Methods. 2010, 7: 335-336. 10.1038/nmeth.f.303. Masella AP, Bartram AK, Truszkowski JM, Brown DG, Neufeld JD: PANDAseq: paired-end assembler for illumina sequences. BMC Bioinformatics. 2012, 13: 31-10.1186/1471-2105-13-31. Magoc T, Salzberg SL: FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics. 2011, 27: 2957-2963. 10.1093/bioinformatics/btr507. Edgar RC: Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010, 26: 2460-2461. 10.1093/bioinformatics/btq461. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R: UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011, 27: 2194-2200. 10.1093/bioinformatics/btr381. 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: 5261-5267. 10.1128/AEM.00062-07. McDonald D, Price MN, Goodrich J, Nawrocki EP, DeSantis TZ, Probst A, Andersen GL, Knight R, Hugenholtz P: An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. Isme J. 2012, 6: 610-618. 10.1038/ismej.2011.139.