Abstract
Deep sequencing of polymerase chain reaction (PCR)-amplified small subunit (16S or 18S) ribosomal RNA (rRNA) genes fragments is commonly employed to characterize the composition and structure of microbial communities. Preparing genomic DNA for sequencing of such gene fragments on Illumina sequencers can be performed in a straightforward, two-stage PCR method, described herein. The protocol described allows for up to 384 samples to be sequenced simultaneously, and provides great flexibility in choice of primers.
Key words
- Polymerase chain reaction
- Microbiome
- Next-generation amplicon sequencing
- 16S ribosomal RNA
- Illumina MiSeq
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References
Ionescu D, Overholt WA, Lynch MD, Neufeld JD, Naqib A et al (2016) Microbial community analysis using high-throughput amplicon sequencing. Manual of environmental microbiology, fourth edition. American Society of Microbiology, Boston, p 2.4. 2-1-2.4 2-26
Green SJ, Venkatramanan R, Naqib A (2015) Deconstructing the polymerase chain reaction: understanding and correcting bias associated with primer degeneracies and primer-template mismatches. PLoS One 10:e0128122
Bybee SM, Bracken-Grissom H, Haynes BD, Hermansen RA, Byers RL et al (2011) Targeted amplicon sequencing (TAS): a scalable next-gen approach to multilocus, multitaxa phylogenetics. Genome Biol Evol 3:1312–1323
Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700
Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA et al (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci 108:4516–4522
Bartram AK, Lynch MD, Stearns JC, Moreno-Hagelsieb G, Neufeld JD (2011) Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end Illumina reads. Appl Environ Microbiol 77:3846–3852
Wu L, Wen C, Qin Y, Yin H, Tu Q et al (2015) Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis. BMC Microbiol 15:125
Krueger F, Andrews SR, Osborne CS (2011) Large scale loss of data in low-diversity illumina sequencing libraries can be recovered by deferred cluster calling. PLoS One 6:e16607
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336
Kennedy K, Hall MW, Lynch MD, Moreno-Hagelsieb G, Neufeld JD (2014) Evaluating bias of Illumina-based bacterial 16S rRNA gene profiles. Appl Environ Microbiol 80:5717–5722
Amaral-Zettler LA, McCliment EA, Ducklow HW, Huse SM (2009) A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes. PLoS One 4:e6372
Vestheim H, Jarman SN (2008) Blocking primers to enhance PCR amplification of rare sequences in mixed samples—a case study on prey DNA in Antarctic krill stomachs. Front Zool 5:12
Green SJ, Minz D (2005) Suicide polymerase endonuclease restriction, a novel technique for enhancing PCR amplification of minor DNA templates. Appl Environ Microbiol 71:4721–4727
Tighe S, Afshinnekoo E, Rock TM, McGrath K, Alexander N et al (2017) Genomic methods and microbiological technologies for profiling novel and extreme environments for the extreme microbiome project (XMP). J Biomol Tech 28:31–39
Acknowledgments
We have been assisted over the years in sequencing amplicons on Illumina MiSeq instruments by Chris Wright and Dr. Hernandez Alvaro of the High-Throughput Sequencing and Genotyping Unit at the University of Illinois at Urbana-Champaign, and we gratefully acknowledge their assistance.
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Naqib, A., Poggi, S., Wang, W., Hyde, M., Kunstman, K., Green, S.J. (2018). Making and Sequencing Heavily Multiplexed, High-Throughput 16S Ribosomal RNA Gene Amplicon Libraries Using a Flexible, Two-Stage PCR Protocol. In: Raghavachari, N., Garcia-Reyero, N. (eds) Gene Expression Analysis. Methods in Molecular Biology, vol 1783. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7834-2_7
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DOI: https://doi.org/10.1007/978-1-4939-7834-2_7
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