Making and Sequencing Heavily Multiplexed, High-Throughput 16S Ribosomal RNA Gene Amplicon Libraries Using a Flexible, Two-Stage PCR Protocol

  • Ankur Naqib
  • Silvana Poggi
  • Weihua Wang
  • Marieta Hyde
  • Kevin Kunstman
  • Stefan J. GreenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1783)


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 



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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ankur Naqib
    • 1
  • Silvana Poggi
    • 1
  • Weihua Wang
    • 1
  • Marieta Hyde
    • 1
  • Kevin Kunstman
    • 1
  • Stefan J. Green
    • 1
    Email author
  1. 1.DNA Services Facility, Research Resources CenterUniversity of Illinois at ChicagoChicagoUSA

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