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