A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes.

Orest Kuzyk,Alexander Loy,Roey Angel,Craig W. Herbold,Claus Pelikan,David Berry,Bela Hausmann

doi:10.3389/fmicb.2015.00731

Abstract

High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology.

Highlights

The gold standard for analyzing and comparing microbial communities across many environmental or medical samples is met with high throughput sequencing of 16S rRNA and functional marker gene amplicons
Current sequencing technologies enable the generation of millions of reads per run, and parallel sequencing of multiple samples can be accomplished through the introduction of a sample-specific short sequence tag at one or both ends of the target gene amplicon during library preparation
Single-step PCR with long fusion primers can lead to differences in amplification efficiency and accuracy between samples, a problem which can be ameliorated to some extent with a two-step PCR procedure in which the large fusion primers are added to PCR

Summary

Introduction

The gold standard for analyzing and comparing microbial communities across many environmental or medical samples is met with high throughput sequencing of 16S rRNA and functional marker gene amplicons. Current sequencing technologies enable the generation of millions of reads per run, and parallel sequencing of multiple samples can be accomplished through the introduction of a sample-specific short sequence tag (barcode, index) at one or both ends of the target gene amplicon during library preparation.

Results

Conclusion