A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

Ko-Hsuan Chen,Hui-Ling Liao,Gregory Bonito,Reid Longley

doi:10.3390/agronomy11071274

Abstract

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

Highlights

The advancement of high-throughput sequencing has transformed our ability to explore microbial diversity across different environments
The overall sequence read output in combination with the ability to multiplex samples makes MiSeq amplicon sequencing more cost-effective than other approaches
In cases where there is a decrease in the PCR efficiency, this can often be attributed to critical issues such as (1) primers binding non- to undesired genomic regions and, failing to amplify the targeted DNA fragment; (2) primers carried over from the previous amplification reactions consumed in the current PCR cycles; and (3) insufficient primertemplate annealing or low template concentration leading to inadequate PCR products

Summary

Introduction

The advancement of high-throughput sequencing has transformed our ability to explore microbial diversity across different environments. The overall sequence read output in combination with the ability to multiplex samples (i.e., pooling PCR products generated from many samples together in one sequencing effort and assigning the reads back to the original samples) makes MiSeq amplicon sequencing more cost-effective than other approaches. If more than 10% unassigned reads are generated during sequencing, it is likely due to mistagging or unsuccessful tagging of barcodes to the intermediate PCR products during library construction. Many existing protocols utilize a one-step PCR for DNA amplicon sequencing [2,3,4]. In this one-step PCR approach, a barcode is linked to a primer that targets a specific genomic region [3].

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Conclusion