Evaluation of ddRADseq for reduced representation metagenome sequencing

PeerJ. 2017 Sep 19;5:e3837. doi: 10.7717/peerj.3837. eCollection 2017.

Abstract

Background: Profiling of microbial communities via metagenomic shotgun sequencing has enabled researches to gain unprecedented insight into microbial community structure and the functional roles of community members. This study describes a method and basic analysis for a metagenomic adaptation of the double digest restriction site associated DNA sequencing (ddRADseq) protocol for reduced representation metagenome profiling.

Methods: This technique takes advantage of the sequence specificity of restriction endonucleases to construct an Illumina-compatible sequencing library containing DNA fragments that are between a pair of restriction sites located within close proximity. This results in a reduced sequencing library with coverage breadth that can be tuned by size selection. We assessed the performance of the metagenomic ddRADseq approach by applying the full method to human stool samples and generating sequence data.

Results: The ddRADseq data yields a similar estimate of community taxonomic profile as obtained from shotgun metagenome sequencing of the same human stool samples. No obvious bias with respect to genomic G + C content and the estimated relative species abundance was detected.

Discussion: Although ddRADseq does introduce some bias in taxonomic representation, the bias is likely to be small relative to DNA extraction bias. ddRADseq appears feasible and could have value as a tool for metagenome-wide association studies.

Keywords: Double digest restriction site associated DNA sequencing; Human gut microbial communities; Metagenome-wide association studies; ddRADseq.

Grant support

This research work was supported in part by the Australian Government through the Australian Research Council, Linkage grant LP150100912 and the AusGEM collaboration between UTS and the Department of Primary Industry of the New South Wales Government, Australia. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.