Gene Capture Coupled to High-Throughput Sequencing as a Strategy for Targeted Metagenome Exploration

Abstract

Next-generation sequencing (NGS) allows faster acquisition of metagenomic data, but complete exploration of complex ecosystems is hindered by the extraordinary diversity of microorganisms. To reduce the environmental complexity, we created an innovative solution hybrid selection (SHS) method that is combined with NGS to characterize large DNA fragments harbouring biomarkers of interest. The quality of enrichment was evaluated after fragments containing the methyl coenzyme M reductase subunit A gene (mcrA), the biomarker of methanogenesis, were captured from a Methanosarcina strain and a metagenomic sample from a meromictic lake. The methanogen diversity was compared with direct metagenome and mcrA-based amplicon pyrosequencing strategies. The SHS approach resulted in the capture of DNA fragments up to 2.5 kb with an enrichment efficiency between 41 and 100%, depending on the sample complexity. Compared with direct metagenome and amplicons sequencing, SHS detected broader mcrA diversity, and it allowed efficient sampling of the rare biosphere and unknown sequences. In contrast to amplicon-based strategies, SHS is less biased and GC independent, and it recovered complete biomarker sequences in addition to conserved regions. Because this method can also isolate the regions flanking the target sequences, it could facilitate operon reconstructions.

Phylogenomic Data Support a Seventh Order of Methylotrophic Methanogens and Provide Insights into the Evolution of Methanogenesis

“…Primers targeting the mtaBC gene cluster designed on the sequences of “ Ca. M. alvus” and M. luminyensis were used to amplify environmental sequences of mtaBC from genomic DNA previously extracted from the Lake Pavin water column (Denonfoux et al 2013) (supplementary methods and table S1, Supplementary Material online). The sequences of mtaB and mtaBC were deposited in GenBank under accession numbers KF302411–KF302419 and KF302420–KF302421, respectively.…”

Targeted capture in evolutionary and ecological genomics

“…The use of one or a few DNA markers for species identification is limited in many ways but remains a common approach in metagenomics (Valentini et al 2009; Portik et al 2012). Targeted capture can provide a high-throughput approach to analyze community composition using species barcodes and to explore functional variation in a community (e.g., by targeting genes underlying specific ecological functions; Denonfoux et al 2013). Gut microbiomes constitute one type of community that has gained substantial interest recently for its potential role governing health and contributing to broader ecological and evolutionary patterns (Muegge et al 2011; The Human Microbiome Project Consortium 2012).…”

The sequence capture by hybridization: a new approach for revealing the potential of mono‐aromatic hydrocarbons bioattenuation in a deep oligotrophic aquifer

“…Sequence capture by hybridization gives quantitative results close to that obtained by qPCR as demonstrates by Denonfoux et al . (). The three contigs obtained by sequence capture and the three bssA amplicons obtained in this study were compared with sequences from pure strains and environmental samples deposited in international databases after having been translated into AA sequences.…”