Gene capture coupled to high-throughput sequencing as a strategy for targeted metagenome exploration.
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.
Centre de Recherche en Nutrition Humaine Auvergne, Clermont Université, Université d'Auvergne, EA 4678, Conception, Ingénierie et Développement de l'Aliment et du Médicament, BP 10448, Clermont-Ferrand 63000, France.
Jérémie
J
Denonfoux
Nicolas
N
Parisot
Eric
E
Dugat-Bony
Corinne
C
Biderre-Petit
Delphine
D
Boucher
Diego P
DP
Morgavi
Denis
D
Le Paslier
Eric
E
Peyretaillade
Pierre
P
Peyret
D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D054892:Metagenome; D017020:Methanosarcina / Q000201:enzymology / Q000235:genetics; D009876:Operon; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics
D004269:DNA, Bacterial; D010088:Oxidoreductases; C023895:methyl coenzyme M methylreductase