PK-189:

Comparison of oligonucleotide and polynucleotide probes for enrichment of target genes from environmental metagenomes by in-solution capture hybridization

Next generation sequencing allows for a rapid acquisition of metagenomic data from environmental samples. Without selection, however, shotgun sequencing often fails to cover the broad spectrum of the diversity of environmental functional genes due to the extremely high complexity of natural microbial communities. To ensure in-depth sequencing, an enrichment of target genes of interest prior to sequencing (i.e. gene targeted metagenomics) is necessary. Recently, enrichment by in-solution capture hybridization has been proposed as a promising approach. Although successful applications to environmental samples have been demonstrated, the probe design strategy and fundamental evaluations of enrichment performance are not yet fully explored. In this study, two different types of probes were adopted for the enrichment of ammonia monooxygenase gene (amoA): a 21-mer oligonucleotide probe targeting a highly conserved region in AmoA, and a polynucleotide probe (~500 bp) amplified from environmental amoA/particulate monooxygenase gene (pmoA) clones. Enrichment performance of the probes was evaluated by hybridizing with a mock community consisting of 10 amoA/pmoA clone sequences in the presence of background E. coli genome fragments. With increasing the hybridization stringency, fold-enrichments of target gene increased in both probes, and more than 10³-fold enrichment was obtained with the oligonucleotide probe whereas the polynucleotide probe could achieve more than 10⁴-fold enrichment. Interestingly, no significant capturing bias was observed with the polynucleotide probe while the captured sequences were highly skewed towards high-GC sequences when the oligonucleotide probe was used. These results show that a design-free, just-PCR-amplified gene fragment can serve as an excellent probe to capture the homologous gene with high specificity and wide coverage of the sequence diversity.

keywords:in-solution capture hybridization,functional gene,metagenome