Thursday, 23 May 2019

Complete genome sequences of pooled genomic DNA from 10 marine bacteria using PacBio long-read sequencing

Song W, Thomas T & Edwards RJ (2019) Complete genome sequences of pooled genomic DNA from 10 marine bacteria using PacBio long-read sequencing. Marine Genomics in press DOI: 10.1016/j.margen.2019.05.002

Abstract

Background

High-quality, completed genomes are important to understand the functions of marine bacteria. PacBio sequencing technology provides a powerful way to obtain high-quality completed genomes. However individual library production is currently still costly, limiting the utility of the PacBio system for high-throughput genomics. Here we investigate how to generate high-quality genomes from pooled marine bacterial genomes.

Results

Pooled genomic DNA from 10 marine bacteria were subjected to a single library production and sequenced with eight SMRT cells on the PacBio RS II sequencing platform. In total, 7.35 Gbp of long-read data was generated, which is equivalent to an approximate 168× average coverage for the input genomes. Genome assembly showed that eight genomes with average nucleotide identities (ANI) lower than 91.4% can be assembled with high-quality and completion using standard assembly algorithms (e.g. HGAP or Canu). A reference-based reads phasing step was developed and incorporated to assemble the complete genomes of the remaining two marine bacteria that had an ANI > 97% and whose initial assemblies were highly fragmented.

Conclusions

Ten complete high-quality genomes of marine bacteria were generated. The findings and developments made here, including the reference-based read phasing approach for the assembly of highly similar genomes, can be used in the future to design strategies to sequence pooled genomes using long-read sequencing.

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