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Volume 10, Issue 2

The long and short of it: benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies Open Access

Abstract

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid assembly approaches that combine long- and short-read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock bacteriophage community of previously sequenced phage genomes and sequenced them using Illumina, Nanopore and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies 41 and 157 % higher than found in Illumina only assemblies, respectively. While the best PacBio assemblies had SNPs at frequencies 12 and 78 % higher than found in Illumina-only assemblies, respectively. Despite high-read coverage, long-read-only assemblies recovered a maximum of one complete genome from any assembly, unless reads were down-sampled prior to assembly. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short-read-only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): bacteriophage , bioinformatics , hybrid assembly , Illumina , long-reads , Nanopore , PacBio , Phage , viral metagenomics and Viromics
Funding
This study was supported by the:
  • Medical Research Council (Award MR/L015080/1)
    • Principle Award Recipient: AndrewMillard
  • Medical Research Foundation National PhD Training Programme in Antimicrobial Resistance Research (Award MRF-145-0004-TPG-AVISO)
    • Principle Award Recipient: RyanCook
© 2024 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2024-02-20
2024-05-19
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The long and short of it: benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies
M Gen 10, 001198 (2024); https://doi.org/10.1099/mgen.0.001198
/content/journal/mgen/10.1099/mgen.0.001198
/content/journal/mgen/10.1099/mgen.0.001198
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