A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses

Elek, Claire K A; Brown, Teagan L.; Viet, Thanh Le; Evans, Rhiannon; Baker, David; Telatin, Andrea; Tiwari, Sumeet K.; Al-Khanaq, Haider; Thilliez, Gaëtan; Kingsley, Robert A.; Hall, Lindsay J.; Webber, Mark A.; Adriaenssens, Evelien M.

doi:10.1101/2023.03.09.531871

“…With the aim of recovering the most fully resolved viral genomes, our findings suggest Illumina is the best approach, whereas for maximising recovered viral diversity ONT is ideal (Table 2). However, previous study of Illumina sequencing for the recovery of viral genomes has uncovered false DTRs at the termini of Illumina sequences arising from repeated assembly artefacts, calling into question the validity of 'complete' viral genomes recovered from Illumina-only assemblies [29]. Similarly, ONT assemblies recovered far more genomes that were ≥90 % complete than Illumina, potentially due to ONT assemblies not being able to resolve DTRs at the phage termini, or perhaps through limitations in the methodology we used to determine whether genomes were complete or not.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, the inclusion of ONT reads has increased the recovery of viral genomes from groundwater metagenomes [28]. Additionally, a hybrid assembly approach has improved the assembly completeness and quality of individual phage genomes belonging to the genus Przondovirus [29], utilising similar methods to those suggested for high-quality bacterial genome assemblies [30]. However, whilst long-read sequencing may aid in the recovery of viral genomes, it is not without limitations and challenges.…”

Section: Introductionmentioning

confidence: 99%

Nanopore and Illumina sequencing reveal different viral populations from human gut samples

Cook,

Telatin,

Hsieh

et al. 2024

Microbial Genomics

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The advent of viral metagenomics, or viromics, has improved our knowledge and understanding of global viral diversity. High-throughput sequencing technologies enable explorations of the ecological roles, contributions to host metabolism, and the influence of viruses in various environments, including the human intestinal microbiome. However, bacterial metagenomic studies frequently have the advantage. The adoption of advanced technologies like long-read sequencing has the potential to be transformative in refining viromics and metagenomics. Here, we examined the effectiveness of long-read and hybrid sequencing by comparing Illumina short-read and Oxford Nanopore Technology (ONT) long-read sequencing technologies and different assembly strategies on recovering viral genomes from human faecal samples. Our findings showed that if a single sequencing technology is to be chosen for virome analysis, Illumina is preferable due to its superior ability to recover fully resolved viral genomes and minimise erroneous genomes. While ONT assemblies were effective in recovering viral diversity, the challenges related to input requirements and the necessity for amplification made it less ideal as a standalone solution. However, using a combined, hybrid approach enabled a more authentic representation of viral diversity to be obtained within samples.

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“…With the aim of recovering the most fully resolved viral genomes, our findings suggest Illumina is the best approach, whereas for maximising recovered viral diversity ONT is ideal (Table 2). However, previous study of Illumina sequencing for the recovery of viral genomes has uncovered false DTRs at the termini of Illumina sequences arising from repeated assembly artefacts, calling into ques4on the validity of "complete" viral genomes recovered from Illumina-only assemblies [27]. Similarly, the increase in predicted viral diversity from ONT sequencing is not necessarily a clear benefit, as further analysis of the data brings the reliability of the predic4ons into ques4on.…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, a long-read LASL approach sequenced on a MinION and paired with Illumina sequencing, dubbed VirION and later improved for VirION2, has also increased the number and completeness of viral genomes from marine samples [25,26]. Addi4onally, a hybrid assembly approach has improved the assembly completeness and quality of individual phage genomes belonging to Przondovirus [27], u4lising similar methods to those suggested for high quality bacterial genome assemblies [28].…”

Section: Introduc:onmentioning

confidence: 99%

Nanopore and Illumina Sequencing Reveal Different Viral Populations from Human Gut Samples

Cook,

Telatin,

Hsieh

et al. 2023

Preprint

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The advent of viral metagenomics, or viromics, has improved our knowledge and understanding of global viral diversity. High-throughput sequencing technologies enable explorations of the ecological roles, contributions to host metabolism, and the influence of viruses in various environments including the human gut microbiome. However, the bacterial metagenomic studies frequently have the advantage. The adoption of advanced technologies like long-read sequencing has the potential to be transformative in refining viromics and metagenomics.Here, we examined the effectiveness of long-read and hybrid sequencing by comparing Illumina short-read and Oxford Nanopore Technology (ONT) long-read sequencing technologies and different assembly strategies on recovering viral genomes from human faecal samples.Our findings showed that if a single sequencing technology is to be chosen for virome analysis, Illumina was preferable due to its superior ability to recover fully resolved viral genomes and minimise erroneous genomes. While ONT assemblies were effective in recovering viral diversity, the challenges related to input requirements and the necessity for amplification made it less ideal as a standalone solution. However, using a combined, hybrid approach enabled a more authentic representation of viral diversity to be obtained within samples.Impact StatementViral metagenomics, or viromics, has revolutionised our understanding of global viral diversity however long-read and hybrid approaches are not yet widespread in this field. Here, we compared the performance of Illumina short-read and Nanopore long-read assembly approaches for recovering fully resolved viral genomes from human faecal samples. We highlight Illumina’s short-read sequencing for recovering fully resolved viral genomes, while acknowledging Oxford Nanopore Technology’s long-read sequencing for capturing broader viral diversity. However, a hybrid approach, utilising both technologies, may mitigate the limitations of one technology alone.Data SummaryAll reads used in this study are available on European Nucleotide Archive (ENA) within the project PRJEB47625.

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“…It is not clear why there are not more viromes sequenced with long-read technologies, as has become commonplace for sequencing of bacterial metagenomes. Even for the sequencing of individual phage isolates there are relatively few studies that have utilized long-reads [28][29][30][31][32]. In part, this is likely because the vast majority of phage genomes can be assembled from short-read Illumina sequences alone [33].…”

Section: Introductionmentioning

confidence: 99%

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

Cook,

Brown,

Rihtman

et al. 2024

Microbial Genomics

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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.

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A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses

Cited by 3 publications

References 97 publications

Nanopore and Illumina sequencing reveal different viral populations from human gut samples

Nanopore and Illumina sequencing reveal different viral populations from human gut samples

Nanopore and Illumina Sequencing Reveal Different Viral Populations from Human Gut Samples

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

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