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

Abstract

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid 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 phage community of previously sequenced phage genomes and sequenced 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 ~4x and 120x higher than found in Illumina only assemblies respectively. While the best PacBio assemblies had SNPs at frequencies ~3.5 x and 12x higher than found in Illumina only assemblies respectively. Despite high read coverage, long-read only assemblies failed to recover a complete genome for any of the 15 phage, down sampling of reads did increase the proportion of a genome that could be assembled into a single contig. 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.

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

“…For example, it is well documented that bacteriophages often have heavily modified DNA, and this may impact on the accurate basecalling of their genomes [78][79][80][81]. It was also previously reported that too much sequence depth is detrimental for ONT virome assembly [35], although downsampling prior to assembly will remove genomes of lower abundance. Furthermore, there are clear differences in ONT assemblers, with no single assembler performing best in all metrics.…”

“…Furthermore, the yields of DNA obtained from virome extractions are typically low and the large input requirements of ONT sequencing can be prohibitive for samples from some environments (typically 1 µg of DNA in 50 µl of buffer). To overcome the input requirements, one of the first ONT virome studies utilised tangential flow filtration to concentrate large volumes of seawater [36], while others have developed a LASL approach [26,27], and MDA [25,35]. However, MDA introduces biases into metagenomic libraries that can lead to the over-representation of viruses with small, circular ssDNA genomes [37][38][39].…”

Refining SARS-CoV-2 Intra-host Variation by Leveraging Large-scale Sequencing Data

“…Bridging this gap poses a notable challenge and offers a valuable opportunity for methodological innovation. This workflow is designed specifically for Illumina sequencing data, favoured for its lower error rate (Fox et al 2014), and is less suitable for nanopore sequencing (Cook et al 2024;Fournelle et al 2024). The latter's high error rate of about 10% complicates the detection of low-frequency emerging mutations.…”

Host interactions of novel Crassvirales species belonging to multiple families infecting bacterial host, Bacteroides cellulosilyticus WH2

“…These isolates were sequenced on different sequencing platforms, including Oxford Nanopore, Illumina Miseq or a combination of both. Nanopore assemblies provided high-quality and complete assemblies, however required polishing the assembly with Illumina reads to correct for frameshift errors that can fragment genes [70–72]. As a result, the 14 complete genomes were classified at the family and genus levels, denoted as three novel species (), while the remaining isolates were grouped as strains of the same species ().…”