Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome

Abstract

The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.

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

“…Sample collection, virome preparation, and Illumina sequencing were performed as part of a previously described comparison of PCR versus PCR-Free DNA library preparation for human faecal viromes [38]. The ethics for the previous study were approved by the University of East Anglia (UEA) Faculty of Medicine and Health Sciences (FMH) Research Ethics Committee (FMH20142015-28), Norwich in 2014, and by the Health Research Authority (HRA) NRES Committee (17/LO/1102; IRAS: 218545) in 2017.…”

“…To gain enough material for ONT sequencing, multiple DNA extracts were performed on aliquots of the same sample and then pooled. For full details, please see the previous publication [38].…”

T7 DNA polymerase treatment improves quantitative sequencing of both double-stranded and single-stranded DNA viruses

“…In the last ten years, many methodological improvements have been put forward, in order to generate informative metagenomics datasets for the viral fraction (or virome) of any type of environmental or biological sample (1)(2)(3)(4)(5). A particular attention has to be paid to the fact that viromes are mixtures of single-stranded (ss) and double-stranded (ds) DNA viruses.…”

Uncovering 1058 Novel Human Enteric DNA Viruses Through Deep Long-Read Third-Generation Sequencing and Their Clinical Impact

“…These low-abundance viral taxa could be retained with PCR-free library preparation, thus potentiating the use of amplification-free methods to investigate "rare" members of the gut virome. 6 Given that most studies analyzed metagenomic reads at low depth, there is currently a lack of high-quality assembled viral genomes, as well as failure in identifying viruses with low abundance in samples. In addition, Illumina sequencing, which generates short reads, and its associated assemblers (eg, megahit 7 and metaSPAde 8 ) have been used frequently.…”