Critical Assessment of Whole Genome and Viral Enrichment Shotgun Metagenome on the Characterization of Stool Total Virome in Hepatocellular Carcinoma Patients

Abstract

Viruses are the most abundant form of life on earth and play important roles in a broad range of ecosystems. Currently, two methods, whole genome shotgun metagenome (WGSM) and viral-like particle enriched metagenome (VLPM) sequencing, are widely applied to compare viruses in various environments. However, there is no critical assessment of their performance in recovering viruses and biological interpretation in comparative viral metagenomic studies. To fill this gap, we applied the two methods to investigate the stool virome in hepatocellular carcinoma (HCC) patients and healthy controls. Both WGSM and VLPM methods can capture the major diversity patterns of alpha and beta diversities and identify the altered viral profiles in the HCC stool samples compared with healthy controls. Viral signatures identified by both methods showed reductions of Faecalibacterium virus Taranis in HCC patients’ stool. Ultra-deep sequencing recovered more viruses in both methods, however, generally, 3 or 5 Gb were sufficient to capture the non-fragmented long viral contigs. More lytic viruses were detected than lysogenetic viruses in both methods, and the VLPM can detect the RNA viruses. Using both methods would identify shared and specific viral signatures and would capture different parts of the total virome.

Targeted Viromes and Total Metagenomes Capture Distinct Components of Bee Gut Phage Communities

“…Here we compare how two common forms of phage community sampling, bioinformatic mining of total metagenomes and targeted sequencing of viromes, influence the phage communities recovered from the guts of two commercially important bee species: Apis mellifera and Bombus impatiens. Similar to the results of previous studies [16][17][18] , we show that sampling method significantly affected alpha diversity, beta diversity, composition, and structure of sampled phage communities. In particular, we show that viromes outperform total metagenomes in terms of the number of phage recovered, but that this can depend on the specific environment being sampled.…”

“…While our results highlight the ability of total metagenomes to recover phage missed by viromes, they also showcase the capacity of viromes to sample a greater diversity of phage. Similar to previous comparisons of viromes and total metagenomes in human gut, soil, and freshwater environments 17,18,29 , our honey bee viromes recovered a substantially larger number of phage than did total metagenomes. Further, the occupancy-abundance relationships examined here and by Medellin et al 16 show that total metagenomes tend to be biased towards sampling the most abundant and prevalent phage, while viromes are more successful at sampling rare phage.…”

“…As such, size filtration may have led some phage which are present in our bee guts to have been excluded from viromes. This size exclusion may explain why we, and previous virome vs total metagenome studies 16–18 , consistently show that some phage are only found in total metagenomes. The particular types of phage which are being excluded likely further explain the total metagenome and virome differences we observed.…”

“…However, whether viromes always outperform total metagenomes is not clear. Work applying these two methods to host-associated (human gut) and low-biomass (deep-sea marine) samples has produced conflicting results 17,18 . One explanation for these discrepancies is the biases associated with total metagenomes and targeted viromes 30,31 .…”

“…As such, insights gained from studying free-living phage communities may not always apply to communities of host-associated phage. Specifically, while the best methods for sampling free-living phage communities have been empirically compared 16,17 , little work has evaluated the best methods for sampling host-associated phage 18 . Moreover, given the importance of microbiomes to host health, understanding phage community dynamics and how phage differ between related animal hosts is a key priority.…”