Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes

Abstract

BackgroundMetagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles.Principal FindingsWe evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7–15% of all reads were classified as “unknown”, depending on the random amplification method.ConclusionThe protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses.

A decade of RNA virus metagenomics is (not) enough

“…While no doubt the best way to enrich for viral like particles, and perhaps to help define a sequence as likely viral in origin, the use of ultracentrifugation is not necessarily required for viral metagenomics. Rather ultracentrifuge is kept for defined "virome" work, especially where there is a high bacterial background such as feces (Kohl et al, 2015;Temmam et al, 2015;Thurber et al, 2009). Indeed a direct headto-head comparisons of ultracentrifugation and filtration for viral particle enrichment found the main benefit of ultracentrifugation to be the removal of host DNA background, which is less of a concern for RNA viral metagenomics if extracted nucleic acids are DNAse treated (Kleiner et al, 2015).…”

Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance

“…An alternative approach for pathogen enrichment in mNGS libraries is the use of a host depletion method. Such methods include differential lysis 37 , the removal of abundant human ribosomal and mitochondrial RNA sequences using antibody hybridization or depletion of abundant sequences by hybridization (DASH) 38 and nuclease treatment before extraction 39,40 . Aside from a post-extraction DNase step, we did not incorporate host depletion as part of MSSPE, given that these methods can be cumbersome, with additional Abbott m2000 RT-PCR assays were used to estimate the titres of HIV and HCV; viral titres for other viruses were estimated using in-house qRT-PCR assays with standard curve analysis.…”

“…d Absolute percentage increase from using random primer only (coverage by SP (%) − coverage by RH (%)); coverage of 40-60% is sufficient for genotypic and phylogenetic inference from partial genome assemblies 27 steps, and difficult to standardize. Furthermore, host depletion methods can bias detection towards specific pathogen types, such as the enrichment of bacteria and fungi with cell walls with differential lysis 37 , or encapsidated viruses with pre-extraction nuclease treatment 39,40 . Host depletion can also be performed at the RNA level by the use of non-human primers to bias against abundant human host ribosomal and/or mitochondrial RNA 41,42 .…”

Screening for Viral Pathogens in African Simian Bushmeat Seized at A French Airport

“…DNA and RNA viromes of sample no STE0011 were prepared as previously described (Temmam et al., ). Briefly, the clarified supernatant was filtered through a 0.45‐ μ m filter (Millipore, Molsheim, France), and free nucleic acids were digested at 37°C for 1 h with the following cocktail of nucleases: 20 U Exonuclease I (New England Biolabs, Évry, France), 25 U Benzonase ® (Merck Millipore, Molsheim, France), 25 U RNase A (Roche Diagnostics, Meylan, France), 20 U Turbo DNase (Life Technologies) and 10 μ l of 10× Turbo DNase buffer.…”