Tools
ToolsHaagmans, Rik, Charity, Oliver J., Baker, Dave, Telatin, Andrea, Savva, George M., Adriaenssens, Evelien M., Powell, Penny P. and Carding, Simon R. (2025) Assessing bias and reproducibility of viral metagenomics methods for the combined detection of faecal RNA and DNA viruses. Viruses, 17 (2). ISSN 1999-4915
Preview |
PDF (viruses-17-00155-v2)
- Published Version
Available under License Creative Commons Attribution. Download (5MB) | Preview |
Abstract
Whole transcriptome amplification (WTA2) and sequence-independent single primer amplification (SISPA) are two widely used methods for combined metagenomic sequencing of RNA and DNA viruses. However, information on the reproducibility and bias of these methods on diverse viruses in faecal samples is currently lacking. A mock community (MC) of diverse viruses was developed and used to spike faecal samples at different concentrations. Virus-like particles (VLPs) were extracted, nucleic acid isolated, reverse-transcribed, and PCR amplified using either WTA2 or SISPA and sequenced for metagenomic analysis. A bioinformatics pipeline measured the recovery of MC viruses in replicates of faecal samples from three human donors, analysing the consistency of viral abundance measures and taxonomy. Viruses had different recovery levels with VLP extraction introducing variability between replicates, while WTA2 and SISPA produced comparable results. In comparing WTA2- and SISPA-generated libraries, WTA2 gave more uniform coverage depth profiles and improved assembly quality and virus identification. SISPA produced more consistent abundance, with a 50% difference between replicates occurring in ~20% and ~10% of sequences for WTA2 and SISPA, respectively. In conclusion, a bioinformatics pipeline has been developed to assess the methodological variability and bias of WTA2 and SISPA, demonstrating higher sensitivity with WTA2 and higher consistency with SISPA.
| Item Type: | Article |
|---|---|
| Additional Information: | Data Availability Statement: The virome metagenomic sequencing data cleaned using the cleanup pipeline are freely available under NCBI BioProject number PRJNA1189570. The code for the analysis pipeline used in this paper is available as a Nextflow pipeline at https://github.com/RHaagmans/mc-spike. Funding Information: This study was supported by the BBSRC Institute Strategic Programme Grant Gut Microbes and Health BB/R012490/1 and its constituent projects BBS/E/F/000PR10353, BBS/E/F/000PR10355, and BBS/E/F/000PR10356 (S.R.C., E.M.A, O.J.C., A.T.). E.M.A was additionally funded by the BBSRC Institute Strategic Programme Food Microbiome and Health BB/X011054/1 and its constituent projects BBS/E/F/000PR13631 and BBS/E/F/000PR13633; and by the BBSRC Institute Strategic Programme Microbes and Food Safety BB/X011011/1 and its constituent projects BBS/E/F/000PR13634, BBS/E/F/000PR13635, and BBS/E/F/000PR13636 (E.M.A). The QIB Sequencing facility and D.B. and G.M.S. were funded by BBSRC Core Capability Grant BB/CCG1860/1. R.H. was supported by PhD studentships jointly funded by Invest in ME Research (UK Charity number 1153730) and the Faculty of Medicine and Health, University of East Anglia. |
| Uncontrolled Keywords: | bacteriophages,eukaryotic viruses,mock community,viral metagenomics |
| Faculty \ School: | Faculty of Medicine and Health Sciences > Norwich Medical School Faculty of Medicine and Health Sciences > School of Health Sciences |
| UEA Research Groups: | Faculty of Medicine and Health Sciences > Research Groups > Gastroenterology and Gut Biology Faculty of Medicine and Health Sciences > Research Centres > Metabolic Health Faculty of Medicine and Health Sciences > Research Centres > Norwich Institute for Healthy Aging |
| Depositing User: | LivePure Connector |
| Date Deposited: | 24 Jun 2025 16:30 |
| Last Modified: | 24 Jun 2025 19:30 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/99710 |
| DOI: | 10.3390/v17020155 |
Downloads
Downloads per month over past year
Actions (login required)
 |
View Item |