Comparison of Experimental Methodologies Based on Bulk-Metagenome and Virus-like Particle Enrichment: Pros and Cons for Representativeness and Reproducibility in the Study of the Fecal Human Virome

Abstract

Metagenomics; Sequencing; Virome

Metagenómica; Secuenciación; Viroma

Metagenòmica; Seqüenciació; Viroma

Studies on the human virome based on the application of metagenomic approaches involve overcoming a series of challenges and limitations inherent not only to the biological features of viruses, but also to methodological pitfalls which different approaches have tried to minimize. These approaches fall into two main categories: bulk-metagenomes and virus-like particle (VLP) enrichment. In order to address issues associated with commonly used experimental procedures to assess the degree of reliability, representativeness, and reproducibility, we designed a comparative analysis applied to three experimental protocols, one based on bulk-metagenomes and two based on VLP enrichment. These protocols were applied to stool samples from 10 adult participants, including two replicas per protocol and subject. We evaluated the performances of the three methods, not only through the analysis of the resulting composition, abundance, and diversity of the virome via taxonomical classification and type of molecule (DNA versus RNA, single stranded vs. double stranded), but also according to how the a priori identical replicas differed from each other according to the extraction methods used. Our results highlight the strengths and weaknesses of each approach, offering valuable insights and tailored recommendations for drawing reliable conclusions based on specific research goals.

This research was funded by Conselleria de Sanitat Universal i Salut Pública, Generalitat Valenciana, grant number CDEI-06/20-E, awarded to V.P.B., as well as by the Asociación Española contra el Cáncer (AECC), project AECC 2017-1485, and the project CIPROM/2021/042 from the Generalitat Valenciana, both awarded to A.M.