Biases in Viral Metagenomics-Based Detection, Cataloguing and Quantification of Bacteriophage Genomes in Human Faeces, a Review

Abstract

The human gut is colonised by a vast array of microbes that include bacteria, viruses, fungi, and archaea. While interest in these microbial entities has largely focused on the bacterial constituents, recently the viral component has attracted more attention. Metagenomic advances, compared to classical isolation procedures, have greatly enhanced our understanding of the composition, diversity, and function of viruses in the human microbiome (virome). We highlight that viral extraction methodologies are crucial in terms of identifying and characterising communities of viruses infecting eukaryotes and bacteria. Different viral extraction protocols, including those used in some of the most significant human virome publications to date, have introduced biases affecting their a overall conclusions. It is important that protocol variations should be clearly highlighted across studies, with the ultimate goal of identifying and acknowledging biases associated with different protocols and, perhaps, the generation of an unbiased and standardised method for examining this portion of the human microbiome.

Optimization and Ev a Luation of Viral Metagenomic Amplification and Sequencing Methods Toward a Genome -le Vel Resolution of the Human Fecal DNA Virome

“…Due to the limitation of low biomass, viral metagenome inevitably requires multiple polymerase chain reaction (PCR) ampli cation procedures to obtain su cient DNA for high-throughput sequencing [17]. For high-delity enzymes approach, the universal ampli cation cycle is 30-40, which may result in severe stochastic or systemic bias in viral genome fragmenting, skew viral abundances, and over-amplify small circular single-stranded DNA (ssDNA) viruses [6,[18][19][20][21]. Such viral ampli cation leads to uneven distribution of read coverage across the viral genome, which prevents genome reconstruction from the sequencing fragments [22,23].…”

Optimisation and Application of a Novel Method to Identify Bacteriophages in Maternal Milk and Infant Stool Identifies Host-Phage Communities Within Preterm Infant Gut

“…Similar to previous studies which applied viral purification protocols using CsCl density gradient ultracentrifugation on term BM ( 14 ), we identify Mimiviridae (a giant group of viruses) in DM from our sequencing data that were not previously reported in BM studies using micron-sized membrane filters ( 16 ). Our proposed method omits the time-consuming CsCl density gradient purification which has previously been reported to have low reproducibility and is not well-suited for high-throughput studies ( 45 , 46 ).…”

Viral biogeography of gastrointestinal tract and parenchymal organs in two representative species of mammals

“…From a technical perspective the study was designed to minimize the biases typically associated with virome analysis 41,46,67 . We used unamplified nucleic acids and assembly-based cataloguing of novel viruses, coupled with quantitation by comparison against a spike-in viral standard.…”