Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome
| Autor: | George M. Savva, Lesley Hoyles, Andrea Telatin, Evelien M. Adriaenssens, Rebecca Ansorge, Thomas Wileman, Shen-Yuan Hsieh, Mohammad A. Tariq, Simon R. Carding, Catherine Booth |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Library preparation
Genome Viral Microbiology Free dna Article Bacteriophage Feces bacteriophage Virology Humans Human virome Bacteriophages Dna viral Cloning Molecular Illumina dye sequencing Gene Library Genetics Genetic diversity virome biology High-Throughput Nucleotide Sequencing Sequence Analysis DNA biology.organism_classification PCR bias QR1-502 Gastrointestinal Microbiome Infectious Diseases Viruses Metagenome Metagenomics Nucleic Acid Amplification Techniques Bacteria |
| Zdroj: | Viruses Volume 13 Issue 10 Viruses, Vol 13, Iss 2093, p 2093 (2021) |
| ISSN: | 1999-4915 |
| Popis: | The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses. |
| Databáze: | OpenAIRE |
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