Next-generation Sequencing of the DNA Virome from Fecal Samples

Cynthia L Monaco; Douglas S Kwon

doi:10.21769/BioProtoc.2159

Next-generation Sequencing of the DNA Virome from Fecal Samples

Bio Protoc. 2017 Mar 5;7(5):e2159. doi: 10.21769/BioProtoc.2159.

Authors

Cynthia L Monaco¹, Douglas S Kwon^{2

3}

Affiliations

¹ Department of Medicine, Washington University School of Medicine, St. Louis, USA.
² Ragon Institute of MGH, MIT, and Harvard, Cambridge, USA.
³ Division of Infectious Diseases, Massachusetts General Hospital and Harvard Medical School, Boston, USA.

Abstract

Herein we describe a detailed protocol for DNA virome analysis of low input human stool samples (Monaco et al., 2016). This protocol is divided into four main steps: 1) stool samples are pulverized to evenly distribute microbial matter; 2) stool is enriched for virus-like particles and DNA is extracted by phenol-chloroform; 3) purified DNA is multiple-strand displacement amplified (MDA) and fragmented; and 4) libraries are constructed and sequenced using Illumina Miseq. Subsequent sequence analysis for viral sequence identification should be sensitive but stringent.

Keywords: Illumina Miseq; MDA; Viral microbiome; Virome; Virus-like particles.

Abstract

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