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

Next-generation Sequencing of the DNA Virome from Fecal Samples.

Author information

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

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