Virome Sequencing of Stool Samples

Abstract

Next-generation sequencing has opened avenues to studying complex populations such as the bacteriome (all bacteria), mycobiome (all fungi), and virome (all viruses in a given sample). Viromes are less often investigated as compared to bacteriomes. The reasons are mostly methodological: because no common pan-viral sequence signature exists, metagenomic sequencing remains the only option. This brings about the need of laborious virus enrichment, multiple signal amplification steps with virtually no possibility of interim quality control, and complicated bioinformatic analysis of the ensuing sequence data. Nevertheless, over the past decade virome sequencing has been enormously successful in identifying new agents in human and animal diseases, and in characterizing viruses in various ecological niches. Recently, virome sequencing has been also employed in studies of non-infectious diseases, which has brought about new challenges of sensitivity, costs, and reproducibility in testing of large sets of samples. Here, we present a detailed protocol that has been utilized in virome studies where hundreds of samples had to be reliably tested in order to assess the association of the stool virome with susceptibility to type 1 diabetes, a non-infectious autoimmune disease.

Key words

Annex 1: Record Sheet for Virome Sequencing (page 1 of 2)

Name of the sequencing plate: __________________________________________.

Date of processing: ____________________Processed by: ____________________.

Index space layout: set AFC-131-2001

A set	1 N701	2 N702	3 N703	4 N704	5 N705	6 N706	7 N707	8 N710	9 N711	10 N712	11 N714	12 N715
A S502
B S503
C S505
D S506
E S507
F S508
G S510
H S511

Index space layout: set DFC-131-2004.

D set	1 N716	2 N718	3 N719	4 N720	5 N721	6 N722	7 N723	8 N724	9 N726	10 N727	11 N728	12 N729
A S513
B S515
C S516
D S517
E S518
F S520
G S521
H S522

The actual experimental plate layout: note both indices, and sample ID.

ind.N7.. ind.S5...smp.id:	1	2	3	4	5	6	7	8	9	10	11	12
A
B
C
D
E
F
G
H

Record Sheet - Virome Sequencing (page 2 of 2).

Name of the sequencing plate: _________________________________________________.

Step	Resulting reaction content	Done date, time	By whom	Notes
3.1	Nucleic acid extracted
3.2.1	First strand synthesized
3.2.2	Second strand synthesized, after Exo/sap treatment
3.2.4	Single primer amplification
3.3.1	Purified product of the amplification
3.3.2	Products of the amplification, diluted to 0.2 ng/μL
3.4.2	Products of tagmentation
3.4.3	Products of limited PCR for indexing (libraries)
3.5.1	Purified libraries
3.5.2	Diluted libraries for KAPA measurement
3.5.4	Libraries normalized to 500 pM
3.5.4	Pool of the normalized libraries
3.6	Sequencing: Instrument ID: ___________ Kit serial #: ______________ BaseSpace ID:____________