A universal genome sequencing method for rotavirus A from human fecal samples which identifies segment reassortment and multi-genotype mixed infection

Abstract

Background: Genomic characterization of rotavirus (RoV) has not been adopted at large-scale due to the complexity of obtaining sequences for all 11 segments, particularly when feces are used as starting material.

Methods: To overcome these limitations, we developed a novel RoV capture and genome sequencing method combining commercial enzyme immunoassay plates and a set of routinely used reagents.

Results: Our approach had a 100% success rate, producing >90% genome coverage for diverse RoV present in fecal samples (Ct < 30).

Conclusions: This method provides a novel, reproducible and comparatively simple approach for genomic RoV characterization and could be scaled-up for use in global RoV surveillance systems.

Trial registration (prospectively registered): Current Controlled Trials ISRCTN88101063 . Date of registration: 14/06/2012.

Keywords: Antibody capture; Co-infection; Genome sequencing; Genomics; Phylogenetics; Reassortment; Rotavirus A.

Fig. 1

The procedure for rotavirus capture and nucleic acid preparation prior to genome sequencing. Flow diagram describing the major protocol steps required for the purification of RoV and the amplification of RoV specific nucleic acid for next generation genome sequencing. Image of Illumina MiSeq obtained from https://assets.illumina.com/content/dam/illumina-marketing/images/systems/miseqdx/web-graphic-miseq-front-comparison-chart.jpg. Clipart image of pipette obtained from http://www.clker.com/clipart-pipette-with-tip.html. Clipart image of 96-well plate obtained from http://cyberuse.com/96-well-plate-template.html. Clipart image of Eppendorf tube obtained from http://www.clker.com/clipart-eppendorf-tube-with-open-cap-1.html and edited in Adobe Illustrator

Fig. 2

Rotavirus nucleic acid enrichment for whole genome sequencing. Circular plots showing the coverage (i.e. the amount of sequence produced across each segment) of each of the 11 RoV segments (highlighted) and the coverage depth of each of the segments after Illumina sequencing for three RoV-positive samples (top, middle, bottom). Plots show coverage and depth following viral capture and random amplification (left, before) and following viral capture and amplification with both random and specific primers (right, after)

Fig. 3

Rotavirus coinfection is characterized by a major and a minor population. Dark grey bars and blue lines indicate the percentage sequence coverage and total number of reads (log scale), respectively, for each of the 11 RoV segments for the majority population present in five samples containing coinfecting RoV populations. Light grey bars and red lines indicate the percentage sequence coverage and total number of reads (log scale), respectively, for each of the 11 rotavirus A segments for the minority rotavirus population present in five samples containing coinfecting rotavirus populations

Fig. 4

Genome constellations suggest intergenotype reassortment in Vietnamese G1P[8] and G2P[4] rotaviruses. A maximum likelihood phylogeny of VP7 gene sequences is shown with genome constellations mapped at the tips of the tree. Standard Wa-like genome segments are indicated by light grey blocks, DS-1-like segments by dark grey blocks. Taxa names in bold indicate Vietnamese sequences from this study; taxa names in red indicate likely reassortant Vietnamese sequences. Asterisks indicate ≥85% bootstrap support at internal nodes of interest

Fig. 5

Phylogenetic incongruity among rotavirus segments confirming genomic reassortment. Diagram outlines G1P[8] VP7-VP4 segments and the DS-1-like backbone segments in four Vietnamese G1P[8] rotaviruses. a Tanglegram showing corresponding VP7 and VP4 segments of G1P[8] and G2P[4] genotypes. b Tanglegram showing phylogenetic incongruity between the VP7 and VP6 segments of the G1P[8] and G2P[4] genotype for four reassortant rotaviruses from Vietnam. Sequences from nonreassortant Vietnamese rotaviruses are indicated by connection with black lines. Sequences from reassortant Vietnamese viruses are indicated by connection with red lines. Segments between which no reassortment is detected (i.e. VP7-VP4) are depicted with reassortant viruses connected by red dashed lines. Asterisks indicate ≥85% bootstrap support at internal nodes of interest