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. 2016 Aug 9:17:579.
doi: 10.1186/s12864-016-2994-6.

Integrated analyses using RNA-Seq data reveal viral genomes, single nucleotide variations, the phylogenetic relationship, and recombination for Apple stem grooving virus

Yeonhwa Jo  1 , Hoseong Choi  1 , Sang-Min Kim  2 , Sun-Lim Kim  2 , Bong Choon Lee  2 , Won Kyong Cho  3   4
Affiliations

Integrated analyses using RNA-Seq data reveal viral genomes, single nucleotide variations, the phylogenetic relationship, and recombination for Apple stem grooving virus

Yeonhwa Jo et al. BMC Genomics. .

Abstract

Background: Next-generation sequencing (NGS) provides many possibilities for plant virology research. In this study, we performed integrated analyses using plant transcriptome data for plant virus identification using Apple stem grooving virus (ASGV) as an exemplar virus. We used 15 publicly available transcriptome libraries from three different studies, two mRNA-Seq studies and a small RNA-Seq study.

Results: We de novo assembled nearly complete genomes of ASGV isolates Fuji and Cuiguan from apple and pear transcriptomes, respectively, and identified single nucleotide variations (SNVs) of ASGV within the transcriptomes. We demonstrated the application of NGS raw data to confirm viral infections in the plant transcriptomes. In addition, we compared the usability of two de novo assemblers, Trinity and Velvet, for virus identification and genome assembly. A phylogenetic tree revealed that ASGV and Citrus tatter leaf virus (CTLV) are the same virus, which was divided into two clades. Recombination analyses identified six recombination events from 21 viral genomes.

Conclusions: Taken together, our in silico analyses using NGS data provide a successful application of plant transcriptomes to reveal extensive information associated with viral genome assembly, SNVs, phylogenetic relationships, and genetic recombination.

Keywords: Apple stem grooving virus; De novo genome assembly; RNA-Seq; Recombination; Single nucleotide variation; Transcriptome.

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Figures

Fig. 1
Fig. 1
Identification of de novo viral genome assembly and SNVs for ASGV from RNA-Seq data. a Genome structure of ASGV isolate Fuji. The conserved domains were identified by the SMART program (http://smart.embl-heidelberg.de/). Abbreviations: MT (Methyltransferase), Hel (Helicase), RNA-dependent RNA polymerase (RdRP), Movement protein (MP), and Coat protein (CP). b Alignment of raw data against genome of ASGV isolate Fuji by BWA was visualized by Tablet program. c Positions of identified SNVs in ASGV-infected apple transcriptome were visualized by Tablet program. d Identified sequence reads from ASGV-free apple sample, which were associated with ASGV by BWA alignment. e BLAST results showing sequence reads from ASGV-free apple sample matched to ASGV genome. f Alignment of raw data using ASGV-infected sRNA data from cultivar GD against reference ASGV genome by BWA was visualized by Tablet program. g Positions of identified SNVs in ASGV-infected apple sRNA transcriptome were visualized by Tablet program. h Alignment of raw data from pear sample against genome ASGV isolate Cuiguan by BWA was visualized by Tablet program. i Positions of identified SNVs of ASGV in pear mRNA transcriptome were visualized by Tablet program
Fig. 2
Fig. 2
Comparison of two de novo assemblers based on number and sizes of assembled contigs and phylogenetic tree of 21 ASGV isolates. Size distribution of identified viral contigs from ASGV-infected apple sample assembled by Trinity (a) and Velvet (b). Size distribution of identified viral contigs from pear sample assembled by Trinity (c) and Velvet (d). The green-, blue-, and red-colored bars indicate ASPV, AGCAV, and ASGV, respectively. The sizes of only the longest and the shortest contigs in each transcriptome are indicated. (e) The phylogenetic tree was constructed based on the genome sequences of 13 ASGV isolates and 8 CTLV isolates. We followed the original annotations for CTLV and PBNLSV, which were highly homologous to ASGV. The accession number and the name of each isolate were indicated. Detailed information for each isolate can be found in Additional file 10
Fig. 3
Fig. 3
Identification of recombination events by RDP4 program. a The positions of identified recombinants were depicted with the respective names of parental sequences. The individual genome of the ASGV isolate was indicated by a different colored bar. The identified recombination events, including number 1 (b) and number 2 (c), were rechecked by the RDP4 program and visualized by plot data with pairwise identity information. Detailed information on the identified recombination events is provided in Table 4
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