Kodoja : a workflow for virus detection in plants using k-mer analysis of RNA-sequencing data

This work was supported by the Biotechnology and Biological Sciences Research Council [BB/N023293/1]. The work of L.T., S.J., S.M. and P.C.was additionally supported by the Scottish Government’s Rural and Environment Science and Analytical Services division (RESAS)RNA-sequencing of plant material allows for hypothesis-free detection of multiple viruses simultaneously. This methodology relies on bioinformatics workflows for virus identification. Most workflows are designed for human clinical data, and few go beyond sequence mapping for virus identification. We present a new workflow (Kodoja) for the detection of plant virus sequences in RNA-sequence data. Kodoja uses k-mer profiling at the nucleotide level and sequence mapping at the protein level by integrating two existing tools Kraken and Kaiju. Kodoja was tested on three existing RNA-seq datasets from grapevine, and two new RNA-seq datasets from raspberry. For grapevine, Kodoja was shown to be more sensitive than a method based on contig building and blast alignments (27 viruses detected compared to 19). The application of Kodoja to raspberry, showed that field-grown raspberries were infected by multiple viruses, and that RNA-seq can identify lower amounts of virus material than reverse transcriptase PCR. This work enabled the design of new PCR-primers for detection of Raspberry yellow net virus and Beet ringspot virus. Kodoja is a sensitive method for plant virus discovery in field samples and enables the design of more accurate primers for detection. Kodoja is available to install through Bioconda and as a tool within Galaxy.PostprintPeer reviewe

Galling and reversion disease incidence in a range of blackcurrant genotypes, differing in resistance to the blackcurrant gall mite (Cecidophyopsis ribis) and blaccurrant reversion disease

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Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids

Rubus yellow net virus (RYNV) infects Rubus species and cultivars worldwide and is an essential component of raspberry veinbanding mosaic (RVBMD), a virus disease complex that causes serious decline in plant vigour and productivity. The virus is transmitted, probably in a semi-persistent manner, by the large raspberry aphid, Amphorophora idaei in Europe, and A. agathonica in North America. The particles of RYNV are bacilliform in shape and measure 80–150 × 25–30 nm, similar to those of badnaviruses. A1.7 kb fragment of the viral DNA was amplified by PCR and then directly sequenced. Analysis of this sequence suggests that RYNV is possibly a distinct species in the genus Badnavirus and is most closely related to Gooseberry vein banding associated virus (GVBAV) and Spiraea yellow leaf spot virus, two other badnaviruses described recently. Using the sequence derived from the PCR-amplified viral DNA fragment, RYNV-specific primers were designed and used in PCR to assay for RYNV in a range of Rubus germplasm infected with RYNV, with other unrelated viruses and virus-like diseases found in Rubus, and in healthy plants. RYNV was detected in all glasshouse cultures of RYNV-infected plants, whether alone or in complex infections with other viruses, but not from healthy Rubus plants, nor from plants infected with other viruses. It was also detected in field-grown raspberry plants with and without symptoms of RVBMD and in raspberry plants infected with RYNV by viruliferous A. idaei. RYNV was also detected by PCR in A. idaei following access feeds on RYNV-infected plants of 1 h or more. PCR failed to amplify DNA from gooseberry infected with GVBAV confirming the specificity of the RYNV analysis. PCR detection of RYNV in dormant raspberry buds allows assays to be made outside the natural growing season, providing a useful application for plant introduction and quarantine programmes

Newly identified RNAs of raspberry leaf blotch virus encoding a related group of proteins

Members of the genus Emaravirus, including Raspberry leaf blotch virus (RLBV), are enveloped plant viruses with segmented genomes of negative-strand RNA, although the complete genome complement for any of these viruses is not yet clear. Currently, wheat mosaic virus has the largest emaravirus genome comprising eight RNAs. Previously, we identified five genomic RNAs for RLBV; here, we identify a further three RNAs (RNA6–8). RNA6–8 encode proteins that have clear homologies to one another, but not to any other emaravirus proteins. The proteins self-interacted in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments, and the P8 protein interacted with the virus nucleocapsid protein (P3) using BiFC. Expression of two of the proteins (P6 and P7) using potato virus X led to an increase in virus titre and symptom severity, suggesting that these proteins may play a role in RLBV pathogenicity; however, using two different tests, RNA silencing suppression activity was not detected for any of the RLBV proteins encoded by RNA2–8

Recent progress towards control of two important viruses and their variants in small fruit crops in Europe

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Genome-wide association study identifies multiple susceptibility loci for diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma subtype and is clinically aggressive. To identify genetic susceptibility loci for DLBCL, we conducted a meta-analysis of 3 new genome-wide association studies (GWAS) and 1 previous scan, totaling 3,857 cases and 7,666 controls of European ancestry, with additional genotyping of 9 promising SNPs in 1,359 cases and 4,557 controls. In our multi-stage analysis, five independent SNPs in four loci achieved genome-wide significance marked by rs116446171 at 6p25.3 (EXOC2; P = 2.33 x 10 ⁻²¹), rs2523607 at 6p21.33 (HLA-B; P = 2.40 x 10 ⁻¹⁰), rs79480871 at 2p23.3 (NCOA1; P = 4.23 x 10 ⁻⁸) and two independent SNPs, rs13255292 and rs4733601, at 8q24.21 (PVT1; P = 9.98 x 10 ⁻¹³ and 3.63 x 10 ⁻¹¹, respectively). These data provide substantial new evidence for genetic susceptibility to this B cell malignancy and point to pathways involved in immune recognition and immune function in the pathogenesis of DLBCL.6 page(s