BaRTv2: A highly resolved barley reference transcriptome for accurate transcript-specific RNA-seq quantification

Abstract

Accurate characterization of splice junctions as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data and enable detailed investigations of transcriptional and post-transcriptional regulation. Using novel computational methods and a combination of PacBio Iso-seq and Illumina short read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset (RTD) from the European 2-row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the splice junctions and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared to an earlier version, BaRTv1.0. The accuracy of transcript level quantification, splice junctions and transcript start and end sites has been validated extensively using parallel technologies and analysis, including high resolution RT PCR and 5 prime RACE. BaRTv2.18 contains 39,434 genes and 148,260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high-quality resource for advanced transcriptomic analyses, including both transcriptional and post-transcriptional regulation, with exceptional resolution and precision.

Identifying Plant Genes Shaping Microbiota Composition in the Barley Rhizosphere

“…Transcript quantification was carried out using Salmon 92 version 1.4.0 using parameters “-l A --seqBias –posBias –validateMappings” with BaRTv2.18 29 as the reference transcriptome. Analysis of RNA-seq quantifications was carried out using a custom modified version of the 3D RNAseq pipeline 93 .…”

“…GO enrichment analysis was carried out using GO terms from the BaRTv2 transcriptome annotation 29 . A fisher's exact test was used to test for significant enrichment.…”

“…Identification and prioritisation of QRMC-3HS candidate genes A total of 59 genes were identified in the BaRTv2 gene/transcript 29 annotation within the bounds of the QRMC-3HS locus (identified as 33,181,340 -36,970,860 bp on chromosome 3H in the cultivar Barke, Supplementary Database 4). Of these, 25 were found to be expressed in the RNA-seq dataset and were prioritised, as they are likely to be expressed in root tissue (Supplementary Database 4).…”

“…We previously demonstrated that wild genotypes and ‘elite’ cultivated varieties host contrasting rhizosphere microbiotas 25,26 . Here, capitalising on an experimental population between barley genotypes at opposing ends of the domestication framework 27 and utilising state-of-the-art genomic 28 and transcriptomic 29 resources we gained novel insights into plant-microbiota interactions. We addressed three interconnected questions.…”

“…We previously demonstrated that wild genotypes and ‘elite’ cultivated varieties host contrasting rhizosphere microbiotas 25, 26 . In this work, capitalising on an experimental population between barley genotypes at opposing ends of the domestication framework 27 and utilising state-of-the-art genomic 28 and transcriptomic 29 resources, we map host genetic determinants of microbiota composition in the rhizosphere, we identify candidate genes putatively underpinning this trait and define genetic variation occurring at those genes.…”