BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq

Abstract

A high-quality, non-redundant barley gene reference transcript dataset and database (Barley Reference Transcripts -BaRTv1.0) has been generated. BaRTv1.0, was constructed from 11 RNA-seq experimental datasets consisting of 808 samples from a range of tissues, cultivars and abiotic treatments. Transcripts were assembled and aligned to the barley cv. Morex reference genome (Mascher et al., 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al., 2011) were used to determine transcript coverage, and high-resolutionRT-PCR to validate alternatively spliced (AS) transcripts of 86 genes in five different organs and tissues. These methods were used as benchmarks to select optimal parameters for transcript assembly with StringTie. Further filtering to remove fragmented, poorly supported and lowly expressed transcripts generated BaRTv1.0, which consists of 60,444 genes with 177,240 transcripts. Overall, BaRTv1.0 contains higher confidence transcripts when compared to the current barley (HORVU) transcriptome; they are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads.We also generated a version of the RTD specifically for quantification of alternative spliced isoforms (BaRTv1.0-Quantification of Alternatively Spliced Isoforms -QUASI) for accurate expression and AS analysis. BaRTv1.0-QUASI overcomes inaccurate quantification due to variation in 5' and 3' UTR ends of transcripts and was used for accurate transcript quantification of RNA-seq data of five barley organs and tissues. Precise transcript quantification allows routine analysis of AS and we identified 20,972 significant differentially expressed genes, 2,791 differentially alternatively spliced genes and 2,768 transcripts with differential transcript usage.

TB1: from domestication gene to tool for many trades

“…The dynamics of expression of TB1 underpin the multiple effects observed for this gene and its orthologues. For example, there is no report of TB1 involvement in plant height in barley, but this is not surprising since its expression is detected only in inflorescences, and not in leaves or stems ( Rapazote-Flores et al , 2019 ). Also, TB-A1 expression is almost absent in wheat stems ( Dixon et al , 2018 ).…”

EoRNA, a barley gene and transcript abundance database

“…Morex pseudomolecules. To access the barley reference transcript dataset a public database and website front-end were constructed to allow researchers to download the reference transcript dataset and interrogate the data via a BLAST search, keyword search or string search using the BaRT or HORVU gene/transcript identifiers (https://ics.hutton.ac.uk/barleyrtd/index.html) (19). The transcripts are arranged as gene models and viewed through GBrowse.…”

“…To create the BaRTv1.0 RTD, transcripts from multiple datasets from a range of tissues, treatments and cultivars were mapped to cv. Morex pseudomolecules to maximise read coverage support for genes and splice junctions (19). BaRTv1.0 is, therefore, a predominantly cv.…”

“…A high confidence barley reference transcript dataset (BaRTv1.0) represented by 60,444 gene models and 177,240 transcript sequences are provided in a database (https://ics.hutton.ac.uk/barleyrtd/index.html) that positions the transcripts on the barley cv. Morex reference genome version 1 (19). The database is fully searchable using either BaRT or HORVU gene names from the Barley cv Morex pseudomolecules, by key word annotation or by BLAST sequence searches.…”

A genome assembly of the barley ‘transformation reference’ cultivar Golden Promise

“…The reference sequence is also available to mine using an integrated gmap search. Transcript annotation is transferred from the BaRT transcriptome dataset 19 and the TRITEX gene annotation 10 , using Gmap (version 2018-03-25) with the following parameters: -f 2 -n 1 --min-trimmed-coverage=0.8 --min-identity=0.9 (both files are available to download from figshare. BaRT: 10.6084/m9.figshare.9705278; TRITEX: 10.6084/m9.figshare.9705125).…”