Improving the quality of barley transcriptome de novo assembling by using a hybrid approach for lines with varying spike and stem coloration.

Abstract

De novo transcriptome assembly is an important stage of RNA-seq data computational analysis. It allows the researchers to obtain the sequences of transcripts presented in the biological sample of interest. The availability of accurate and complete transcriptome sequence of the organism of interest is, in turn, an indispensable condition for further analysis of RNA-seq data. Through years of transcriptomic research, the bioinformatics community has developed a number of assembler programs for transcriptome reconstruction from short reads of RNA-seq libraries. Different assemblers makes it possible to conduct a de novo transcriptome reconstruction and a genome-guided reconstruction. The majority of the assemblers working with RNA-seq data are based on the De Bruijn graph method of sequence reconstruction. However, specif ics of their procedures can vary drastically, as do their results. A number of authors recommend a hybrid approach to transcriptome reconstruction based on combining the results of several assemblers in order to achieve a better transcriptome assembly. The advantage of this approach has been demonstrated in a number of studies, with RNA-seq experiments conducted on the Illumina platform. In this paper, we propose a hybrid approach for creating a transcriptome assembly of the barley Hordeum vulgare isogenic line Bowman and two nearly isogenic lines contrasting in spike pigmentation, based on the results of sequencing on the IonTorrent platform. This approach implements several de novo assemblers: Trinity, Trans-ABySS and rnaSPAdes. Several assembly metrics were examined: the percentage of reference transcripts observed in the assemblies, the percentage of RNA-seq reads involved, and BUSCO scores. It was shown that, based on the summation of these metrics, transcriptome meta-assembly surpasses individual transcriptome assemblies it consists of.