Trans2express – de novo transcriptome assembly pipeline optimized for gene expression analysis

Abstract

BackgroundAs genomes of many eukaryotic species, especially plants, are large and complex, their de novo sequencing and assembly is still a difficult task despite progress in sequencing technologies. An alternative to genome assembly is the assembly of transcriptome, the set of RNA products of the expressed genes. While a bunch of de novo transcriptome assemblers exists, the challenges of transcriptomes (the existence of isoforms, the uneven expression levels across genes) complicates the generation of high-quality assemblies suitable for downstream analyses.ResultsWe developed Trans2express – a web-based tool and a pipeline of de novo hybrid transcriptome assembly and postprocessing based on rnaSPAdes with a set of subsequent filtrations. The pipeline was tested on Arabidopsis thaliana cDNA sequencing data obtained using Illumina and Oxford Nanopore Technologies platforms and three non-model plant species. The comparison of structural characteristics of the transcriptome assembly with reference Arabidopsis genome revealed the high quality of assembled transcriptome with 86.1% of Arabidopsis expressed genes assembled as a single contig. We tested the applicability of the transcriptome assembly for gene expression analysis. For both Arabidopsis and non-model species the results showed high congruence of gene expression levels and sets of differentially expressed genes between analyses based on genome and based on the transcriptome assembly.ConclusionsWe present Trans2express – a protocol for de novo hybrid transcriptome assembly aimed at recovering of a single transcript per gene. We expect this protocol to promote the characterization of transcriptomes and gene expression analysis in non-model plants and web-based tool to be of use to a wide range of plant biologists.