However, reconstruction of all full-length transcripts from short reads with considerable sequencing error rates poses substantial computational challenges 4: ( 1) some transcripts have low coverage, whereas others are highly expressed ( 2) read coverage may be uneven across the transcript’s length, due to sequencing biases ( 3) reads with sequencing errors derived from a highly expressed transcript may be more abundant than correct reads from a lowly expressed transcript ( 4) transcripts encoded by adjacent loci can overlap and thus can be erroneously fused to form a chimeric transcript ( 5) data structures need to accommodate multiple transcripts per locus, due to alternative splicing and ( 6) sequences that are repeated in different genes introduce ambiguity.

In principle, such data can allow us to identify all expressed transcripts 3, as complete and contiguous mRNA sequence from the transcription start site to the transcription end, for multiple alternatively spliced isoforms. Recent advances in massively-parallel cDNA sequencing (RNA-Seq) provide a cost-effective way to obtain large amounts of transcriptome data from many organisms and tissue types 1, 2. Our approach provides a unified and general solution for transcriptome reconstruction in any sample, especially in the complete absence of a reference genome. In all cases, Trinity performs better than other available de novo transcriptome assembly programs, and its sensitivity is comparable to methods relying on genome alignments. Trinity fully reconstructs a large fraction of the transcripts present in the data, also reporting alternative splice isoforms and transcripts from recently duplicated genes. Here, we present the Trinity methodology for de novo full-length transcriptome reconstruction, and evaluate it on samples from fission yeast, mouse, and whitefly – an insect whose genome has not yet been sequenced. Current approaches for transcript reconstruction from such data often rely on aligning reads to a reference genome, and are thus unsuitable for samples with a partial or missing reference genome.

Massively-parallel cDNA sequencing has opened the way to deep and efficient probing of transcriptomes.

Downloading Trinity Fusion