Impact of sequencing depth and technology on de novo RNA-Seq assembly

BACKGROUND: RNA-Seq data is inherently nonuniform for different transcripts because of differences in gene expression. This makes it challenging to decide how much data should be generated from each sample. How much should one spend to recover the less expressed transcripts? The sequencing technology used is another consideration, as there are inevitably always biases against certain sequences. To investigate these effects, we first looked at high-depth libraries from a set of well-annotated organisms to ascertain the impact of sequencing depth on de novo assembly. We then looked at libraries sequenced from the Universal Human Reference RNA (UHRR) to compare the performance of Illumina HiSeq and MGI DNBseq™ technologies. RESULTS: On the issue of sequencing depth, the amount of exomic sequence assembled plateaued using data sets of approximately 2 to 8 Gbp. However, the amount of genomic sequence assembled did not plateau for many of the analyzed organisms. Most of the unannotated genomic sequences are single-exon transcripts whose biological significance will be questionable for some users. On the issue of sequencing technology, both of the analyzed platforms recovered a similar number of full-length transcripts. The missing “gap” regions in the HiSeq assemblies were often attributed to higher GC contents, but this may be an artefact of library preparation and not of sequencing technology. CONCLUSIONS: Increasing sequencing depth beyond modest data sets of less than 10 Gbp recovers a plethora of single-exon transcripts undocumented in genome annotations. DNBseq™ is a viable alternative to HiSeq for de novo RNA-Seq assembly.