Evaluation of transcript assembly in multiple porcine tissues suggests optimal sequencing depth for RNA-Seq using total RNA library

RNA sequencing (RNA-Seq) libraries are prepared by either selecting poly(A) messenger RNAs (mRNA-Seq) or by depleting total RNA of highly abundant ribosomal RNAs (total RNA-Seq). The ribosomal RNA (rRNA) depletion protocols offer an attractive option for novel transcript discovery, as they facilitate the simultaneous characterization of polyadenylated and non-polyadenylated RNAs, including non-coding RNAs. However, the cost associated with total RNA-Seq is much greater than that of mRNA-Seq. Hence, the determination of an optimal target sequencing depth for total RNA-Seq would assist researchers in optimizing the cost-effectiveness of their experiments. In this study, we evaluate the appropriate depth of sequencing needed for transcriptome profiling in total RNA-Seq using a random sampling method to generate varying levels of sequencing depth in three different porcine tissues. As expected, our results indicated that the depth of sequencing has the greatest effect on the identification and quantification of lowly expressed transcripts. We propose that a depth of 80 M reads per library is desirable to identify and quantify expression of transcripts across the genome. The protocol used in this study can be utilized to determine optimal sequencing depth in other tissues and/or species.