Choice of Coverage
For this analysis, we assessed the relative impact of reducing coverage from 35 to 25, 20, 15, and 10 M reads on DEG agreement. Our main findings were that (i) compared to read length when coverage was kept constant (35 M reads), coverage had a larger influence on DEG agreement when read length was kept constant (151 nt); (ii) the relationship was modulated by the strength of the biological signal; (iii) for the strongest biological signals (Day 7 and 14), an approximate linear relationship was observed with a 5% drop in agreement with each drop in 5 M sequencing depth (down to 80% agreement with 10 M reads); and (iv) for the weaker signal at Day 2, a non-linear relationship was observed with the biggest drop seen between 15 M and 10 M, indicating that weaker biological signals require higher coverage than stronger signals to produce robust DEG results.In order to assess the impact of coverage on accurately covering genes expressed in the PBMC transcriptome, based on pooled data, we generated a “PBMC transcriptome compendium” comprised of 26,172 genes. Cumulatively, our results demonstrated that, even at relatively deep sequencing depth of 30 M, a significant percentage of the gene content expressed in PBMCs was not detectable (17-44% depending on read count filtering). This limitation in turn negatively impacts DEG detection contributing to an increased false negative rate.