How had cleaning impacted read counts?

The following plots compare 4 samples that are representative of the rest of the dataset.

STAR CPMs using raw data on y-axis vs STAR CPMs using cleaned data on x-axis.

Salmon CPM values using raw data on y-axis vs Salmon using cleaned data on x-axis.

How does quantification method impact read counts?

Salmon CPM values using cleaned reads on y-axis vs STAR CPM values using cleaned reads on x-axis.

Note the pattern of genes with low expression with STAR and high expression with Salmon.

MDS plot, STAR raw counts:

MDS plot, STAR cleaned counts:

MDS plot, Salmon raw counts

MDS plot, Salmon cleaned counts

Top 10 genes with STAR on cleaned reads

##                        logFC   AveExpr    adj.P.Val
## ENSMUSG00000020608 -2.487484  7.859413 2.740380e-10
## ENSMUSG00000049103  2.163930  9.880300 3.953575e-09
## ENSMUSG00000052212  4.552076  6.191089 3.953575e-09
## ENSMUSG00000027508 -1.899053  8.113147 4.914713e-09
## ENSMUSG00000051177  3.186426  4.986113 4.914713e-09
## ENSMUSG00000042700 -1.813750  6.084302 5.114391e-09
## ENSMUSG00000050335  1.112010  8.961475 6.293314e-09
## ENSMUSG00000038147  1.689965  7.138663 6.293314e-09
## ENSMUSG00000038807 -1.562053  9.003764 6.635062e-09
## ENSMUSG00000020437 -1.226829 10.305956 6.635062e-09

Top 10 genes with Salmon

##                        logFC  AveExpr    adj.P.Val
## ENSMUSG00000052212  4.544736 5.821098 4.785833e-08
## ENSMUSG00000049103  2.146572 9.265595 5.872193e-08
## ENSMUSG00000020387 -4.533149 3.027947 7.530002e-08
## ENSMUSG00000089929 -3.614906 6.375914 8.599109e-08
## ENSMUSG00000037185 -1.611175 8.943865 8.599109e-08
## ENSMUSG00000037820 -4.186695 6.277701 1.003892e-07
## ENSMUSG00000020437 -1.206067 9.498283 1.003892e-07
## ENSMUSG00000027215 -2.654346 7.216311 1.003892e-07
## ENSMUSG00000021990 -2.666258 6.892383 1.003892e-07
## ENSMUSG00000023827 -2.106487 6.587921 1.003892e-07

Totals genes DE at adj.P.val < 0.05

STAR + without cleaning : 5060.

STAR + with cleaning : 5418.

Salmon + without cleaning: 4074.

Salmon + with cleaning: 4287.

Overlap in DEGs at adj.P.val < 0.05

Overlap in top 100 DEGs (sorted by P value)

Conclusions

• STAR and Salmon show differences in quantification of low expressed genes.
• This does not seem to have much impact on the relative distances between samples or differential expression (note that low expressed genes are often filtered out before analysis).
• Pick one approach for a project and go with it.
• Don’t run multiple analyses and pick the one that gives you the most DE genes.