"high" quality RNA but poor RNAseq results

Question

[CrazyB]

Wonder if anyone else on this forum has similar experience with high quality RNA, defined by DV200 metrics, but poor RNAseq results, defined by poor concordance of RNAseq results from different RNAseq platforms.

The samples in question are extracted RNA from FFPE samples. As RIN values are generally low for RNA from FFPE samples, Illumina recommended using DV200 metrics. For unknown reasons, we have a few samples that have superior scores by DV200 metrics but perform poorly by different RNAseq platforms (poor data concordance). In contrast, samples that are of "lower" DV200 values (lower than those superior scorers - most samples are in this "lower" range of DV200 value) have okay performance (decent concordance) by different RNAseq platforms.

Any thoughts ?

Answer 1

[Charles Warden]

Have you tried using RSeQC to confirm gene body coverage and/or TIN scores?

Samples with lower RIN scores should have lower TIN scores (at least if there is a clear difference, like going from RIN of 9 to RIN of 3).

For example, if there is one sample of each, it might be good to confirm that there wasn't a sample swap.