Hello,

We have switched to performing our RNA expression anlaysis to NextSeq500 and will now be including a NTC (no template control) into the sequencing for QC. However, this is my first time dealing with NTC and RNA-seq. I am used to dealing with them in qRT-PCR reactions.

How would I go about treating gene expression observations in the NTC in comparison to the other samples in the run? For example:

Gene X TPM in NTC = 323

Gene X TPM in Sample1 = 646

Gene X TPM in Sample2 = 1033

Gene X TPM in Sample3 = 11

Gene X TPM in Sample4 = 7

There has clearly been some kind of contamination of Gene X into the NTC, but for down stream analysis how would I go about determining the true TPM value of Gene X in my samples knowing there is expression of this gene in the NTC?

Any advice would be greatly appreciated.

Cheers!