Kallisto : Compare TPM between samples from different illumina sequencers (HiSeq2000 and NextSeq500)

Question

[Nicolas Rosewick]

Hi,

I've a bunch of RNA-Seq samples sequenced on a HiSeq2000 (2x100bp) and an other bunch sequenced on a NextSeq500 (2x150bp and 2x76 depending on the samples). The sequencing depth varies between 25M to 60M on average. All libraires were prepared using the same kit (ribo-zero). Only the sequencing kit changes between the HiSeq and the NextSeq.

I applied kallisto on all these samples and now want to check the expression of a specific gene across the samples. Is it correct to use the TPM from kallisto to compare the samples ; or should I use an other metric : or maybe add an additional per-sample normalization step ?

Thanks

Answer 1

[andrew.j.skelton73]

Can you absorb the effect of sequencer in your design matrix, or does that make it not full rank? TPM is probably the most stable metric to use for a quick eyeball (especially better than R/FPKM). There's the tximport method which allows you to read in Kallisto results and leverage the bootstraps so it's ready for DESeq2

[Nicolas Rosewick]

Indeed I could do a PCA plot a see if my samples cluster by sequencer (or not). I'll try it.

Question : I didn't apply bootstrap (-b option in kallisto). Should I ?

[andrew.j.skelton73]

I've not tested it's effect in DESeq2 to be honest, but I did notice the other day when looking through the tximport package, that there's a parameter called varReduce, which I think uses the bootstraps. You'll be fine not using bootstraps.

[Nicolas Rosewick]

I tested a PCA plot using the TPM and it seems ok to compare samples sequenced on different sequencer (at least in my case).