RNAseq read pairs orientation issue with assembling

Question

[emilyellis13]

Hi all,

I'm assembling a bunch of RNAseq data, and just having issue with one sample. This sample has 4 F and 4 R reads, each 2G. I did not have any trouble during trimming with TrimGalore. I then unzipped each F read into a single F file, likewise for the R.

I first tried to assemble in Trinity, and now in rnaSPAdes, and get the following warnings. How can I check which of the 8 reads are mis-oriented? I think one of the individual reads is mis-labeled - i.e. labeled R when it should be F, but not sure how to tell which one is. Thanks in advance!

Emily

• 1:48:09.216 12G / 28G WARN General (pair_info_count.cpp : 336) Unable to estimate insert size for paired library #0
• 1:48:09.216 12G / 28G WARN General (pair_info_count.cpp : 342) None of paired reads aligned properly. Please, check orientation of your read pairs.
• 1:49:33.965 12G / 28G WARN General (repeat_resolving.cpp : 63) Insert size was not estimated for any of the paired libraries, repeat resolution module will not run.

[GenoMax]

Can you post header examples for the files displaying this error? You are using reads/files interchangeably but I assume it is actually R1/R2 files in 4 pieces each?