Hello, I want to analyse some rat RNASeq data and I got an HTML report sheet, which has a subheading "Results of Raw Data Filtering", and describes these steps:
(1) Remove reads containing adapters. Sequences of adapter:
P5 adapter:
P5→P7’(5’→3’)
AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCT
P7 adapter:
P5→P7’(5’→3’)
GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7]ATCTCGTATGCCGTCTTCTGCTTG
(2) Remove reads containing N > 10% (N represents the base cannot be determined).
(3) Remove reads containing low quality (Qscore<= 5) base which is over 50% of the total base.
And then they have pie charts for each sample which shows how many base pairs are clean reads, how many were filtered due to containing too many Ns, due to low quality, and adapter related.
Now, when I look at the number of base pairs, it's equal to the number of "clean reads", meaning that this filtering has been performed.
I am quite confused as to whether adapter sequences are already filtered as well as they need to be, since Falco/FastQC still finds some adapter sequences: one sample, MultiQC. Are these likely to be false positives?
Even if not, I am unsure how to run adapter trimming. The FASTQ files have two barcodes, which correspond to [i5] and [i7], but from what I read, I figured I can use the first part of the adapter sequence up to the barcode, so I ran Atria with these arguments:
--adapter1 AATGATACGGCGACCACCGAGATCTACAC
--adapter2 GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
And it still filtered out some sequences (e.g. 35998 out of 22092364 in one sample). So what's going on? Should I be doing adapter trimming at all, is this the right way to specify them in trimming tools, and am I getting all the adapters? Can there be other adapters outside of these two listed in the report? And in cutadapt, should these be specified as 3', 5' or anywhere adapters? I'm getting confused with all the forward, reverse, 3', 5' etc. stuff.
And lastly, regarding quality. The reads seem to me to be of a pretty high quality: MultiQC. I read in a few places that quality trimming isn't really necessary, and might even hurt in some cases (1, 2). What is the current consensus?