Hi Everyone, I am new to RNAseq data analysis. I have a couple hundred sRNAseq data. We used NEBNEXT small RNA kit. We used NEBNEXT SR Primer (Forward) and ScriptSeq Index Primer (Reverse) to form about 147 bp products. Since I already have the data sequenced, I am assuming the first thing I need to do is get adapters trimmed? We used illumina sequencing and here I found the list of all adapters. Now I am really confused whether I have to make my own adaptor files (or maybe not) if I decide to use tools like trimmomatic? Another question I have is about the interpretation of FASTQC plot. I have this image here https://ibb.co/bFDsZ5 which shows 'X' mark in Adapter Content (or does the X mean there is no adapter?) on the left, then there is an illumina small RNA3' Adapter on the base of the plot (the solid blue line) and also the red curve that goes from 9 to 35 position in read (bp). Can someone please help me understand this. Thanks
Take a look at documentation from the kit you used. There may be specific steps recommended as far as trimming etc goes before you do any of the following.
You definitely have Illumina's universal adapters in there (indicating that a major fraction of your reads had inserts smaller than you probably expected). Those you can remove by using any standard trimming program (bbduk, trimmomatic etc).
If you have paired end reads and enough reads have inserts shorter than the read length then you can identify adapter by using bbmerge from BBMap suite as:
bbmerge.sh in1=r1.fq.gz in2=r2.fq.gz outa=adapters.faThank you for the clear answer.