trim_galore - when to stop trimming?

[rvann]

I've just used trim_galore (arguments below) to trim the adapter sequences off of fastq files from Illumina hiseq 4000 run TruSeq prep. Cutadapt seemed to work well, running it in the default mode to auto-detect adapters and remove them, as well as remove any bases with phred score < 5, but my fastQC reports for some files show that Illumina Single End PCR primer or TruSeq Adapter, Index 7, remain in certain samples (0.15 % and 0.53 %, respectively).

Do I have to run cutadapt again and feed it these specific sequences to remove? I have many samples and searching through each report for specific adapters to remove in a second cutadapt run is not ideal.

Was I not stringent enough in trimming?

Do I need to get rid of the remaining contaminants to perform differential gene expression analysis?

trim_galore --paired -q 5 -o /output/path/ --fastqc_args "--outdir /fastqc/output/path/" sample_R1.fastq.gz sample_R2.fastq.gz

[Brian Bushnell]

I suggest including all Illumina adapters in the first pass, rather than trying to autodetect and then running a second pass to clean the stuff that was not removed. All standard Illumina adapters are contained in the file "bbmap/resources/adapters.fa" in the BBMap suite.

[fkrueger]

Hi rvann,

sorry for replying so late. For adapter trimming it is most important to capture and remove read through adapters which obviously worked fine. If FastQC still picks up certain indexed adapter you will most likely find that these are full-length adapter sequence (which are missing the A at the start) and are thus not removed during the adapter removal process. Full length adapter sequences won't align to your genome of interest anyway and are normally not a problem. Cheers, Felix