Trimming question + analysis of specific regions about methylseq

[ewels]

Hi @kokyriakidis,

TrimGalore! already trims low-quality bases I think. Have a look at the FastQC reports from after the trimming to check the read qualities (these FastQC results are not shown in the MultiQC report, you need to look in results/trim_galore/fastqc.

[FelixKrueger]

Trim Galore removes poor qualities from the 3' end of sequence, but does not do this from the 5' end (as it is never really needed). If you have some special kind of BS-seq data I'd be happy to look at your FastQC report (the HTML file), to see if I have any recommendations.

On a related note, a close colleague of mine has recently written a tool to analyse raw sequencing files and try to figure out which kind of bisulfite sequencing experiment had been performed. The tool (Charades) is still kind of in alpha mode, but you are very welcome to give it a try! https://github.com/ChristelKrueger/Charades

[FelixKrueger]

Regarding your second question: I haven't looked at the ENCODE-DCC pipeline myself, but you could either

• align the data genome-wide and just look at your genes of interest, or
• get the sequence of the genes of interest (and maybe some surrounding sequence) and use that as a custom genome

The latter approach will be a a lot quicker, but it might not be as accurate for repetitive regions within your genes of interest, and you will have to adjust the positions if you want to bring it back in line with other genome coordinates.

[kokyriakidis]

@FelixKrueger
Thank you very much! You can check the fastq in this link:

These are the initial files:
https://wetransfer.com/downloads/1b780edcfca5363f09e74202cca1ebd020190325114013/08a8a71624bbad7ecf6db365789c066520190325114013/affc1f

These are after TrimGalore with default settings:
https://wetransfer.com/downloads/a88486a3a7c15ac09042a291f60673b220190325114819/e96bc14c81defea887f22aa21369c0e820190325114819/c7af3f

[FelixKrueger]

Whoah, that looks quite wild indeed. Judging by the sequence composition, it doesn't look like a standard sequencing protocol, it rather looks like amplicon sequencing to me. Any chance this is targetting the PTPN22 gene, and nothing else? I would probably proceed with a standard trim_galore --paired file1 file2 command, followed by a default directional alignment, and see what you get. Cheers, Felix

[kokyriakidis]

It is targeting PTPN22 but also the lns-1 gene