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Trim Geoduck RRBS libraries #260

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sr320 opened this Issue May 11, 2018 · 6 comments

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sr320 commented May 11, 2018

per Bismark recommendations


Technique 5' Trimming 3' Trimming Mapping Deduplication Extraction
RRBS --rrbs (R2 only) --rrbs (R1 only) ⬜️ (--ignore_r2 2)

SPECIFIC LIBRARY/KIT NOTES

RRBS

RRBS is a specialised technique to only look at CpG rich regions of the genome by using the restriction enzyme MspI (please see this RRBS Guide for some more specifics regarding data processing). For reasons explained in the RRBS-guide, the second last position of all reads before reading into the Illumina adapter exhibits an artificially (not methylated) methylation state as a result of the end-repair reaction. The option --rrbs within Trim Galore removes 2 extra bases whenever adapter contamination has been detected. This 3' end trimming that needs to be carried out for single-end runs or Read 1 of paired-end libraries. Read 2 of paired-end libraries is however not affected by this 3' bias, but instead the first couple of positions on the 5' end of Read 2 suffer from the read-through problem as Read 1 (Read 2 is a mere copy of Read 1), so Read 2 needs to have the first 2 bp removed instead. As of the current development version of Trim Galore (v0.4.2_dev; 12/16/2016) the option --rrbs removes: - 2 bp from the 3' end of single-end and Read 1 of paired-end reads in addition to adapter contamination, and - 2 bp from the 5' end of Read 2 of paired-end reads

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kubu4 commented May 16, 2018

Trimmed files are here (*.fq.gz):

Notebook:

Will run FastQC/MultiQC on pre-trimmed files for comparison. Will add to this issue when complete.

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sr320 commented May 16, 2018

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kubu4 commented May 16, 2018

Yeah, I noticed that. But, if we (meaning any researcher performing NGS) always have to trim the first/last 10 -15bp, then why bother with the TrimGalore settings for specific sequencing types?

Would we expect some sort of bias that's related to the restriction digestions that could explain this?

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kubu4 commented May 16, 2018

OMG!

I ran this with the --non-directional setting (despite having caught and "fixed").

This is the incorrect setting, as the libraries are directional!

Going to restart.

😠

@kubu4 kubu4 reopened this May 16, 2018

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kubu4 commented May 16, 2018

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