- I finished addressing reviewer comments! The manuscript is almost finalized for resubmission.
- I presented my preliminary results at PCSGA!
- Worked on in
compGO and DAVID, but my methods are most likely wrong
- Didn’t make much headway writing because the gene enrichment stuff was confusing enough
- Address minor reviewer comments
- Address major comments
- Try using multivariate methods to explore relationship between environmental data and protein expression data
- Upload proteomics data to online databases
- Reformat the paper repository
- Finish extracting broodstock DNA
- Identify sequencing protocol
- Prepare samples for bisulfite sequencing
methylKit to solidfy analysis parameters
- Present more solid findings to Eastern oyster methylation project group
- Update methods and results
- Fix gene enrichment methods
from the responsible grad student https://ift.tt/2xWEVis
~/tools/bismark_v0.17.0/bismark relative/path/to/prepared/genome –bowtie2 –score_min L,0,-0.6 -1 blah_R1.trim.gz -2 blah_R2.trim.gz -p 12 –bam
Of all the commands here, the –score_min stands out as the unexplainable non-default. This exact command was suggested by the program creator in one of the forum posts (http://seqanswers.com/forums/showthread.php?t=40496) when someone complained they obtained very low mapping rates for their data.
“you haven’t mentioned the read length or the genome you are working with, is it a good assembly or still quite rough? Do you expect many SNPs? Do you expect insertions or deletions in the reads? In any case I would try running Bismark in Bowtie 2 mode (–bowtie2), which allows mapping with InDels and also has a function (–score_min) for the number of mismatches and/or InDels that scales with the read length. This is set very stringently by default (L,0,-0.2), but could be relaxed somewhat to see if this helps your mapping efficiency. As a guideline, –score_min L,0,-0.2 would allow ~3 mismatches for a 100bp read, –score_min L,0,-0.6 would allow up to 10 mismatches and/or Indels. If you just want to test these things you can use the -u option to just use a subset of the reads.”
–score-min controls how “loose” the mapping should be–and from testing, I can vouch that this cutoff strikes a good balance between sensitivity and accuracy. –score_min is a Bowtie2 parameter, read its docs if you want to know what it means EXACTLY. To pre-empt a bit of confusion if you actually DO read the docs, Bowtie2’s default –score-min is L,-0.6,-0.6 for end-to-end mapping. This default setting is OVERRIDDEN by bismark–bismark sets this parameter as L,0,-0.2 if one does not override its defaults. We’re basically overriding bismark’s defaults to bring it more in line with bowtie2’s default setting.
In this pipeline, –score_min is the BIGGEST determinant of the results. This is the one thing I’ll optimise if my results look weird. If mapping rates are really low (< 20%), I suggest using L,0,-0.9 or L,0,-1.2.