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I know the definition of directional and non-directional libraries but I can't figure out which one I actually made. I did whole genome bisulfite sequencing and I used illumina TruSeq adaptors rather than the PE adaptors that are in most preps. I am trying to do analysis with trim_galore then Bismark but I think I need to specify directional or non-directional. I just want to compare methylation at specific sites between 2 samples. I think I made non-directional libraries. This causes a problem in trim-galore; so could I just not specify and the default would be directional. Would that adversely affect my data? I only seem to be able to find info on directionality for RNAseq libraries on the forums so hoping someone could help.
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Hi shawpa,
If you used TruSeq adapters then you library should be directional. There are a couple of ways to find this out:
- if you align your data in directional mode and get very low mapping efficiencies (~30-40%), you might want to try --non_directional. If that increases the mapping efficiency to ~60-80%, the library was indeed non-directional (you could use -u 1000000 to quickly try this)
- if you align the data in non-directional mode and you see that most alignments came from the OT and OB strands, then your library was directional. In this case run it again in the default (= directional) mode to exclude misalignments. If you see OT, OB and CTOT and CTOB alignments with a ratio of roughly 1:1:1:1, your library was non-directional.
- in any case, the library type should not matter for WGS BS-Seq with trim-galore, since the --non_directional option is intended for RRBS only.
Hope this helps,
Felix -
So I mapped in directional mode (default) and then in non-directional mode. I got the exact same mapping efficiency number. The alignment to specific strand in non-directional was :
Number of sequence pairs with unique best (first) alignment came from the bowtie output:
CT/GA/CT: 40374 ((converted) top strand)
GA/CT/CT: 15 (complementary to (converted) top strand)
GA/CT/GA: 27 (complementary to (converted) bottom strand)
CT/GA/GA: 39961 ((converted) bottom strand)
Based on what you said about the ratios, I think it is directional. Can you confirm?Comment
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Absolutely, these libraries are directional and should be run in the default, i.e. directional, mode. Good luck!Comment
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Hi Felix,
Just out of interest,
If TruSeq adapters are used , I presume they are going to be directional anyways, as the original adaptors are ligated to OT abd OB strands only.
But one tries to do non-directional. how will you ligate the adaptors to CTOT and CTOB and is there settings in HiSeq200 to specify that you want sequencing from all 4 strands.
I am thinking a dirty way would be- as one knows the sequence of the original adaptor sequence and they are methylated, so its possible to know after BS conversion and PCR amplification what will be the sequence of the adaptors in the complimentary strands. and use that information?
Whats the process actually.Comment
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Hi Aniruddha,
Yes I think you would need a second round of PCR so that you can end up with the read 1 adapter on the complementary reads as well. I have never thought it through how you could make TruSeq libraries non-directional, mainly since it makes everything so much more complicated and won't allow you to assess methylation in an allele-specific manner... If it was my experiment I would certainly try to make sure that the libraries are directional!Comment
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yeah.. I woulnt do non-directional myself.....for methylation I dont see much advantage of the method.. thanks..Comment
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one of the advantages you get from a directional protocol is that you should be able to address the SNPs, which is a source of bias in single base methylation estimation, and at the same time get extra information about your DNA for the same price.Comment
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Thanks fkrueger for the explanation and suggestion. It helped.Comment
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