Confused by --nOT and --nOB and the examples provided in the issues section

[Flope]

Hi,

I am confused by the definition of --nOT and --nOB and some examples provided by Devon here.

My starting reads are 150bp. I trimmed the reads before alignment using pretty standard trimming to remove possible adapter sequences and low-quality bases. The average length of the reads is now ~135bp. So, for MethylDackel extract, I was planning to use --nOT and --nOB to define the inclusion bases.

--nOT INT,INT,INT,INT Like --OT, but always exclude INT bases from a given end from inclusion, regardless of the length of an alignment. This is useful in cases where reads may have already been trimmed to different lengths but still nonetheless contain a certain length bias at one or more ends.

My understanding of these options (and as somebody else mentioned in another thread) is that if I want to exclude 5bp from each end of the reads, I would use this:

--nOT 5,5,5,5

However, in #102, Devon mentions: “Assuming you want to exclude the first 10 bases produced by the sequencer, the --nOT 10,0,0,140 --nOB 10,0,0,140 would do it (presuming you originally had 150 base reads).”

Was Devon confused with --OT and --OB, or am I not understanding how --nOT and --nOB work?

Thanks!

[dpryan79]

I did a bad job when writing the documentation for this. What I wrote in #102 is correct, I'll make a mental note to clarify this in the documentation.

[Flope]

thanks for the answer but there is still an issue...

How would we trim reads of different lengths using -nOT and -nOB when reads are of different lengths due to initial trimming (e.g., trimmomatic)? Specially at the end of the reads?

If original reads of 150bp gets trimmed down to different lengths. How can we exclude 5bp of the original read 3' (last bases)?

If #102 explanation is correct, then the main documentation is also incorrect?

In the alignment section, it says:
....Thus, to ignore the 5 bases on either end of alignments on the original top (OT) strand, one would specify --nOT 5,5,5,5. Thus, it is irrelevant how long each alignment is, the outermost 5 bases will always be ignored.