[Samtools-help] BC tag question

[Richard C. <rco...@bc...>]

Hi all,

We are working on an application in our wet lab where we introduce 
random hexamers into our adapters to allow us to differentiate between 
PCR duplicates and fragments that were present in multiple copies of 
starting material.      The workflow for processing the sequence is that 
we trim the first and last six bases off of our single-ended reads and 
use those bases as our "FP" or "fragment provenance".   When we want to 
mark the duplicates we only want to mark reads as duplicates if they 
align to the same position AND they have the same FP.   Reads aligning 
to the same position with different FPs should not marked as duplicates.

If I'm not mistaken, I now see in the Picard docs that I can  supply a 
BARCODE_TAG to MarkDuplicates to mark the duplicates as I describe 
above.   My question to the group is if it is more appropriate to use 
the BC tag, or to use some custom tags to hold the hexamer sequences 
associated with each read.   I expect that the BC tag should be reserved 
for the classical sample multiplexing application (many libraries in a 
pool).

If you are still reading I have another question for you:
-Since our reads are single-ended and we have two separate hexamer 
sequences (one from each end of the read)...would you recommend 
concatenating them into one sequence (perhaps with a delimiter) and 
keeping them in one tag, or separating them into two tags?  Would there 
be any issues in Picard if we have single ended sequencing but supply 
two different barcode-like tags?

thanks,
Richard


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