I'm using the QIIME1 scripts extract_barcodes.py to extract barcodes from a dual-indexed MiSeq library, and then demultiplex my reads using split_libraries_fastq.py. I have been having trouble correctly using a couple of parameters relating to reverse complementing barcodes.

When extracting the barcodes with extract_barcodes.py, I use --rev_comp_bc1 (as directed by the individual in charge of sequencing). Then when I demultiplex the reads with split_libraries_fastq.py, sometimes I need to include the parameter --rev_comp_mapping_barcodes to get the script to work, and sometimes I don't. I'm not sure why, unless the barcodes I'm receiving in my mapping files are sometimes written in different orientations (I don't write them).

I think the parameter --rev_comp_bc1 is self-explanatory, but could someone explain what --rev_comp_mapping_barcodes does and when to use it?
To make things simple we can assume that the two barcodes are written from 5' to 3' in the mapping file.

  • $\begingroup$ I dowvoted this question despite being well write because I don't see any reference to what have you done to understand the differences between the parameters. Did you look up to the documentation? Have you asked the responsible of the sequencing why sometimes is like this? When you update the question I will remove the downvote. $\endgroup$
    – llrs
    Jan 16 '18 at 21:22
  • $\begingroup$ Of course I have. There's no point in being critical. You obviously don't know the answer so why bother. $\endgroup$
    – user2197
    Jan 17 '18 at 1:27
  • $\begingroup$ Sorry I was so critical. I did some effort to answer your question and then realized you didn't disclose if you where aware of the documentation and how it hadn't helped you. I was a bit disappointed to have spend some time for nothing, that's why I decided to leave the comment to prevent other to invest more time to answer your question in pages and papers you are already aware. I hope you find the answer somehow. Best wishes $\endgroup$
    – llrs
    Jan 17 '18 at 7:56

As the help page says:

Reverse complement barcode in mapping before lookup (useful if barcodes in mapping file are reverse complements of golay codes) [default: False]

As the help page of extract_barcode says:

Reverse complement barcode 1 before writing [default: False]

You seem to be doing two complete different things. extract_barcode.py is designed to

format fastq sequence and barcode data so they are compatible with split_libraries_fastq.py

While the first one is for:

demultiplexing of Fastq sequence data where barcodes and sequences are contained in two separate fastq files

So it seems that depending on the machine used or the previous processing you are given with the barcode in another file or together. I would advice to ask the person who gave you this data.


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