Barcode demultiplexing after albacore

[rrwick]

Barcode demultiplexing after albacore

[rrwick]

Hi Olga,

This was an interesting one!

Porechop can only determine Albacore's barcodes if it is given a whole Albacore directory for its input (-i). Then for each fastq it finds in the directory, it looks for the barcode name in the path. For example, /barcode01/ in the path for this fastq:
workspace/pass/barcode01/fastq_runid_4409128d0616a0c2bb18e7405515d7fa445a812b_0.fastq
Porechop does not use the info in the read header (though that's not a bad idea - I'll consider adding it). So I suspect you ran Porechop only on the one file, not on the whole Albacore directory.

If my assumption is correct, Porechop never knew what the Albacore barcode call was. If you run Porechop like this porechop -i albacore_dir -b output_dir, then I think it will put the read in the 'none' bin, as you expected.

What I found interesting about this case is this: Albacore got the barcode correct (barcode02) and Porechop got it wrong (barcode01).

Here's the alignment of barcode01 to your read's start:

TCATGCTTCGTTCAGTTACGTATTGCTTCAGTTCGTTTACATGAAAGTCGTCTGTGT-TTT-CGCATTTATCGAC
                                        | |||||| ||| |||| |||  |           
----------------------------------------AAGAAAGTTGTCGGTGTCTTTGTG-----------

And here's the alignment of barcode02:

TCATGCTTCGTTCAGTTACGTATTGCTTCAG-TT-CGTTTACATGAAAGTCGTCTGTGTTTTCGCATTTATCGAC
                           || | || ||||    ||  ||||||||||                  
---------------------------TC-GATTCCGTT----TG-TAGTCGTCTGT------------------

Barcode01 indeed looks like a better match (which is why Porechop called it as such), but I can see from the flanking sequences that the barcode01 alignment is in the wrong place and the barcode02 alignment is in the right place! In a rapid kit, the barcode is preceded by ...CGTATTGCT and followed by GTTTTCGCA..., which matches the barcode02 alignment.

It's an interesting case, and it makes me wonder if and how Porechop should be considering those flanking sequences. But I'm not too worried about it - I suspect it's an unusual case and Porechop would 'none' bin the read because of the Albacore disagreement.

I hope that clears it up. I'm going to close this issue now, but if something's still not right or you still have questions, please let me know!

Ryan