Does demux apply any quality threshold to the index reads?

[Nicholas_Bokulich]

Hi @rrohwer and @colinbrislawn!

@rrohwer

• Does demux require an exact match?

Yes — currently it does not support golay barcodes or mismatches, but error correction would be a great feature to add at some point (we have an open issue)

@rrohwer

• Does demux apply any quality filter to the index.fastq file before deciding if it’s a match?

No

@rrohwer

The reason I want to understand this is that applying a quality filter to the index sequences (the barcodes) has been shown to be an effective way to reduce cross-talk errors

Thanks for including that paper! We had discussed that paper, and some others, this discussion:

Discussion: methods for removing contaminants and cross-talk

Just to add further to this discussion, I've previously used decontam prior to it being incorporated into QIIME 2, and whilst I'm a huge fan of the principles it's built around, it didn't work fantastically for my samples. I will caveat that as I did only attempt the prevalence method with 2 negative controls (one kit negative and one sequencing negative) for around 20 samples, which is likely too low a number of controls to have any real statistical power, I'm not sure what the ideal number of …

so yes I have been considering implementing that method at some point but have no eta.

@rrohwer

So how did demux interpret the low quality bases in the index fastq?

As far as I know it does not and this is a very good point, that low-quality base scores could lead to mis-mapped reads. There are 2 things to consider regarding this: 1) usually quality is good in the barcode reads (there are exceptions!), and quality does not drop off in the same way that it does for very long reads; 2) usually the barcoding schemes require 2+ differences between each barcode so that a single error will by default cause that read to be dropped. It will require 2 errors for a barcode to incorrectly map, and 2 errors in that short read will be very rare.

But I will let @gregcaporaso chime in if he has anything to add regarding the philosophy behind the design of demux.