Analyzing only the forward sample with mixed orientation reads

[Nicholas_Bokulich]

Hi @aiaoz ,

@aiaoz

We are actually on our way to develop a tool to fix this problem by sorting the pairs into the correct files. While googling I saw that this is something that happens when the wrong library preparation method is used by the sequencing center.

We already have a method in the QIIME 2 plugin RESCRIPt for reorienting mixed-orientation reads — but it currently only works on FASTA data, not FASTQ. Making that edit in RESCRIPt might be easier than a de novo method. VSEARCH also has an orient method that operates on FASTQ, so this should work out of the box.

QIIME 2's q2-cutadapt plugin also allows demultiplexing/re-orientation of mixed-orientation barcoded reads. I think this should work for your case. You can see some relevant discussion here:

Discarded results produced by q2-cutadapt's trim-paired when working with mixed-orientation reads

Summary We recently discovered a bug related to the q2-cutadapt plugin's demux-paired command that, in certain situations, can unnecessarily discard a significant portion of reads, when the command is run with the --p-mixed-orientation flag. Impact This issue impacts the following versions of q2-cutadapt: 2020.6 2020.8 If you think you may have been impacted please check your provenance at https://view.qiime2.org for the versions listed above to verify that you ran demux-paired with the mi…