raw paired sequences were mixed

I found that the raw paired sequences were mixed, there should only have the sequences guided by 515F primer in the raw-r1.fq, and there should only have the sequences guided by 806R primer in the raw-r2.fq, respectively.

The sequences was obtained from illumina Miseq PE300.

I asked the sequencing company for details but they cannot explain the reason of this mixed situation.

So I wonder if anyone have found this problem too, and please tell me the possible reason and solutions. Thank you very much.

Hello @yhuizhen,

Yes, lots of other people have also had this problem, but the good news is that the cutadapt plugin now supports mixed orientation reads!

Adding --p-mixed-orientation to your qiime cutadapt demux-paired command should allow you to import these reads as shown in the cutadapt tutorial.

Let us know if you have any questions!


Thanks for you reply and suggestions. That’s peaceful to know mixure is commom.
and I am trying the plugin. :smiling_face_with_three_hearts:

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hi,Colin, I don’t find the correct way to identify the mixed sequences using cutadapt in qiime2. Could you show me one example??
In my raw r1.fq, there are 515F primer from the far left, and 806R primer from the seventh base. The first 6 bases may be the adapter.
attached one snapshot below:

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Good afternoon,

I’m not sure that’s the best way to handle this? I though the direction / orientation of these reads was mixed, but if the reads contain both primers, I’m not sure the best way to work with that.

(I’ve made this post public because other folks will have more experience than me working with this sort of data.)

I know @devonorourke has worked with interesting primer sets before. Let’s see if they have any ideas about how best to import your data.


Well, Colin, I’ll keep on learning the details on cutadapt official document to try.
Thanks for your kindness.
And I’ll ask Devon O’rourke for help.