Multiple runs and splitting up an existing demux artifact


I am having some trouble with regards to multiple runs too. I have fastq files from Illumina MiSeq (Demultiplexed, paired end). Importing them was trouble free - but I did not catch that each run needs to be analyzed in DADA2 separately. Therefor I would love to filter my SampleData[PairedEndSequencesWithQuality] file so I get one file for each run. Is this even possible?

If not do I have to import my fastq files as separate processes, run demux summarize to find how best to crop my sequences for each run and then proceed with DADA2 x 4 in my case? And following this question -when one operates with more than 1 run - should choice of sequence length be the same for all reads to be merged eventually?

And out of curiosity: Is it possible in DADA2 to add a mapping alternative so one can run the process taking runs into account?

Hi @stangedal!

Unfortunately not really.

That is correct!

Because your data is paired-end, you don’t need to worry about it as your primer-pair will ensure that you get the same ASVs (assuming they overlap) since all of your sequences will start from the same forward primer and end at the same reverse primer. (If your data was single end, you would want to trim in a uniform way so that the final ASVs were all comparable.)

If I understand what you are suggesting correctly, you’d provide some metadata column that would indicate a run number/group and q2-dada2 would execute DADA2 on each run, then give you the final merged output? The only problem I see with that is there isn’t an obvious way to supply the parameters for each run, since they will probably vary by run (although in the single-end case, you’d have to match the basic truncation parameters anyways).

Thank you for responding so quickly. Too bad - it sure would be great being able to filter the SampleData[PairedEndSequencesWithQuality] file :smiley: I will share the information with my co workers so they can avoid making the same mistake as I did. Maybe I could suggest it being mentioned in the Moving Pictures tutorial and even in the plug in as a heads up?

You understand me correctly on the mapping alternative in DADA2. I guess it would demand for the demux summarizer to handle an input from the mapping file - allowing you to see and pick parameters for each run and have this incorporated in a mapping file for the DADA2 step…

So - I will delete my files and start over with 4 uploaded runs!

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