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dereneaton / ipyrad

Interactive assembly and analysis of RAD-seq data sets
http://ipyrad.readthedocs.io
GNU General Public License v3.0
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Ipyrad excluding samples during demultiplexing stage #548

Closed ikb001 closed 4 months ago

ikb001 commented 4 months ago

My dataset includes two 96 well plates containing 185 samples in total. I had a single barcode file which was supposed to be used in demultiplexing both plates so I modified the barcode file to include sample names for the specific plates. Ipyrad then ran the demultiplexing stage and excluded 98 samples from my dataset saying there was no match for those samples. Kindly find attached the barcode files used in demultiplexing my reads. GBS_barcodes.ApeKI-1-96-A.txt GBS_barcodes.ApeKI-1-96-B.txt

I would be glad if any help or advice could be offered to me in making sure this does not occur again especially when I merge them finally.

isaacovercast commented 4 months ago

Did you run each plate in ipyrad step 1 independently? I assume so.

Can you post the s1_*.txt stats files from each of the _fastqs directories?

ikb001 commented 4 months ago

Yes, I did. I have attached copies of the s1 stats files to my reply. s1_demultiplex_stats - B.txt s1_demultiplex_stats-A.txt

isaacovercast commented 4 months ago

The stats for plate A look mostly fine except sample E10_FCR-3 which has 0 reads because it's barcode is identical to another sample:

E10_FCR-3   GTCAA
F10_FCR-4   GTCAA

Plate B doesn't make sense to me because there are samples in the stats file from plate A (all the ones with 0 total reads). Both the barcodes files only have 95 samples (as they should), so i'm not sure which barcode file you used to generate the plate B stats, but it seems to have been a composite of some kind. Plate A and B need to be run independently in step 1, using the barcodes files you sent me, which look fine except for the duplicates. then you can merge the plates prior to step 2. Let me know if you can figure out what's happening with plate b and i can try to help more.

ikb001 commented 4 months ago

Thank you very much for this insight! I think the error may have happened in the process of me copying the barcodes. I have since corrected that for plate A. For Plate B, I am not quite sure what the issue might be either. But I am repeating the step to see if it turns out the same. I will update the thread on the outcome. Thank you!

ikb001 commented 4 months ago

properly structuring the barcode files worked. Thank you very much for the help.