Value Error: Mismatched sequence IDs with Demux command


I'm trying to run demux on my paired end sequences and I'm getting this error:

ValueError: Mismatched sequence ids: HWI-M00888:107:000000000-AARVV:1:1101:13665:2184, HWI-M00888:107:000000000-AARVV:1:1102:13703:23899, and HWI-M00888:107:000000000-AARVV:1:1102:13703:23899.

When I go back and look at the sequence files themselves and search for the listed IDs they are found in both the forward and reverse read file. Also, the last two ids are the same. I'm not sure how to fix this problem since the ids are actually there.

These are the commands I ran:
qiime tools import --type EMPPairedEndSequences --input-path QIIME2/emp-paired-end-seqs/ --output-path QIIME2/emp-paired-end-seqs.qza

This importing command worked without a problem. The input path contains the required barcodes.fastq.gz, forward.fastq.gz, and reverse.fastq.gz.

qiime demux emp-paired --i-seqs QIIME2/emp-paired-end-seqs.qza --m-barcodes-file mapping_all1.txt --m-barcodes-category BarcodeSequence --o-per-sample-sequences demux.qza

Here is the error log file from the demux command.
demux_error.txt (1.1 KB)


I was trying to edit my original post but it won’t let me so I’m adding this reply.

*Edit: the repeated ids are not found in the barcode file so I removed those, but the first ID is in all three files. Still getting the error, but now it’s adding new ids that weren’t a problem before: HWI-M00888:107:000000000-AARVV:1:1102:13685:23919, and HWI-M00888:107:000000000-AARVV:1:1102:13685:23919

Hi @saatkinson!

It sounds like your barcodes.fastq.gz isn’t matching the order (or is missing sequences) of the other two files. Are you certain that you have the right index/barcode file? Also, would you be able to post the first ~10 or so records from each of the 3 files? It seems like there is a problem with the data itself.


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Hi @ebolyen!

Just looking at the first 10 records of each file, you are correct the barcodes are not in the same order as the forward/reverse read files. I'm positive that they are the correct barcodes, I've been able to run the data through both QIIME1 (a year ago) and QIIME2 using single end importing a couple months ago (before I found the paired end import ability). Do I need to re-order the barcode file?

reverse_10.fastq.txt (5.5 KB)
barcodes_10.fastq.txt (845 Bytes)
forward_10.fastq.txt (5.5 KB)

Yep all 3 files need to have the same order.

Was the order different that time? It should have complained in the same way between the barcodes and the forward reads.

Last time I used the MergedReads file the sequencing center returned (it’s a merged file of the forward and reverse reads). I just checked and this file has the same order as the barcodes file. I also realized that the sequencing center actually merged the forward and reverse reads for me, not just merged the two files.

Sorry for the confusion. I think I can just import these merged sequences as single end reads and be golden!

Thanks for your help!!


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