Have trouble with demux

I use Miseq to get my pair end reads and use qiime2-2018.6 to demux my data. But it is always error with
Plugin error from demux:

Mismatched sequence ids: M03609:80:000000000-C5RR6:1:1101:19503:1363, M03609:80:000000000-C5RR6:1:1101:8683:1333, and M03609:80:000000000-C5RR6:1:1101:8683:1333

Debug info has been saved to /tmp/qiime2-q2cli-err-8vouccie.log

I use qiime1 to get the barcode.fq file and the command is below
extract_barcodes.py -f forward.fastq -r reverse.fastq
-m mappingfile.txt
-o pairedbarcode
-c barcode_paired_end --bc1_len 8 --bc2_len 8 -a --attempt_read_reorientation


Hi @ucassee,
The QIIME 1 command must be dropping those sequences from one or the other sequence file that it outputs.

Since it is a very small number of sequences, I recommend just looking for those records in each sequence file and deleting those lines. That’s what the QIIME 1 command should be doing, anyway: deleting mismatched sequences. Somehow it must be missing a few.

If you get more errors, please be sure to post the full error message. You can run your commands with the --verbose flag added to the end, or open the log file mentioned here:

Thanks for your help~
Now I always use qiime1 (extract_barcodes.py ) to get barcode.fq file, which is a little bit complicated to switch between qiime1 and qiime2 . I wonder whether there is another good way to get a barcode.fq file?

It looks like you have dual-indexed reads so unfortunately there is not a way to do this in QIIME 2 right now.

Talk to your sequencing center — the best way to do things is to have the sequencing center take care of demultiplexing if you provide them a list of barcodes. Illumina’s onboard software is designed to support dual-indexed barcodes and other barcoding designs, so can handle a lot of this before you even see the data.

I agree, it is inconvenient to switch between the two, especially now that QIIME 1 is deprecated and no longer supported. We will eventually add support for dual-indexed reads in QIIME 2 but I am sorry I do not have an ETA on that.

1 Like

An off-topic reply has been split into a new topic: How to handle mixed orientation reads

Please keep replies on-topic in the future.

Once you use qiime1 to get the barcode.fq file, then once you use that file in qiime2 there are no more issues with demux and remaining workflows?

it is okay to use barcode.fq file to run qiime2 with the remanining workflows. Do you use the sequences file with cut-off barcode after qiimme1 or the original sequences file ?


1 Like

This topic was automatically closed 31 days after the last reply. New replies are no longer allowed.