Have trouble with demux


(ucassee) #1

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


(Nicholas Bokulich) #2

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:

(ucassee) #3

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?

(Nicholas Bokulich) #4

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.

(Nicholas Bokulich) #5

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.

(Sabrina) #6

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?

(ucassee) #7

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 ?