New to Qiime2 Mismatched sequence ids:

[kulandai1 (Balamurugan)]

HI
I am new to Qiime2 i have just installed qiime2 and it tried atacama tutorial and it work well. but when i used my sample data i found error in demux emp-paried end. Anyone can help me in this i have past the commad and error. I try a lot help plz

 qiime2-2017.11) drsbalamurugan@drsbalamurugan-Inspiron-1440:~/qiime2-atacama-tutorial$ qiime demux emp-paired \
>   --m-barcodes-file sample-metadata.tsv \
>   --m-barcodes-category BarcodeSequence \
>   --i-seqs emp-paired-end-sequences.qza \
>   --o-per-sample-sequences demux \
>   
Plugin error from demux:

  Mismatched sequence ids: HISEQ:504:HCJ2CBCXY:2:1101:3521:3486, HISEQ:504:HCJ2CBCXY:2:1101:2578:2121, and HISEQ:504:HCJ2CBCXY:2:1101:2578:2121

Debug info has been saved to /tmp/qiime2-q2cli-err-th6avgf2.log

[thermokarst]

Hmm! Sorry to hear that @kulandai1. Let’s dissect that error message:

'Mismatched sequence ids: BARCODE_HEADER_ID, FORWARD_HEADER_ID, and REVERSE_HEADER_ID'

So if I fill in this “formula”:

BARCODE_HEADER_ID = 'HISEQ:504:HCJ2CBCXY:2:1101:3521:3486'
FORWARD_HEADER_ID = 'HISEQ:504:HCJ2CBCXY:2:1101:2578:2121'
REVERSE_HEADER_ID = 'HISEQ:504:HCJ2CBCXY:2:1101:2578:2121'

Whoa! Something looks mighty strange with that barcode ID. Would you be able to share your emp-paired-end-sequences.qza and sample-metadata.tsv? If you are unable to publicly share feel free to send a DM. If you are unable to share your data at all, can you please rerun the command, with --verbose included, and copy all of the output?

Thanks!

[megladds (Megan)]

Hello,
I am having the same issue and I looked at all of my files and it seems as if one sample that is present in the forward and reverse files is not present in the barcodes file.
Is there a reason for this and am I able to fix it?
I attached both my files below

Thanks!

Mapping_1-41_reverse_primer.txt (4.2 KB)

[thermokarst]

Hi @megladds - looks like only your metadata was attached - would you be able to share your sequences artifact, too? Feel free to send along as a direct message to me if you are unable to post publicly. Thanks!

[megladds (Megan)]

Hello,

I emailed you using the email on your github page, but here is the google drive link as well (it should be accessible by you).
(how would I direct message you on this forum?)

-Megan

https://drive.google.com/open?id=1ONXaWYmxG7WNtv_ecDYJUJkczjWC0neD

[thermokarst]

Thanks @megladds! I cracked open your files, and found the following first three ids in each file:

barcodes:

@M01522:85:000000000-AU4UD:1:1101:19131:1345 2:N:0:2
@M01522:85:000000000-AU4UD:1:1101:16978:1376 1:N:0:2
@M01522:85:000000000-AU4UD:1:1101:18361:1378 2:N:0:2

forward:

@M01522:85:000000000-AU4UD:1:1101:19131:1345 1:N:0:2
@M01522:85:000000000-AU4UD:1:1101:18045:1364 1:N:0:2
@M01522:85:000000000-AU4UD:1:1101:16978:1376 1:N:0:2

reverse:

@M01522:85:000000000-AU4UD:1:1101:19131:1345 2:N:0:2
@M01522:85:000000000-AU4UD:1:1101:18045:1364 2:N:0:2
@M01522:85:000000000-AU4UD:1:1101:16978:1376 2:N:0:2

Those should all match up, but you can see here, you are missing the second record (@M01522:85:000000000-AU4UD:1:1101:18045:1364 1:N:0:2) in your barcode file.

I then went ahead and checked the line count in each file (they should all be the same):

$ wc -l barcodes.fastq
 17047564 barcodes.fastq
$ wc -l forward.fastq
 17611740 forward.fastq
$ wc -l reverse.fastq
 17611740 reverse.fastq

Looks like you are missing some records in your barcodes file! I would suggest contacting your sequencing facility (or whoever prepared these files for you) and see if they have any thoughts.

Good luck and keep us posted!

---

PS - you can send a DM to someone by clicking on their username/avatar and then clicking the blue “Message” button.

[thermokarst]

@kulandai1,

I took a look at your files, and you also are missing records in your barcodes (see my message above to @megladds).

$ wc -l barcodes.fastq
 1252816 barcodes.fastq
$ wc -l forward.fastq
 1448300 forward.fastq
$ wc -l reverse.fastq
 1448300 reverse.fastq

As I suggested above, I would recommend contacting your sequencing facility to see if they have any ideas. Keep us posted!