qiime demux emp-paired error with 4 input files: R1 R2 I1 I2, Barcode header lines do not contain description fields but sequence header lines do.

Technical Support
,
1

I am running Qiime 2 version 2019.1.0.

I have been working with Illumina R1, R2, I1 and I2 files as my input data. I have reviewed this post
https://forum.qiime2.org/t/dmx-with-4-initial-read-files-i1-i2-r1-r2/4190/3 and have performed the following:

Merged the I1 and I2 index files into one by simply concatenating the sequence and quality lines. For example, using this I1 read and I2 read:

    @M70271:95:000000000-C7WK3:1:1101:11188:1742 1:N:0:1
    TCGACGTC
    +
    -,,,8+8,

    @M70271:95:000000000-C7WK3:1:1101:11188:1742 2:N:0:1
    TCTTCTTT
    +
    -,,,,6,6

I create this merged read:

    @M70271:95:000000000-C7WK3:1:1101:11188:1742
    TCGACGTCTCTTCTTT
    +
    -,,,8+8,-,,,,6,6

I removed all primers from my R1 and R2 reads using cutadapt.

I created a metadata file and verified that it is properly formatted using keemei. My metadata file looks like this:
#SampleID BarcodeSequence
83WellA1-16S TAAGGCGACTCTCTAT
83WellA2-16S CGTACTAGCTCTCTAT
83WellA3-16S AGGCAGAACTCTCTAT

I then imported my forward.fastq.gz, reverse.fastq.gz and merged barcodes.fastq.gz using this command:
qiime tools import
–type EMPPairedEndSequences
–input-path /test/trimmed_files
–output-path /test/trimmed_files/emp-pair-seqs.qza

Next I ran qiime demux emp-paired with this command:

qiime demux emp-paired
–i-seqs /test/trimmed_files/emp-pair-seqs.qza
–m-barcodes-file /test/trimmed_files/Qiiime_multiplexed_barcode_mapping_file.txt
–m-barcodes-column BarcodeSequence
–o-per-sample-sequences demux.qza
–verbose

And that produces this error:

Traceback (most recent call last):
File “/hpc/apps/qiime2-2019.1/install/lib/python3.6/site-packages/q2cli/commands.py”, line 274, in call
results = action(**arguments)
File “</hpc/apps/qiime2-2019.1/install/lib/python3.6/site-packages/decorator.py:decorator-gen-422>”, line 2, in emp_paired
File “/hpc/apps/qiime2-2019.1/install/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 231, in bound_callable
output_types, provenance)
File “/hpc/apps/qiime2-2019.1/install/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 365, in callable_executor
output_views = self._callable(**view_args)
File “/hpc/apps/qiime2-2019.1/install/lib/python3.6/site-packages/q2_demux/_demux.py”, line 331, in emp_paired
for barcode_record, forward_record, reverse_record in seqs:
File “/hpc/apps/qiime2-2019.1/install/lib/python3.6/site-packages/q2_demux/_demux.py”, line 194, in iter
'Barcode header lines do not contain description fields ’
ValueError: Barcode header lines do not contain description fields but sequence header lines do.

Plugin error from demux:

Barcode header lines do not contain description fields but sequence header lines do.

See above for debug info.

At this point, I do not see what the issue is. What could be the problem here?

2

the barcode header lines do not match the sequence header lines. What do the corresponding sequence header lines look like?

3

My sequence headers look like this:

@M70271:95:000000000-C7WK3:1:1101:11188:1742 1:N:0:1

@M70271:95:000000000-C7WK3:1:1101:11188:1742 2:N:0:1

The only difference between the sequence headers and the merged barcode header is that the characters after the space are missing. I left them off when merging the barcode sequences because it doesn't seem to make sense to keep them. If I did keep them, which one would I keep? The R1 header ending or the R2 header ending? Or should I make a new header ending?

4

I think that may be the mistake. I am not 100% sure, but I think the EMP format probably expects the barcode sequences to match the header lines of the forward reads. Do you want to give that a try and let me know what happens?

5

Thank you for taking the time to look at this. I got it working but had already started on my solution before your most recent reply.
I ended up adding this ending to all of my concatenated barcode fastq records: 3:N:0:1. This is very similar to what you suggested (add 1:N:0:1 to the barcodes header line so it matches the R1 reads).

I saw a reference to barcode R3 files in this post https://forum.qiime2.org/t/clarify-on-demux-emp-paired/5565 which mentioned

(barcodes should be in R3 ). It will use the order of R3 to work out which sequences from R1 and R2 belongs to which samples.

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