Qiime vsearch join-pairs error

Hi I’m new to qiime2 and having trouble with qiime vsearch join-pairs.
This is the commad and the error I got.

qiime vsearch join-pairs --i-demultiplexed-seqs paired-end-demux.qza --o-joined-sequences demux-joined.qza

Plugin error from vsearch:
Command ‘[‘vsearch’, ‘–fastq_mergepairs’, ‘/var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-a13po6pp/53743d83-7f36-40e8-9029-450453314b27/data/A11_10_L001_R1_001.fastq.gz’, ‘–reverse’, ‘/var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-a13po6pp/53743d83-7f36-40e8-9029-450453314b27/data/A11_11_L001_R2_001.fastq.gz’, ‘–fastqout’, ‘/var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/q2-SingleLanePerSampleSingleEndFastqDirFmt-okkwlcbq/A11_2_L001_R1_001.fastq’, ‘–fastq_ascii’, ‘33’, ‘–fastq_minlen’, ‘1’, ‘–fastq_minovlen’, ‘10’, ‘–fastq_maxdiffs’, ‘10’, ‘–fastq_qmin’, ‘0’, ‘–fastq_qminout’, ‘0’, ‘–fastq_qmax’, ‘41’, ‘–fastq_qmaxout’, ‘41’]’ returned non-zero exit status 1

Thank you for your help in advance!

Hi @Jeongsu_Kim!

Could you re-run with --verbose or attach the debug log-file which is printed at the end of the error?

Also, what version of QIIME 2 are you running?

Thanks!

I tried --verbose but I got an error again..
I am attaching the error message below.

qiime vsearch join-pairs --verbose --i-demultiplexed-seqs paired-end-demux.qza --o-joined-sequences demux-joined.qza
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: vsearch --fastq_mergepairs /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-5e1m62dz/53743d83-7f36-40e8-9029-450453314b27/data/A1_0_L001_R1_001.fastq.gz --reverse /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-5e1m62dz/53743d83-7f36-40e8-9029-450453314b27/data/A1_1_L001_R2_001.fastq.gz --fastqout /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/q2-SingleLanePerSampleSingleEndFastqDirFmt-i1o16g9g/A1_0_L001_R1_001.fastq --fastq_ascii 33 --fastq_minlen 1 --fastq_minovlen 10 --fastq_maxdiffs 10 --fastq_qmin 0 --fastq_qminout 0 --fastq_qmax 41 --fastq_qmaxout 41

vsearch v2.7.0_macos_x86_64, 8.0GB RAM, 4 cores

Merging reads 100%
199077 Pairs
190464 Merged (95.7%)
8613 Not merged (4.3%)

Pairs that failed merging due to various reasons:
792 too few kmers found on same diagonal
4 potential tandem repeat
1727 too many differences
6080 alignment score too low, or score drop to high
10 staggered read pairs

Statistics of all reads:
250.82 Mean read length

Statistics of merged reads:
454.51 Mean fragment length
13.02 Standard deviation of fragment length
0.62 Mean expected error in forward sequences
0.70 Mean expected error in reverse sequences
0.87 Mean expected error in merged sequences
0.44 Mean observed errors in merged region of forward sequences
0.53 Mean observed errors in merged region of reverse sequences
0.97 Mean observed errors in merged region
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: gzip /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/q2-SingleLanePerSampleSingleEndFastqDirFmt-i1o16g9g/A1_0_L001_R1_001.fastq

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: vsearch --fastq_mergepairs /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-5e1m62dz/53743d83-7f36-40e8-9029-450453314b27/data/A10_8_L001_R1_001.fastq.gz --reverse /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-5e1m62dz/53743d83-7f36-40e8-9029-450453314b27/data/A10_9_L001_R2_001.fastq.gz --fastqout /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/q2-SingleLanePerSampleSingleEndFastqDirFmt-i1o16g9g/A10_1_L001_R1_001.fastq --fastq_ascii 33 --fastq_minlen 1 --fastq_minovlen 10 --fastq_maxdiffs 10 --fastq_qmin 0 --fastq_qminout 0 --fastq_qmax 41 --fastq_qmaxout 41

vsearch v2.7.0_macos_x86_64, 8.0GB RAM, 4 cores

Merging reads 100%
80574 Pairs
77990 Merged (96.8%)
2584 Not merged (3.2%)

Pairs that failed merging due to various reasons:
165 too few kmers found on same diagonal
640 too many differences
1778 alignment score too low, or score drop to high
1 staggered read pairs

Statistics of all reads:
250.92 Mean read length

Statistics of merged reads:
451.85 Mean fragment length
12.60 Standard deviation of fragment length
0.59 Mean expected error in forward sequences
0.74 Mean expected error in reverse sequences
0.85 Mean expected error in merged sequences
0.37 Mean observed errors in merged region of forward sequences
0.46 Mean observed errors in merged region of reverse sequences
0.83 Mean observed errors in merged region
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: gzip /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/q2-SingleLanePerSampleSingleEndFastqDirFmt-i1o16g9g/A10_1_L001_R1_001.fastq

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: vsearch --fastq_mergepairs /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-5e1m62dz/53743d83-7f36-40e8-9029-450453314b27/data/A11_10_L001_R1_001.fastq.gz --reverse /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-5e1m62dz/53743d83-7f36-40e8-9029-450453314b27/data/A11_11_L001_R2_001.fastq.gz --fastqout /var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/q2-SingleLanePerSampleSingleEndFastqDirFmt-i1o16g9g/A11_2_L001_R1_001.fastq --fastq_ascii 33 --fastq_minlen 1 --fastq_minovlen 10 --fastq_maxdiffs 10 --fastq_qmin 0 --fastq_qminout 0 --fastq_qmax 41 --fastq_qmaxout 41

vsearch v2.7.0_macos_x86_64, 8.0GB RAM, 4 cores

Merging reads 99%

Fatal error: Invalid line 139048 in FASTQ file: Sequence and quality lines must be equally long
Plugin error from vsearch:

Command '['vsearch', '--fastq_mergepairs', '/var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-5e1m62dz/53743d83-7f36-40e8-9029-450453314b27/data/A11_10_L001_R1_001.fastq.gz', '--reverse', '/var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/qiime2-archive-5e1m62dz/53743d83-7f36-40e8-9029-450453314b27/data/A11_11_L001_R2_001.fastq.gz', '--fastqout', '/var/folders/vh/l4ckn9311hx8yrvvbfpnh6zh0000gn/T/q2-SingleLanePerSampleSingleEndFastqDirFmt-i1o16g9g/A11_2_L001_R1_001.fastq', '--fastq_ascii', '33', '--fastq_minlen', '1', '--fastq_minovlen', '10', '--fastq_maxdiffs', '10', '--fastq_qmin', '0', '--fastq_qminout', '0', '--fastq_qmax', '41', '--fastq_qmaxout', '41']' returned non-zero exit status 1

Oh! I’m running version qiime2-2018.2

Hi @Jeongsu_Kim, thanks for the details!

Here is the important part of the error message:

This means that in either A11_10_L001_R1_001.fastq.gz (forward reads) or A11_11_L001_R2_001.fastq.gz (reverse reads), there is a FASTQ record starting on line 139048 that is invalid. It appears that the record doesn’t have the same number of quality scores as the corresponding sequence’s length, which is a requirement of the FASTQ format.

These corrupted FASTQ files would have been imported into paired-end-demux.qza. Can you decompress (i.e. gunzip) those two files listed above and check that the number of quality scores are exactly the same as the length of the corresponding sequence? Running qiime tools validate paired-end-demux.qza may also help with debugging this.

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