Problems with Manifest Import

I am running qiime2-2019.1.

I am trying to import my files that went through Trimmomatic processing for trimming and then joined using pandaseq into Qiime via:

qiime tools import \

–type ‘SampleData[SequencesWithQuality]’
–input-path Manifest-TM.csv
–output-path Mouse-TM-demux.qza
–input-format SingleEndFastqManifestPhred33

I am getting the following error:
There was a problem importing Manifest-TM.csv:

/tmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-hl8_tpf5/G63_48_L001_R1_001.fastq.gz is not a(n) FastqGzFormat file:

Header on line 5 is not FASTQ, records may be misaligned

I will tell you that G63_48_L001_R1_001.fastq.gz is not a file that I am trying to input.

I have created a manifest file with all my files and paths with correct paths. Am I getting this area because I have already joined my paired reads from Trimmomatic?



Hi there @Micro1984!

For sure — that is the new filename for sample G63. I suspect if you open up the source file for G63 you will find the issue referred to in the second record of the file (so, lines 5-8).

You are getting this area because at least one file (for sample G63) has malformed fastq records in it. It is possible that trimmomatic is responsible.

Taking a step back, why did you pre-join? That isn’t the issue here, but could explain why the record is corrupt. I ask because if you are planning on q2-dada2 for denoising, that method requires that the reads be unjoined prior to running — DADA2 will merge reads for you.


I was wondering if the problem was actually with Pandaseq. I was doing my merging with that program and then I was going to use Deblur.

:man_shrugging: I'm sorry, I'm not really sure. - without more information or data, the best I can suggest is what I mentioned above. The problem might be caused by an upstream tool, or, it could be a problem with the data transfer from your sequencing center. If you want to run zcat THE_FILE_FOR_G63.fastq.gz | head -n 30 and return the results here that would be helpful, but, I suspect the solution is going to lie in changing something in the pre-QIIME 2 steps applied to these data. :t_rex:

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