Problem importing ok fastq in qiime1, error reported : Header on line 9 is not fastq

Hi guys,

I am running a metaanalisys and some of my data, which were working well on QIIME1, cannot be imported. Error reported is Header on line 9 is not fastq, records must be misaligned (see image below)

However I have use these files in the past without any issues. Te error refers to the section of the file I have underlined in the image attached, which I think looks ok.

My Set up: qiime2-2018.6 on fedora

My code:

qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path "/home/jmseoane/bioinfo/JMS/SharedFolder/Illumina/ArchALL/manifestAr.csv" --source-format PairedEndFastqManifestPhred33 --output-path "/home/jmseoane/bioinfo/JMS/SharedFolder/Illumina/ArchALL/ResultsAr/demux-paired-end.qza"

If I remmove these specific files I get the same error from others from the same run. I attach a couple of them in the link below if anyone wants to try to reproduce the error, just need to change the path in the manifest file.

Thank you very much in advance for any help and your work with QIIME2! This is an awesome open source soft and a great community!

Best regards,


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Hi @JoseM,

First of all, I’m so sorry for the delayed response :frowning:

Thank you for the well described issue and attached screenshots/data!

I was able to pull your example data down, but I’m unable to reproduce the issue. After changing the paths, those files import successfully with the manifest (2018.8).

You attached command and screenshot all looks correct, so I’m at a loss for why you are seeing that error.

In fact, that error only happens when the @ is missing (source here). As we can see from the screenshot, that’s clearly not the case.

Is it at all possible your files have gotten mixed up somehow and so the files QIIME 2 is looking at are the wrong ones?

Once again, very sorry for how long it took me to respond :frowning:

Thank you Evan! No worries about the timing, you are extremely helpful for this community!

From our side, another person found the same problem with the exact same file. We will try to run it in another computer with your VM just in case. Removing this specific sample from our dataset “solves” the issue and we can run the rest of the analysis, but just wanted to understand why it fails (specially now that I know it works for you, feeling like such a rookie!)

Thank you very much for taking a look anyways!

Best regards,


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