Importing data with 2 x 2 fastq.gz files for each sample

[JMurase (Jun MURASE)]

Hi, I am a new QIIME2 user and facing a problem to import the Casava 1.8 paired-end demultiplexed fastq files. Some of my samples were sequenced twice due to the insufficient data obtained in the first run and eventually have 4 (2 x 2) files like:
Ig3517_16S_ACTCGCTA-TCTCTCCG_L001_R1_001.fastq.gz
Ig3517_16S_ACTCGCTA-TCTCTCCG_L001_R1_101.fastq.gz
Ig3517_16S_ACTCGCTA-TCTCTCCG_L001_R2_001.fastq.gz
Ig3517_16S_ACTCGCTA-TCTCTCCG_L001_R2_101.fastq.gz

When I run the import tool, I got the error message:

There was a problem importing unifarm16S:

Unrecognized file (unifarm16S/Ig3525_16S_GGAGCTAC-TCTCTCCG_L001_R1_101.fastq.gz) for CasavaOneEightSingleLanePerSampleDirFmt.

Then I collected the files of the second run with the names of *101.fastq.gz:
Ig3517_16S_ACTCGCTA-TCTCTCCG_L001_R1_101.fastq.gz
Ig3517_16S_ACTCGCTA-TCTCTCCG_L001_R2_101.fastq.gz
and run the import tool. But I got another problem:

There was a problem importing test:

Missing one or more files for CasavaOneEightSingleLanePerSampleDirFmt: ‘.+_.+_L[0-9][0-9][0-9]_R[12]_001\.fastq\.gz’

I really appreciate your help to fix the problem.

[thermokarst (Matthew Ryan Dillon)]

Hey there @JMurase!

Some of my samples were sequenced twice due to the insufficient data obtained in the first run and eventually have 4 (2 x 2) files like:

If they were sequenced twice, then it sounds like that happened in multiple runs. I would import each run separately (that way none of your samples should be duplicated in any given run), perform QA/QC (dada2, deblur, otu clustering, etc), then merge the resulting sequences and table. You can find a good example of this in the FMT tutorial.

Good luck, hope that helps, and let us know if you get stuck or have any questions!

[JMurse (Jun MURASE)]

Thank you so much for your quick advice.
Actually that was exactly what I tried; I collected the files of the second run (represented by the names with “*101.fastq.gz”) to perform the separate analysis, but I got stuck with importing them. The error message sounds the files should be renamed to *001.fastq.gz" before importing. correct?

Then I collected the files of the second run with the names of *101.fastq.gz:

Ig3517_16S_ACTCGCTA-TCTCTCCG_L001_R1_101.fastq.gz

Ig3517_16S_ACTCGCTA-TCTCTCCG_L001_R2_101.fastq.gz

and run the import tool. But I got another problem:

There was a problem importing test:

Missing one or more files for CasavaOneEightSingleLanePerSampleDirFmt: ‘.+_.+_L[0-9][0-9][0-9]_R[12]_001.fastq.gz’

[Nicholas_Bokulich]

The error message sounds the files should be renamed to *001.fastq.gz" before importing. correct?

Yes, that appears correct.

Alternatively, you may be able to import these with their current names by using the fastq manifest format for importing.

I hope that helps!

[JMurse (Jun MURASE)]

Thanks a lot for your help!