Problems with Manifest Import

[Micro1984 (Audra Crouch)]

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?

Thanks

Audra

[thermokarst]

Hi there @Micro1984!

@Micro1984

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

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).

@Micro1984

Am I getting this area because I have already joined my paired reads from Trimmomatic?

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.

[Micro1984 (Audra Crouch)]

Hey,

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.

[thermokarst]

@Micro1984

I was wondering if the problem was actually with Pandaseq

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.