Trouble importing manifest (.tsv) file & --verbose not working

I honestly have no idea why this doesn’t work, but I keep getting an error when trying to import my single ended, non-EMP or Cassava .fastq files by running this command:

qiime tools import
–type SampleData[SequencesWithQuality]
–input-path seqs.tsv
–output-path seqs.qza
–input-format SingleEndFastqManifestPhred33V2 \

The error that is given is:

There was a problem importing seqs.tsv:

seqs.tsv is not a(n) SingleEndFastqManifestPhred33V2 file:

Filepath on line 1 and column “absolute-filepath” could not be found (./Desktop/Hines/FASTQ_Seqs/HCl-1_R1.fastq) for sample “HCl-1”.

When I try to use the --verbose command, I get this error:

Usage: qiime tools import [OPTIONS]
Import data to create a new QIIME 2 Artifact. See https://docs.qiime2.org/
for usage examples and details on the file types and associated semantic
types that can be imported.

Options:
–type TEXT The semantic type of the artifact that will be
created upon importing. Use --show-importable-types
to see what importable semantic types are available
in the current deployment. [required]
–input-path PATH Path to file or directory that should be imported.
[required]
–output-path ARTIFACT Path where output artifact should be written.
[required]
–input-format TEXT The format of the data to be imported. If not
provided, data must be in the format expected by the
semantic type provided via --type.
–show-importable-types Show the semantic types that can be supplied to
–type to import data into an artifact.
–show-importable-formats
Show formats that can be supplied to --input-format
to import data into an artifact.
–help Show this message and exit.

There was a problem with the command:
(1/1?) no such option: --verbose

I am including my manifest file that has been cleared by Keemei as well as a handful of my sequence files (.fastq).

seqs.tsv (21.6 KB)
HCl-1_R1.fastq (688 Bytes) HCl-2_R1.fastq (690 Bytes) HCl-3_R1.fastq (690 Bytes) HCl-4_R1.fastq (690 Bytes) HCl-5_R1.fastq (688 Bytes) HCl-6_R1.fastq (690 Bytes) HCl-7_R1.fastq (692 Bytes) HCl-9_R1.fastq (689 Bytes) HCl-10_R1.fastq (693 Bytes)

These sequences are from Sanger sequencing, so I know from this link that I have a Phred33 offset.

Why isn’t this a SingleEndManifestPred33V2 file?? Why can’t these files be found? I know the directory string is correct. Should I type out every single bit of that file path (e.g. MacHD/Users/Desktop/Hines/…)?

I’m running out of time & getting frustrated. Any help is greatly appreciated.

Hi! I never used manifest format before, but

Did you try to use absolute pathway like
/home/USER_NAME/Desktop/Hines/FASTQ_Seqs/HCl-1_R1.fastq

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Hi @jhines1! I agree with @timanix - the error message indicates that the file can’t be found:

When I look at the filepath you provided, I see that it is a “relative filepath”, not an “absolute filepath”. Check out the importing tutorial for more details (there is a link there describing the difference between absolute and relative filepaths):

https://docs.qiime2.org/2019.7/tutorials/importing/#fastq-manifest-formats

Thanks, but Keemei doesn’t know anything about manifest files, it is just treating that manifest file like a QIIME 2 Metadata file, which are two different things.

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I was finally able to sit down & work out the filepath issue, which seemed to resolve the initial issue, but now when I try to import my manifest file I getting the warning:

There was a problem importing seqs.tsv:

/var/folders/zc/csj0fb595j98l9vn8xybjdr40000gp/T/q2-SingleLanePerSampleSingleEndFastqDirFmt-lp2cavnw/LCl-85_212_L001_R1_001.fastq.gz is not a(n) FastqGzFormat file:

Quality score length doesn’t match sequence length for record beginning on line 5.

So, I checked out the file & there was one extra character in the quality score (e.g. 316) vs the sequence (e.g. 315). It seems that this is an issue for some. This person had the same issue due to a joining of FASTA & quality scores with a converter. That got me to thinking about the way Windows & Mac (or Unix) code their line breaks/endings, as you have mentioned to me before.

Using BBEdit, I have gone through every fastq file :persevere: in the folder holding my sequences & switched the line break types to Mac (CR) & made sure that each seq/fastq file had only four lines. All of them had an extra space that caused the file to have 5 lines, though only four lines had any data/info. I’m thankful I only have ~425 sequences.

Having done all of that, I am still getting this error. I have checked the number of characters in both the quality scores as well as the sequences themselves & they have an identical number of characters. So, I’m really not sure why this is still an issue. Would it help at all to change the line break type to Unix (LF)? I can’t imagine that would be the case, but I’m completely lost on this issue.

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@jhines1 has continued this discussion here:

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