Error in Importing data in qiime2

Question

[Vaishnavi]

Hello All, I am trying to provide an input of my amplicon sequencing files which are in .fq format for qiime2 and I am getting error

There was a problem importing fastqcpwo:

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

Can someone please help me to resolve this?

[andres.firrincieli]

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

[Vaishnavi]

Hi thanks for your reply I attempted the single lane cassava command but it gave same error I am trying to redirect to your link on how to create manifest file but I am not able to that can you please help me with that?

[andres.firrincieli]

I am not able to that

What do you mean?

Can you show the command line used to import the data and the name of the fastq files?

[Vaishnavi]

Hi so I made a manifest file attaching its pic below and tried to run it but I am getting a different error now.
sample-id forward-absolute-filepath reverse-absolute-filepath
sample1 $PWD/fastqcpwo/sample1_R1.fastq.zip $PWD/fastqcpwo/sample1_R2.fastq.zip This is my manifest file I am trying with one sample.

![This is my command line snapshot 1

Command: qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path manifest-pwonew.tsv --output-path paired-end-demuxpwo.qza --input-fo rmat PairedEndFastqManifestPhred33V2

Error: Traceback (most recent call last): File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/q2cli/builtin/tools.py", line 157, in import_data artifact = qiime2.sdk.Artifact.import_data(type, input_path, File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/qiime2/sdk/result.py", line 277, in import_data return cls._from_view(type_, view, view_type, provenance_capture, File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/qiime2/sdk/result.py", line 305, in _from_view result = transformation(view, validate_level) File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/qiime2/core/transform.py", line 73, in transformation other.validate(new_view) File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/qiime2/core/transform.py", line 143, in validate view.validate(level) File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/qiime2/plugin/model/directory_format.py", line 173, in validate getattr(self, field)._validate_members(collected_paths, level) File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/qiime2/plugin/model/directory_format.py", line 103, in _validate_members self.format(path, mode='r').validate(level) File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/qiime2/plugin/model/file_format.py", line 26, in validate self._validate_(level) File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/q2_types/per_sample_sequences/_format.py", line 284, in _validate_ self._check_n_records(record_count_map[level]) File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/site-packages/q2_types/per_sample_sequences/_format.py", line 244, in _check_n_records for i, record in file_: File "/home/vaishnavi/anaconda3/envs/qiime2-2022.2/lib/python3.8/encodings/ascii.py", line 26, in decode return codecs.ascii_decode(input, self.errors)[0] UnicodeDecodeError: 'ascii' codec can't decode byte 0x95 in position 10: ordinal not in range(128)

An unexpected error has occurred:

'ascii' codec can't decode byte 0x95 in position 10: ordinal not in range(128)

See above for debug info.

[andres.firrincieli]

I think the problem is that qiime2 does not support .zip files.

Unzip the fastq files and try again

[Vaishnavi]

Hey so I did as you said and now I am getting this error

qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path manifest-pwonew.tsv --output-path paired-end-demuxpwo.qza --input-fo rmat PairedEndFastqManifestPhred33V2 There was a problem importing manifest-pwonew.tsv:

/tmp/q2-SingleLanePerSamplePairedEndFastqDirFmt-51qdmy8j/sample1_1_L001_R2_001.fastq.gz is not a(n) FastqGzFormat file:

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

[andres.firrincieli]

sample1_1_L001_R2_001.fastq.gz is not a(n) FastqGzFormat file:

Why the file now has a gz extension? What did you do with the original fastq.zip files?

Can you show me the output of:

zcat sample1_1_L001_R2_001.fastq.gz | head

[Vaishnavi]

Hi, So I just converted .zip files into gz, I am not able to understand your meaning from output should I add
zcat sample1_1_L001_R2_001.fastq.gz | head in input somewhere. Because when I put this in sample1_1_L001_R2_001.fastq.gz in as my path for sample 1 in manifest file I am getting above error

[andres.firrincieli]

So I just converted .zip files into gz

By doing so it seems that your fastq are now corrupted and the command zcat sample1_1_L001_R2_001.fastq.gz was to check the integrity of the file by looking at the first lines of the fastq

Let's start from the begining. Your fastq files are compressed (zip). QIMEII accept two kind of files: fastq or fastq.gz (fastq compressed file). You do not need to convert your fastq.zip files into gz. Just simply convert (decompress) the fastq.zip files into fastq

[Vaishnavi]

Hi there sorry for late response I was facing some issues with my system. So should just place my fastq into location rather than .gz format and add the path of that to the manifest.

[Vaishnavi]

Hi there, So there was problem with input path I resolved it. I successfully converted it into .qza Thanks