Problem importing my data in qiime 2

Hello!!

I have a problem importing my data

I used this command

qiime tools import   

–type ‘SampleData[PairedEndSequencesWithQuality]’
–input-path manifest.csv
–output-path paired-end-demux.qza
–input-format PairedEndFastqManifestPhred33

And I get this error message:

There was a problem importing manifest.csv:

manifest.csv is not a(n) PairedEndFastqManifestPhred33 file:

File referenced on line 2 could not be found (/usr/home/biopatic/querejeta/mydata/amplicons_paired/CONTROL_S47_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired)

My manifest.csv file looks like this:

sample-id, absolute-filepath, direction
CONTROL_S47_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CONTROL_S47_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired,forward
CONTROL_S47_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CONTROL_S47_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired,reverse
CV10_S34_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CV10_S34_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired,forward
CV10_S34_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CV10_S34_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired,reverse

And my fastq file like this

@M00598:163:000000000-AP6WK:1:1101:14859:1150 2:N:0:27
ATACTAATTTATCGGTTACTTTATTTAACAATACTTTATTTTAAGGAAGT
+
[email protected]<[email protected]<[email protected]

Has anyone any idea of what could be the mistake?

Thanks!!

Nat

Hi @Natalie ,
Welcome to the forum!
I believe the problem is that your manifest file is a .csv when it needs to be a .tsv, as per the importing tutorial. Simply save your original file as .tsv and that should fix it up.

Yep, as @Mehrbod_Estaki said, you can either convert to the new TSV format, or, you could stay with the CSV, and use the old format.

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Thank you, both. But it is still not working for me. If I use the new format, in .tsv, which looks like this:

sample-id forward-absolute-filepath reverse-absolute-filepath
CONTROL_S47_L001 /usr/marinaq/Desktop/IRBI/petrels_metab/amplicons_paired/CONTROL_S47_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq /usr/home/biopatic/querejeta/mydata/amplicons_paired/CONTROL_S47_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq
CV10_S34_L001 /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV10_S34_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV10_S34_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq
CV11_S35_L001 /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV11_S35_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV11_S35_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq
CV12_S36_L001 /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV12_S36_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV12_S36_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq
CV13_S37_L001 /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV13_S37_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV13_S37_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq
CV14_S38_L001 /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV14_S38_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV14_S38_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq
CV15_S39_L001 /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV15_S39_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV15_S39_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq
CV16_S40_L001 /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV16_S40_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq /usr/home/biopatic/querejeta/mydata/amplicons_paired/CV16_S40_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq

I get this mistake:

There was a problem importing manifest2.tsv:

manifest2.tsv is not a(n) PairedEndFastqManifestPhred33 file:

Found header on line 1 with the following labels: [‘sample-id\tforward-absolute-filepath \treverse-absolute-filepath’], expected: [‘sample-id’, ‘absolute-filepath’, ‘direction’]

And if I use the old one in .tsv:

sample-id, absolute-filepath, direction
CONTROL_S47_L001,/usr/marinaq/Desktop/IRBI/petrels_metab/amplicons_paired/CONTROL_S47_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq,forward
CONTROL_S47_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CONTROL_S47_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq,reverse
CV10_S34_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CV10_S34_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq,forward
CV10_S34_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CV10_S34_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq,reverse
CV11_S35_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CV11_S35_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq,forward
CV11_S35_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CV11_S35_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq,reverse
CV12_S36_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CV12_S36_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq,forward
CV12_S36_L001,/usr/home/biopatic/querejeta/mydata/amplicons_paired/CV12_S36_L001_R2_001.16S2R_5’-3’.fq.trimmed.paired.fastq,reverse

I get this mistake:

There was a problem importing manifest.tsv:

manifest.tsv is not a(n) PairedEndFastqManifestPhred33 file:

File referenced on line 2 could not be found (/usr/marinaq/Desktop/IRBI/petrels_metab/amplicons_paired/CONTROL_S47_L001_R1_001.16S1F_5’-3’.fq.trimmed.paired.fastq).

Thanks for your help.

Best,

Nat

Hi @Natalie!

For the first error:

This is because you are using the wrong input-format — it should be PairedEndFastqManifestPhred33V2, not PairedEndFastqManifestPhred33 (please see the import guide).

For the second:

A minor comment on this, this file is not a TSV (tab-separated), it is a CSV (comma-separated).

The error message is pretty clear here — the manifest was not able to find the file listed at that path. Please double check that this location is correct. One observation — it is exceptionally unusual for your home directory to be under /usr - is that actually the case?

I remember that it is required your input-file-path must be an absolute pathway, for instance, $HOME/Desktop/… . Or it would not find your files.

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