importing fastq data: forward.fastq.gz not found

Hii,
I have files in following format and want to analyse using qiime2 metagenome 2024.5.
123_R1.fastq.gz
123_R2.fastq.gz
234_R1.fastq.gz
234_R2.fastq.gz
881_R1.fastq.gz
881_R2.fastq.gz
339_R1.fastq.gz
339_R2.fastq.gz

I get error while importing it that says forward.fastq.gz not found.

May I get correct commands to run it?

Hello,
could you post your full qiime tools import command please?
Then it will be much easier to debug your problem.

(Edit: thnx Nicholas for the split)

1 Like

Hii @tripitakit
Thanks for reply. Here is qiime tools import output:

Last login: Thu Jun 20 21:05:21 on ttys000
-zsh
(base) bhagwanrekadwad@MicrobeAI ~ % -zsh
zsh: command not found: -zsh
(base) bhagwanrekadwad@MicrobeAI ~ % cd Desktop
(base) bhagwanrekadwad@MicrobeAI Desktop % conda activate qiime2-metagenome-2024.5
(qiime2-metagenome-2024.5) bhagwanrekadwad@MicrobeAI Desktop % qiime tools import
--type EMPPairedEndSequences
--input-path metagenome
--output-path metagenome.qza
There was a problem importing metagenome:

Missing one or more files for EMPPairedEndDirFmt: 'forward.fastq.gz'

(qiime2-metagenome-2024.5) bhagwanrekadwad@MicrobeAI Desktop %

Hi @Bhagwan
Looks likes like you are trying to import using EMP formating but your data looks like it is de-multiplexed already (you have files for each sample and not a 3 files for forward_reads, reservse_reads, barcodes respectively).

I think you are going to want to follow the steps documented here for importing with a manifest: Importing data β€” QIIME 2 2024.5.0 documentation

Hope this helps!
:turtle:

3 Likes

Hi @cherman2
I tried suggestions. Still, there is an issue. So my sequences imported to and able to create paired-end.qza. But, later command failed.

(qiime2-metagenome-2024.5) bhagwanrekadwad@MicrobeAI ~ % cd Desktop/shotgun
(qiime2-metagenome-2024.5) bhagwanrekadwad@MicrobeAI shotgun % qiime demux summarize
--i-data paired-end-demux-shotgun.qza
--o-visualization demux.qzv
Saved Visualization to: demux.qzv
(qiime2-metagenome-2024.5) bhagwanrekadwad@MicrobeAI shotgun % qiime demux emp-paired
--m-barcodes-file sample-metadata.tsv
--m-barcodes-column sample-id
--p-rev-comp-mapping-barcodes
--i-seqs paired-end-demux-shotgun.qza
--o-per-sample-sequences demux-full.qza
--o-error-correction-details demux-details.qza
Usage: qiime demux emp-paired [OPTIONS]

Demultiplex paired-end sequence data (i.e., map barcode reads to sample ids)
for data generated with the Earth Microbiome Project (EMP) amplicon
sequencing protocol. Details about this protocol can be found at
Protocols and Standards : earthmicrobiome

Inputs:
--i-seqs ARTIFACT EMPPairedEndSequences
The paired-end sequences to be demultiplexed.
[required]
Parameters:
--m-barcodes-file METADATA
--m-barcodes-column COLUMN MetadataColumn[Categorical]
The sample metadata column containing the per-sample
barcodes. [required]
--p-golay-error-correction / --p-no-golay-error-correction
Perform 12nt Golay error correction on the barcode
reads. [default: True]
--p-rev-comp-barcodes / --p-no-rev-comp-barcodes
If provided, the barcode sequence reads will be
reverse complemented prior to demultiplexing.
[default: False]
--p-rev-comp-mapping-barcodes / --p-no-rev-comp-mapping-barcodes
If provided, the barcode sequences in the sample
metadata will be reverse complemented prior to
demultiplexing. [default: False]
--p-ignore-description-mismatch / --p-no-ignore-description-mismatch
If enabled, ignore mismatches in sequence record
description fields. [default: False]
Outputs:
--o-per-sample-sequences ARTIFACT
SampleData[PairedEndSequencesWithQuality]
The resulting demultiplexed sequences. [required]
--o-error-correction-details ARTIFACT ErrorCorrectionDetails
Detail about the barcode error corrections. [required]
Miscellaneous:
--output-dir PATH Output unspecified results to a directory
--verbose / --quiet Display verbose output to stdout and/or stderr during
execution of this action. Or silence output if
execution is successful (silence is golden).
--example-data PATH Write example data and exit.
--citations Show citations and exit.
--help Show this message and exit.

              There were some problems with the command:                  

(1/2) Invalid value for '--m-barcodes-file': There was an issue with
retrieving column 'sample-id' from the metadata.
(2/2) Invalid value for '--i-seqs': Expected an artifact of at least type
EMPPairedEndSequences. An artifact of type
SampleData[PairedEndSequencesWithQuality] was provided.
(qiime2-metagenome-2024.5) bhagwanrekadwad@MicrobeAI shotgun % qiime demux emp-paired
--m-barcodes-file sample-metadata.tsv
--m-barcodes-column sample-id
--p-rev-comp-barcodes
--i-seqs paired-end-demux-shotgun.qza
--o-per-sample-sequences demux-full.qza
--o-error-correction-details demux-details.qza
Usage: qiime demux emp-paired [OPTIONS]

Demultiplex paired-end sequence data (i.e., map barcode reads to sample ids)
for data generated with the Earth Microbiome Project (EMP) amplicon
sequencing protocol. Details about this protocol can be found at
Protocols and Standards : earthmicrobiome

Inputs:
--i-seqs ARTIFACT EMPPairedEndSequences
The paired-end sequences to be demultiplexed.
[required]
Parameters:
--m-barcodes-file METADATA
--m-barcodes-column COLUMN MetadataColumn[Categorical]
The sample metadata column containing the per-sample
barcodes. [required]
--p-golay-error-correction / --p-no-golay-error-correction
Perform 12nt Golay error correction on the barcode
reads. [default: True]
--p-rev-comp-barcodes / --p-no-rev-comp-barcodes
If provided, the barcode sequence reads will be
reverse complemented prior to demultiplexing.
[default: False]
--p-rev-comp-mapping-barcodes / --p-no-rev-comp-mapping-barcodes
If provided, the barcode sequences in the sample
metadata will be reverse complemented prior to
demultiplexing. [default: False]
--p-ignore-description-mismatch / --p-no-ignore-description-mismatch
If enabled, ignore mismatches in sequence record
description fields. [default: False]
Outputs:
--o-per-sample-sequences ARTIFACT
SampleData[PairedEndSequencesWithQuality]
The resulting demultiplexed sequences. [required]
--o-error-correction-details ARTIFACT ErrorCorrectionDetails
Detail about the barcode error corrections. [required]
Miscellaneous:
--output-dir PATH Output unspecified results to a directory
--verbose / --quiet Display verbose output to stdout and/or stderr during
execution of this action. Or silence output if
execution is successful (silence is golden).
--example-data PATH Write example data and exit.
--citations Show citations and exit.
--help Show this message and exit.

              There were some problems with the command:                  

(1/2) Invalid value for '--m-barcodes-file': There was an issue with
retrieving column 'sample-id' from the metadata.
(2/2) Invalid value for '--i-seqs': Expected an artifact of at least type
EMPPairedEndSequences. An artifact of type
SampleData[PairedEndSequencesWithQuality] was provided.
(qiime2-metagenome-2024.5) bhagwanrekadwad@MicrobeAI shotgun %

Hi @Bhagwan,
Once you import using a manifest, you already have a demux.qza so you don't need to run qiime demux emp-paired.

Here I can see that you got a demux.qzv so you should be able to use that to decided trim and trunc values for dada2.

You should be able to just continue on with dada2 and beyond!

I hope that helps!
:turtle:

3 Likes

Just a quick thought, rename all of your raw data file to exactly the format shown in the picture tutorial. It’s been a while for me but the numbers of spaces in the prefix is lengthy.

Hii... @cherman2
Thank you for showing me the right path. Dada2 may take a long time to complete due to the large amount of data in my files. I will ping you after dada2 analysis.

@propolis
Thanks for advice. I have relabelled my sample ids.

Bhagwan

2 Likes