How to import individual fastq files into qiime2

Hi all. I’m VERY new to QIIME2 although I am competent with QIIME1. I have a data set consisting of individual fastq files (generated by Illumina) with trimmed barcodes and the corresponding mapping file (Qiime1). I want to begin analysis using qiime2-2018.2 but I am uncertain as to how to get started. Should I convert the fastq to fasta and qual in qiime1, catenate the fastas and import the resulting file? Or should I import the indidual fastq into qiime2 and start from there? I would appreciate any guidance. Thank you in advance

Hi @chauvell1,

Glad you’ve made the leap to Qiime2! Have you had a chance to look through the importing tutorials? It covers this topic very nicely. It sounds like you have demultiplexed files which you can import using the manifest approach. Have a try there and let us know if you run into any issues.
Good luck!


I'm still having trouble importing the demultiplexed R1 and R2 fastq.gz. These individual files as well as the manifest (attached) are in the directory ~/Desktop/Cef_seqs/. Can you help?

Here is the error message:
(qiime2-2018.2) rauls-imac:Cef_seqs raulcano$ qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path Cef-seqs-manifest.csv --output-path Cef_paired-end-demux.qza --source-format PairedEndFastqManifestPhred64
/Users/raulcano/miniconda3/envs/qiime2-2018.2/lib/python3.5/site-packages/q2_types/per_sample_sequences/ UserWarning: Importing of PHRED 64 data is slow as it is converted internally to PHRED 33. Working with the imported data will not be slower than working with PHRED 33 data.
Traceback (most recent call last):
File "/Users/raulcano/miniconda3/envs/qiime2-2018.2/lib/python3.5/site-packages/q2cli/", line 116, in import_data
File "/Users/raulcano/miniconda3/envs/qiime2-2018.2/lib/python3.5/site-packages/qiime2/sdk/", line 214, in import_data
return cls.from_view(type, view, view_type, provenance_capture)
File "/Users/raulcano/miniconda3/envs/qiime2-2018.2/lib/python3.5/site-packages/qiime2/sdk/", line 239, in _from_view
result = transformation(view)
File "/Users/raulcano/miniconda3/envs/qiime2-2018.2/lib/python3.5/site-packages/qiime2/core/", line 59, in transformation
new_view = transformer(view)
File "/Users/raulcano/miniconda3/envs/qiime2-2018.2/lib/python3.5/site-packages/q2_types/per_sample_sequences/", line 346, in _9
File "/Users/raulcano/miniconda3/envs/qiime2-2018.2/lib/python3.5/site-packages/q2_types/per_sample_sequences/", line 268, in _fastq_manifest_helper
File "/Users/raulcano/miniconda3/envs/qiime2-2018.2/lib/python3.5/site-packages/q2_types/per_sample_sequences/", line 153, in _parse_and_validate_manifest
'%s' % path)
FileNotFoundError: A path specified in the manifest does not exist or is not accessible: /Users/raulcano/Desktop/Cef_seqs/-Set3-1_S60_L001_R1_001.fastq.gz

An unexpected error has occurred:

A path specified in the manifest does not exist or is not accessible: /Users/raulcano/Desktop/Cef_seqs/-Set3-1_S60_L001_R1_001.fastq.gz

See above for debug info.
(qiime2-2018.2) rauls-imac:Cef_seqs raulcano$

I am also attaching the maifest file (below)
Cef-seqs-manifest.csv (7.6 KB)

Hi @chauvell1,

You’re close! Here is the important line from the manifest tutorial to pay attention to:

In the fastq manifest formats, a manifest file maps sample identifiers to fastq.gz or fastq absolute filepaths

You just need to change the file location from your manifest file from the relative paths you have at the moment to absolute-paths.


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