importing demultiplexed Illumina paired read data

I am looking for a tutorial to import data from a number or paired reads fastq files.

  • I only have a very basic sample metadata reporting the sample id’s and names
  • I did not yet figured out how and what the manifest should be (dead links in the forum pages)
  • I do not have the matching barcodes.fastq.gz files and no idea how to get them

provided this dummy example for two samples, could someone please help me create the necessary metadata / manifest to import the reads and start analysis

my sample_metadata.tsv is currently

sample-id subject
#q2:types categorical
Exp_A1 normal_rep1
Exp_A9 normal_rep2

my fastq files have already be demultiplexed and are named:


Is there hope !

Hi @ splaisan,
Sounds like you have everything you need to get your files imported into Qiime2. Since your files are already demultiplexed you won’t need the barcodes.fastq.gz file. Have you seen the manifest format section of the importing tutorial yet? Also note that your metadata file can double as a manifest file by simply adding the 2 extra columns needed which give the location of your files in your computer. Once you have your manifest file prepared, you can import with something like:

qiime tools import \
  --type 'SampleData[PairedEndSequencesWithQuality]' \
  --input-path sample_metadata.tsv \
  --output-path pe-demux.qza \
  --input-format PairedEndFastqManifestPhred33V2

Hope that helps!
Also, could you please point us towards the dead links you mentioned so that we can fix them if possible. Thanks!


thanks a lot, this is exactly what I was looking for.

I made a fastq_manifest.tsv as instructed then used it to import with the following code

qiime tools import \
--type 'SampleData[PairedEndSequencesWithQuality]' \
--input-path fastq_manifest.tsv \
--output-path pe-demux.qza \
--input-format PairedEndFastqManifestPhred33V2

I previously found the following page (Importing paired end demultiplexed MiSeq data) which is an older page explaining how to make a manifest but where links end dead as it was written for an old version of qiime. This is what I named dead link in my post.

thanks a lot, I can now proceed…

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