Importing Demultiplexed Sequences

QIIME2 works with its own artifact format (.qza). The first step is importing your demultiplexed FASTQ files into this format using a manifest file that maps sample IDs to file paths.

1. Get the Manifest Files

Copy the manifest files for both sequencing runs into your working directory:

cp /pl/active/courses/2025_summer/CSU_2025/q2_workshop_final/QIIME2/manifest_run2.txt .
cp /pl/active/courses/2025_summer/CSU_2025/q2_workshop_final/QIIME2/manifest_run3.txt .

2. Get the Demultiplexed Reads

Copy the FASTQ read directories for both runs:

cp -r /pl/active/courses/2025_summer/CSU_2025/q2_workshop_final/QIIME2/reads_run2 .
cp -r /pl/active/courses/2025_summer/CSU_2025/q2_workshop_final/QIIME2/reads_run3 .

3. Import Run 2

qiime tools import \

--type \

--input-format \

--input-path manifest_run2.txt \

--output-path demux_run2.qza

Hint Params

Check →

You are importing demultiplexed paired-end FASTQ files using a manifest. The type encodes what kind of sequencing data this is; the format tells QIIME2 how the manifest file is structured and what quality encoding the reads use.

--type, QIIME2 semantic type. For demultiplexed paired-end FASTQ: SampleData[PairedEndSequencesWithQuality]

--input-format, Manifest format + quality encoding. PairedEndFastqManifestPhred33V2 means a manifest with forward and reverse paths per sample, Phred+33 encoded.

4. Visualize Run 2

qiime demux summarize \
  --i-data demux_run2.qza \
  --o-visualization demux_run2.qzv

Viewing .qzv files

Drag and drop any .qzv file onto view.qiime2.org to explore it interactively. The demux summarize visualization shows per-sample read counts and per-position quality score distributions, use these to choose truncation lengths for DADA2.

Review both run visualizations using this checklist before moving on:

Demux Visualization Review Reset

Per-sample read counts look reasonableNo samples with suspiciously low reads Forward read quality identifiedNote the position where Q scores drop below Q30 Reverse read quality identifiedReverse reads typically drop in quality sooner than forward Truncation lengths chosen for DADA2Both runs use 150 bp in this workshop

5. Import Run 3

qiime tools import \

--type \

--input-format \

--input-path manifest_run3.txt \

--output-path demux_run3.qza

Hint Params

Check →

Same data type and manifest format as run 2. The only difference is the manifest file path and output name.

--type, QIIME2 semantic type. For demultiplexed paired-end FASTQ: SampleData[PairedEndSequencesWithQuality]

--input-format, Manifest format + quality encoding. PairedEndFastqManifestPhred33V2 means a manifest with forward and reverse paths per sample, Phred+33 encoded.

6. Visualize Run 3

qiime demux summarize \
  --i-data demux_run3.qza \
  --o-visualization demux_run3.qzv

Outputs

File	Type	Description
demux_run2.qza	Artifact	Imported sequences, run 2
demux_run2.qzv	Visualization	Summary of run 2 read quality
demux_run3.qza	Artifact	Imported sequences, run 3
demux_run3.qzv	Visualization	Summary of run 3 read quality

Next: Denoising with DADA2