# Humann (v3.9) returning 100% unaligned reads on demo data **URL:** \ **Category:** HUMAnN\ **Created:** [December 18, 2025, 3:51pm UTC](https://forum.biobakery.org/t/humann-v3-9-returning-100-unaligned-reads-on-demo-data/8691 "2025-12-18T15:51:45Z")\ **Posts on this page:** 2\ **Page:** 1
**Author:** ![HUIW0006](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/huiw0006/32/3487_2.png) [@HUIW0006](https://forum.biobakery.org/u/HUIW0006)\ **Post date:** [December 18, 2025, 3:51pm UTC](https://forum.biobakery.org/t/humann-v3-9-returning-100-unaligned-reads-on-demo-data/8691/1 "2025-12-18T15:51:45Z")
Hi, As the title says, I tried running humann3 on the demo data but found 100% unaligned reads. I’m guessing that this shouldn’t be the case? **My command is:** humann --input demo.fastq.gz --output demo\_fastq **The output is:** Running bowtie2 … Total bugs from nucleotide alignment: 0 Total gene families from nucleotide alignment: 0 Unaligned reads after nucleotide alignment: 100.0000000000 % Running diamond … Aligning to reference database: uniref90\_201901b\_full.dmnd CRITICAL ERROR: Error executing: /home/user/miniconda3/envs/humann3/bin/diamond blastx --query /home/user/metagenomics/results/humann\_demo/demo\_humann\_temp/demo\_bowtie2\_unaligned.fa --evalue 1.0 --threads 1 --top 1 --outfmt 6 --db /home/user/metagenomics/databases/humann/uniref/uniref90\_201901b\_full --out /home/user/metagenomics/results/humann\_demo/demo\_humann\_temp/tmpja7rf3jq/diamond\_m8\_rm\_2z37c --tmpdir /home/user/metagenomics/results/humann\_demo/demo\_humann\_temp/tmpja7rf3jq Would greatly appreciate any help or advice. Thanks! ---
**Author:** ![franzosa](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/franzosa/32/3511_2.png) [@franzosa](https://forum.biobakery.org/u/franzosa)\ **Post date:** [January 9, 2026, 8:27pm UTC](https://forum.biobakery.org/t/humann-v3-9-returning-100-unaligned-reads-on-demo-data/8691/2 "2026-01-09T20:27:03Z")
Make sure you are running the version of the demo specific for your HUMAnN version. They are highly specialized for efficiency, so (for example) the v3 demo input probably won’t produce any output on the v4 demo databases.