Humann3 only generated temp folder and no 3 main output files

Hi members in the Huttenhower Lab,

When running humann v3.6, there were no 3 main output files and only temp folder generated, shown as below

The error says:

#CPU threads: 6
Scoring parameters: (Matrix=BLOSUM62 Lambda=0.267 K=0.041 Penalties=11/1)
Temporary directory: /nobackup/denxyu/zijian/metaphlan4/01_mpa/humann_output/merged_trimmed_006D_humann_temp/tmpbu6dzawx
Percentage range of top alignment score to report hits: 1
Opening the database… [0.164s]
Database: /nobackup/denxyu/new_humann3/humann_databases/uniref/uniref90_201901b_full (type: Diamond database, sequences: 87296736, letters: 29247941583)
Block size = 2000000000
Opening the input file… [0.043s]
Opening the output file… [0s]
Loading query sequences… Error: Error reading input stream at line 6: Invalid character (*) in sequence

CRITICAL ERROR: Error executing: /nobackup/denxyu/miniconda/envs/humann/bin/diamond blastx --query /nobackup/denxyu/zijian/metaphlan4/01_mpa/humann_output/merged_trimmed_006H_humann_temp/merged_trimmed_006H_bowtie2_unaligned.fa --evalue 1.0 --threads 6 --top 1 --outfmt 6 --db /nobackup/denxyu/new_humann3/humann_databases/uniref/uniref90_201901b_full --out /nobackup/denxyu/zijian/metaphlan4/01_mpa/humann_output/merged_trimmed_006H_humann_temp/tmpykvlw7q0/diamond_m8_odufijgj --tmpdir /nobackup/denxyu/zijian/metaphlan4/01_mpa/humann_output/merged_trimmed_006H_humann_temp/tmpykvlw7q0

Error message returned from diamond :
diamond v2.1.6.160 (C) Max Planck Society for the Advancement of Science
Documentation, support and updates available at
Please cite: Nature Methods (2021)

When checking the *_bowtie2_unaligned.fa file, there is *. I have sucessfully run humann, but failed with a different batch of sequencing data. Could you please give me some suggestions? Thank you very much in advance.

* will sometimes occur as a STOP codon in protein sequences but I don’t think it should be occurring in your sequencing reads? Can you share a portion of the reads file containing the *(s)? That appears to be what is tripping up DIAMOND.

Hi Franzosa,
Thank you very much for your reply.
I double-checked the data and found that * was generated during cutadapt step. After discarding lines with *, HUMANN was running successfully. Appreciate your response again.