Clair3-RNA is a small variant caller for long-read RNA sequencing (lrRNA-seq) data. Clair3-RNA supports ONT R10.4.1 and R9.4.1 complementary DNA sequencing (cDNA) and direct RNA sequencing (dRNA). dRNA sequencing support the ONT latest SQK-RNA004 kit data for variant calling. Clair3-RNA also supports PacBio Sequel and PacBio MAS-Seq RNA sequencing data.

References:

• Zheng, Z., Yu, X., Chen, L. et al. Clair3-RNA: a deep learning-based small variant caller for long-read RNA sequencing data. Nat Commun 16, 11553 (2025).

• Clair3-RNA on GitHub

• Module Name: clair3-rna (see the modules page for more information)
• Multithreaded with the -t/--threads option.
• This application produces HTML reports. You can use hpcdrive to view these reports on your local workstation.
• Environment variables set
  • CLAIR3RNA_HOME
  • CLAIR3RNA_DATA
• Example files in $CLAIR3RNA_DATA

Allocate an interactive session and run the program.
Sample session (user input in bold):

[user@biowulf]$ sinteractive -c4
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn3144 are ready for job

[user@cn3144 ~]$ module load clair3-rna

[user@cn3144 ~]$ run_clair3_rna \
  --bam_fn ${CLAIR3RNA_DATA}/ont/HG004_chr1_demo.bam \
  --ref_fn ${CLAIR3RNA_DATA}/ont/GRCh38_no_alt_chr1.fa \
  --output_dir out \
  --threads ${SLURM_CPUS_PER_TASK} \
  --platform "ont_guppy_drna002" \
  --region chr1:816000-828000 \
  --tag_variant_using_readiportal

[user@cn3144 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$

Create a batch input file (e.g. clair3-rna.sh). For example:

#!/bin/bash
set -e
module load clair3-rna

run_clair3_rna \
  --bam_fn ${CLAIR3RNA_DATA}/ont/HG004_chr1_demo.bam \
  --ref_fn ${CLAIR3RNA_DATA}/ont/GRCh38_no_alt_chr1.fa \
  --output_dir out \
  --threads ${SLURM_CPUS_PER_TASK} \
  --platform "ont_guppy_drna002" \
  --region chr1:816000-828000 \
  --tag_variant_using_readiportal

Submit this job using the Slurm sbatch command.

sbatch --cpus-per-task=# [--mem=#] clair3-rna.sh

Create a swarmfile (e.g. clair3-rna.swarm). For example:

run_clair3_rna \
  --bam_fn sample1.bam \
  --ref_fn ${CLAIR3RNA_DATA}/ont/GRCh38_no_alt_chr1.fa \
  --output_dir out1 \
  --threads ${SLURM_CPUS_PER_TASK} \
  --platform "ont_guppy_drna002" \
  --region chr1:816000-828000 \
  --tag_variant_using_readiportal
run_clair3_rna \
  --bam_fn sample2.bam \
  --ref_fn ${CLAIR3RNA_DATA}/ont/GRCh38_no_alt_chr1.fa \
  --output_dir out2 \
  --threads ${SLURM_CPUS_PER_TASK} \
  --platform "ont_guppy_drna002" \
  --region chr1:816000-828000 \
  --tag_variant_using_readiportal
run_clair3_rna \
  --bam_fn sample3.bam \
  --ref_fn ${CLAIR3RNA_DATA}/ont/GRCh38_no_alt_chr1.fa \
  --output_dir out3 \
  --threads ${SLURM_CPUS_PER_TASK} \
  --platform "ont_guppy_drna002" \
  --region chr1:816000-828000 \
  --tag_variant_using_readiportal

Submit this job using the swarm command.

swarm -f clair3-rna.swarm [-g #] -t # --module clair3-rna