Quality control of Long-read transcriptomes.

SQANTI3 is the newest version of the SQANTI tool that merges features from SQANTI and SQANTI2, together with new additions. SQANTI3 is the first module of the Functional IsoTranscriptomics (FIT) framework, which also includes IsoAnnot and tappAS.

References:

• Pardo-Palacios, F.J., Arzalluz-Luque, A. et al. SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms. Nat Methods (2024).
• Keil, N., Monzó, C., McIntyre, L., Conesa, A. SQANTI-reads: a tool for the quality assessment of long read data in multi-sample lrRNA-seq experiments. BioRxiv (2024).

• SQANTI3 on GitHub

The command line interface changed in version 5.4. See Running SQANTI3 from the wrapper. Although the individual sqanti3_*.py commands remain available, there were also changes to their command-line arguments. The example below illustrates that usage.

• Module Name: sqanti3 (see the modules page for more information)
• Multithreaded using the -t/--cpus flag. It will attempt to use 10 cpus by default (as of time of writing / v5.3.6), so please set this flag accordingly.
• This application produces HTML reports. You can use hpcdrive to view these reports on your local workstation.
• Environment variables set
  • SQANTI3_HOME
• Example files in $SQANTI3_HOME/examples
• Reference data in $SQANTI3_HOME/data

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

[user@biowulf]$ sinteractive --cpus-per-task 8
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 sqanti3

[user@cn3144 ~]$ ln -s  $SQANTI3_HOME/data . # program expects to find other files in a relative path here

[user@cn3144 ~]$ 
    sqanti3_qc.py \
    --isoforms data/UHR_chr22.gtf \
    --refGTF data/reference/gencode.v38.basic_chr22.gtf \
    --refFasta data/reference/GRCh38.p13_chr22.fasta \
    --CAGE_peak data/ref_TSS_annotation/human.refTSS_v3.1.hg38.bed \
    --polyA_motif_list data/polyA_motifs/mouse_and_human.polyA_motif.txt \
    -o UHR_chr22 \
    -fl data/UHR_abundance.tsv \
    --short_reads data/UHR_chr22_short_reads.fofn \
    --cpus $SLURM_CPUS_PER_TASK \
    --report both

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

Create a batch input file (e.g. sqanti3.sh). For example:

#!/bin/bash
set -e
module load sqanti3

sqanti3_qc.py \
   --isoforms data/UHR_chr22.gtf \
   --refGTF data/reference/gencode.v38.basic_chr22.gtf \
   --refFasta data/reference/GRCh38.p13_chr22.fasta \
   --CAGE_peak data/ref_TSS_annotation/human.refTSS_v3.1.hg38.bed \
   --polyA_motif_list data/polyA_motifs/mouse_and_human.polyA_motif.txt \
   -o UHR_chr22 \
   -fl data/UHR_abundance.tsv \
   --short_reads data/UHR_chr22_short_reads.fofn \
   --cpus $SLURM_CPUS_PER_TASK \
   --report both

Submit this job using the Slurm sbatch command.

sbatch [--cpus-per-task=#] [--mem=#] sqanti3.sh

Create a swarmfile (e.g. sqanti3.swarm). For example:

sqanti3_qc.py --isoforms isoforms1.gtf --refGTF ref_annotation.gtf --refFasta ref_genome.fa -o sample1 -d out1 -t $SLURM_CPUS_PER_TASK
sqanti3_qc.py --isoforms isoforms2.gtf --refGTF ref_annotation.gtf --refFasta ref_genome.fa -o sample2 -d out1 -t $SLURM_CPUS_PER_TASK
sqanti3_qc.py --isoforms isoforms3.gtf --refGTF ref_annotation.gtf --refFasta ref_genome.fa -o sample3 -d out1 -t $SLURM_CPUS_PER_TASK
sqanti3_qc.py --isoforms isoforms4.gtf --refGTF ref_annotation.gtf --refFasta ref_genome.fa -o sample4 -d out1 -t $SLURM_CPUS_PER_TASK

Submit this job using the swarm command.

swarm -f sqanti3.swarm [-g #] [-t #] --module sqanti3