Error Scaffolding Using RagTag

[neupanebpn]

We performed high-fidelity (HiFi) whole genome sequencing of two wheat cultivars, Madsen and Pritchett, using the PacBio Revio Circular Consensus Sequencing (CCS) platform. The high-accuracy long reads were first assembled into contigs using Hifiasm. Post-assembly, we conducted quality control and completeness assessments using tools such as BUSCO and Gfastats. For downstream scaffolding, we employed RagTag using the high-quality genome of the wheat cultivar ‘Attraktion’ as the reference assembly.

However, I’m facing challenges with my reference-guided scaffolding project using RagTag and could use your insights. Madsen and Pritchett has nearly identical BUSCO scores (C: 99.7% [S: 2.0%, D: 97.7%], F: 0.2%, M: 0.1%, n: 4896, E: 0.4%). Madsen has 4424 contigs, and Pritchett has 2754, both assembled with Hifiasm. The genomes are about 14Gb big.

I successfully scaffolded Madsen using RagTag, but Pritchett consistently fails with the same SLURM script and pipeline. For Pritchett, the job runs for ~7 days, reports as “completed,” but produces no ragtag.scaffold.fasta. The ragtag.scaffold.asm.paf.log is not complete and gets terminated at same point everytime. Error says:


Traceback (most recent call last):
File “/home/…/bin/ragtag_scaffold.py”, line 577, in <module>
main()
File “/home/…/bin/ragtag_scaffold.py”, line 420, in main
al.run_aligner()
File “/home/…/BPN/lib/python3.10/site-packages/ragtag_utilities/Aligner.py”, line 128, in run_aligner
run_oe(self.compile_command(), self.out_file, self.out_log)
File “/home/…/lib/python3.10/site-packages/ragtag_utilities/utilities.py”, line 73, in run_oe
raise RuntimeError(“Failed : minimap2 -x asm5 -t 24 … > ragtag.scaffold.asm.paf 2> ragtag.scaffold.asm.paf.log”)

The Slurm Job I gave was:

#SBATCH --partition=abc
#SBATCH --cpus-per-task=24
#SBATCH --mem=1500000
#SBATCH --time=14-00:00:00
ragtag.py scaffold “$REF” “$QUERY” -o “$OUT” -t 24 -u Troubleshooting Steps:

1. Ran minimap2 manually on Pritchett’s reference (attraktion.fasta) and query (pt2_busco.fa); it generated a 442 MB .paf file in ~21 hours. Came to know that RagTag does not use pregenerated paf file.
2. Tested RagTag on a Pritchett subset (~409 Mbp, 10 contigs); it succeeded in ~10 hours, placing 9/10 sequences (~402 Mbp).
3. Someone suggested that with large genomes, minimap2 might struggle due to multi-indexing issues that can slow things down or cause memory overload. They recommended indexing the reference with minimap2 using -I 20G (which should be suitable for wheat) and then passing the prebuilt .mmi index directly to RagTag as if it were a FASTA file. I followed this approach — created the .mmi file and used it in RagTag — but unfortunately, it still didn’t resolve the issue with Pritchett.
4. Used SLURM settings: bigmem, 24 CPUs, 1.5 TB memory, 14-day limit, BPN environment (RagTag v2.1.0)