souporcell: Error: bcftools sort returned non-zero exit status 255

I’m getting a strange error related to bcftools sort, any ideas what this could be due to?

Thank you.

#! /bin/bash

set -euxo pipefail
ml load singularity

demuxafy_dir="/path/to/demuxafy"
data_dir="/path/to/data"

singularity exec \
-B "$demuxafy_dir" \
-B "$data_dir" \
$demuxafy_dir/Demuxafy.sif \
souporcell_pipeline.py \
-t 8 \
-f $demuxafy_dir/refdata-gex-GRCh38-2020-A/fasta/genome.fa \
-i $data_dir/souporcell/test/possorted_genome_bam_subsampled.bam \
-b $data_dir/souporcell/test/subsampled_barcodes.tsv \
-o $data_dir/souporcell/test/demuxafy_out/SAM24434291 \
-k 3 \

running freebayes
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r KI270442.1:0-392061
freebayes -f/path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr11:1721089-135086622
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr14:6463670-107043718
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr18:11301817-80373285
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r KI270727.1:0-448248
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r KI270728.1:0-1872759
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r KI270729.1:0-280839
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr10:0-133797422
.
.
.
.
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr8:0-117819804
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr13:0-114364328
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa  -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr17:0-83257441
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa  -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chrY:0-57227415
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa  -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr6:0-60502916
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa  -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr4:0-44786890
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa  -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr18:0-11301817
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa  -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr20:0-17586227
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa  -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr11:0-1721089
freebayes -f /path/to/demuxafy/refdata-gex-GRCh38-2020-A/fasta/genome.fa  -iXu -C 2 -q 20 -n 3 -E 1 -m 30 --min-coverage 20 --pooled-continuous --skip-coverage 100000 -r chr14:0-6463670

merging vcfs
Checking the headers and starting positions of 35 files
Traceback (most recent call last):
  File "/opt/souporcell/souporcell_pipeline.py", line 586, in <module>
    final_vcf = freebayes(args, bam, fasta)
  File "/opt/souporcell/souporcell_pipeline.py", line 474, in freebayes
    subprocess.check_call(['bcftools', 'sort', args.out_dir + "/souporcell_merged_vcf.vcf"], stdout = vcfout, stderr = vcferr)
  File "/opt/conda/envs/py36/lib/python3.6/subprocess.py", line 311, in check_call
    raise CalledProcessError(retcode, cmd)
subprocess.CalledProcessError: Command '['bcftools', 'sort', 'path/to/souporcell/test/demuxafy_out/SAM24434291/souporcell_merged_vcf.vcf']' returned non-zero exit status 255.