minimap error using sc_long_pipeline.py

Hi @LuyiTian , Could you please comment on my error below?

Running code:

for i in test; do /FLAMES/python/sc_long_pipeline.py --gff3 hg38v99.Cellranger.genes.gtf --infq $i.demultiplexed.fq.gz --outdir FLAMES_Output/$i --genomefa hg38v99.Cellranger.genome.fa --config_file /FLAMES/config_sclr_nanopore_default.json --minimap2_dir /Software/anaconda_py2/bin/  >$i.log 2>&1 & done

Error:

Use config file: config_sclr_nanopore_default.json

Parameters in configuration file:

comment : this is the default config for nanopore single cell long read data using 10X RNA-seq kit. use splice annotation in alignment.

global_parameters

    has_UMI : True

    generate_raw_isoform : False

isoform_parameters

    Min_sup_pct : 0.2

    MAX_SPLICE_MATCH_DIST : 10

    random_seed : 666666

    Min_cnt_pct : 0.001

    MAX_DIST : 10

    Min_sup_cnt : 5

    MAX_TS_DIST : 120

    Max_site_per_splice : 3

    strand_specific : -1

    remove_incomp_reads : 4

    min_fl_exon_len : 40

pipeline_parameters

    do_transcript_quantification : True

    do_read_realignment : True

    do_genome_alignment : True

    do_isoform_identification : True

transcript_counting

    min_tr_coverage : 0.4

    min_read_coverage : 0.4

realign_parameters

    use_annotation : True

alignment_parameters

    no_flank : False

    use_junctions : True

output directory not exist, create one:

FLAMES_Output/test

Input parameters:

    gene annotation: hg38v99.Cellranger.genes.gtf

    genome fasta: hg38v99.Cellranger.genome.fa

    input fastq: test.demultiplexed.fq.gz

    output directory: FLAMES_Output/test

    directory contains minimap2: /Software/anaconda_py2/bin/

### align reads to genome using minimap2 2021-01-30 12:48:05

Traceback (most recent call last):

  File "/FLAMES/python/sc_long_pipeline.py", line 213, in <module>

    sc_long_pipeline(args)

  File "/FLAMES/python/sc_long_pipeline.py", line 159, in sc_long_pipeline

    minimap2_align(args.minimap2_dir, args.genomefa, args.infq, tmp_bam, no_flank=config_dict["alignment_parameters"]["no_flank"], bed12_junc=tmp_bed if config_dict["alignment_parameters"]["use_junctions"] else None)

  File "/FLAMES/python/minimap2_align.py", line 37, in minimap2_align

    print subprocess.check_output([align_cmd], shell=True, stderr=subprocess.STDOUT)

  File "/Software/anaconda_py2/lib/python2.7/subprocess.py", line 223, in check_output

    raise CalledProcessError(retcode, cmd, output=output)

subprocess.CalledProcessError: Command '['/Software/anaconda_py2/bin/minimap2 -ax splice -t 12 --junc-bed FLAMES_Output/test/tmp.splice_anno.bed12 --junc-bonus 1  -k14 --secondary=no hg38v99.Cellranger.genome.fa test.demultiplexed.fq.gz | samtools view -bS -@ 4 -m 2G -o FLAMES_Output/test/tmp.align.bam -  ']' returned non-zero exit status 1

LuyiTian / FLAMES

minimap error using sc_long_pipeline.py #13