demultiplex failure

[Salvobioinfo]

Running command line: python guideseq.py all -m manifest_1.yaml
after demultiplexing begins I receive: IOError: [Errno 24] Too many open files: ...

Upon inspection of the demultiplexed output directory, there is one file for every barcode.
I saw other issue like mine, but others solutions don't work for me.
My manifest file is:

reference_genome: /solid/Reference/GRCh38/GRCh38.d1.vd1.fa
output_folder: /tank/home/salvo/KATIA/OUTPUT

bwa: /solid/Programs/bwa-0.7.17/bwa
bedtools: /solid/Programs/bedtools2/bin/bedtools

demultiplex_min_reads: 1000

undemultiplexed:
    forward: /tank/USB/Undetermined_S0_L001_R1_001.fastq.gz
    reverse: /tank/USB/Undetermined_S0_L001_R2_001.fastq.gz
    index1: /tank/USB/Undetermined_S0_L001_I1_001.fastq.gz
    index2: /tank/USB/Undetermined_S0_L001_I2_001.fastq.gz

samples:
    control_a
        target:  
        barcode1: TTCTGCCT
        barcode2: CTCTCTAT
        description: Hek-MECP2_CTRLpos


    Hek-MECP2pos:
        target: GATTTTGACTTCACGGTAACTGG
        barcode1: TCGCCTTA
        barcode2: TAGATCGC
        description: Hek-MECP2pos


    control_b:
        target:  
        barcode1: GCTCAGGA
        barcode2: CTCTCTAT
        description: Hek-Hek-MECP2_CTRLneg


    Hek-MECP2neg:
        target: GATTTTGACTTCACGGTAACTGG
        barcode1: CTAGTACG
        barcode2: TAGATCGC
        description: Hek-MECP2neg

Any idea what is going on here?
Salvatore

[Salvobioinfo]

I modified manifest file in:

reference_genome: /solid/Reference/GRCh38/GRCh38.d1.vd1.fa
output_folder: /tank/home/salvo/KATIA/OUTPUT

bwa: bwa
bedtools: bedtools

demultiplex_min_reads: 100000

undemultiplexed:
    forward: /tank/USB/Undetermined_S0_L001_R1_001.fastq.gz
    reverse: /tank/USB/Undetermined_S0_L001_R2_001.fastq.gz
    index1: /tank/USB/Undetermined_S0_L001_I1_001.fastq.gz
    index2: /tank/USB/Undetermined_S0_L001_I2_001.fastq.gz

samples:
    control:
        target:  
        barcode1: GCTCAGGA
        barcode2: CTCTCTAT
        description: Control


    HekMECP2neg:
        target: GATTTTGACTTCACGGTAACTGG
        barcode1: CTAGTACG
        barcode2: TAGATCGC
        description: Hek-MECP2ne

But I obtained always same results.

[staciawyman]

If you increase the "demultiplex_min_reads" even more? I set mine to 50000.

[Zethson]

@martinaryee @vedtopkar It's unfortunately clearly broken. It would be highly appreciated if you could take a look at the demultiplexing step again.

I have the same errors as @Salvobioinfo .

[Salvobioinfo]

@Zethson In the first our attempt the issue was due to the sequencing problems, Indeed I tried to demultiplex our data with different tools and only one sample has a good reads amount.
Did you try it ?
To validate if these problem are linked with our seqeuncing step, I ran guideseq with good samples obtained from other people, and tool worked well.