miRDeep2 "no file 'precursors.fa_stack' found" error.

Question

[Yashrajsinh Jadeja]

Hello everyone!

I was trying to run miRDeep2 for a project on novel miRNA discovery from transcriptomic data, however, this error pops up when I try to run the "mirdeep2.pl" script with appropriate parameters.

The error:

#excising precursors
#excising precursors
started: 15:50:33
excise_precursors_iterative_final.pl /home/lab7/Project/newgenome.fa mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/reads_vs_refdb.arf_parsed.arf mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/precursors.fa mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/precursors.coords -1
No file mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/precursors.fa_stack found

Am I messing something up with my parameters? My command is as follows:

date && time miRDeep2.pl ~/Project/reads_collapsed.fa ~/Project/newgenome.fa ~/Project/reads_vs_refdb.arf ~/Project/new_human_mature.fasta none ~/Project/new_human_stem.fasta -t Human -g -1 hsa -v

Note: The " -g -1 " parameter was added after I tried running the script without it, because I saw it on an online forum. Either way, the script with default "-g" parameter or "-g -1" fails to run.

Also, here is the entire dialogue on the terminal from start to end:

(Syntax: newgenome.fa - reference genome file

reads_collapsed.fa - deep sequencing reads collapsed by mapper.pl

reads_vs_refdb.arf - mapped file created by mapper.pl

new_human_mature.fasta - FASTA file containing mature miRNAs from mirBase

new_human_stem.fasta - FASTA file containing precursor/stem sequences from mirBase)

date && time miRDeep2.pl ~/Project/reads_collapsed.fa ~/Project/newgenome.fa ~/Project/reads_vs_refdb.arf ~/Project/new_human_mature.fasta none ~/Project/new_human_stem.fasta -t Human -g -1 hsa -v
Wed Aug 28 15:49:37 EST 2019


    #####################################
    #                                   #
    # miRDeep2.0.1.2                    #
    #                                   #
    # last change: 22/01/2019           #
    #                                   #
    #####################################

    miRDeep2 started at 15:49:37


    #Starting miRDeep2
    #Starting miRDeep2
    /home/lab7/Project/Softwares/mirdeep2-master/bin/miRDeep2.pl /home/lab7/Project/reads_collapsed.fa /home/lab7/Project/newgenome.fa /home/lab7/Project/reads_vs_refdb.arf /home/lab7/Project/new_human_mature.fasta none /home/lab7/Project/new_human_stem.fasta -t Human -g -1 hsa -v

    miRDeep2 started at 15:49:37


    mkdir mirdeep_runs/run_28_08_2019_t_15_49_37

    Use of uninitialized value $tmps in split at /home/lab7/Project/Softwares/mirdeep2-master/bin/miRDeep2.pl line 350, <IN> line 1.
    Use of uninitialized value in pattern match (m//) at /home/lab7/Project/Softwares/mirdeep2-master/bin/miRDeep2.pl line 354, <IN> line 1.
    Use of uninitialized value $tmps in split at /home/lab7/Project/Softwares/mirdeep2-master/bin/miRDeep2.pl line 377.
    #testing input files
    #testing input files
    started: 15:50:33
    sanity_check_mature_ref.pl /home/lab7/Project/new_human_mature.fasta


    ended: 15:50:33
    total:0h:0m:0s

    sanity_check_reads_ready_file.pl /home/lab7/Project/reads_collapsed.fa

    started: 15:50:33

    ended: 15:50:33
    total:0h:0m:0s

    started: 15:50:33
    sanity_check_genome.pl /home/lab7/Project/newgenome.fa


    ended: 15:50:33
    total:0h:0m:0s

    started: 15:50:33
    sanity_check_mapping_file.pl /home/lab7/Project/reads_vs_refdb.arf


    ended: 15:50:33
    total:0h:0m:0s

    started: 15:50:33
    sanity_check_mature_ref.pl /home/lab7/Project/new_human_stem.fasta


    ended: 15:50:33
    total:0h:0m:0s

    started: 15:50:33
    Quantitation of expressed miRNAs in data


    #Quantitation of known miRNAs in data
    quantifier.pl -p /home/lab7/Project/new_human_stem.fasta -m /home/lab7/Project/new_human_mature.fasta  -r /home/lab7/Project/reads_collapsed.fa  -t Human -y 28_08_2019_t_15_49_37 -k  

    ended: 15:50:33
    total:0h:0m:0s

    started: 15:50:33
    rna2dna.pl /home/lab7/Project/new_human_mature.fasta > mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/new_human_mature.fasta

    rna2dna.pl /home/lab7/Project/new_human_stem.fasta > mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/new_human_stem.fasta


    ended: 15:50:33
    total:0h:0m:0s

    #parsing genome mappings
    #parsing genome mappings
    parse_mappings.pl /home/lab7/Project/reads_vs_refdb.arf -a 0 -b 18 -c 25 -i 5 > mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/reads_vs_refdb.arf_parsed.arf

    started: 15:50:33

    ended: 15:50:33
    total:0h:0m:0s

    #excising precursors
    #excising precursors
    started: 15:50:33
    excise_precursors_iterative_final.pl /home/lab7/Project/newgenome.fa mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/reads_vs_refdb.arf_parsed.arf mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/precursors.fa mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/precursors.coords -1
    No file mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/precursors.fa_stack found

    real    0m57.660s
    user    0m13.274s
    sys 0m3.129s

Answer 1

[Rituriya]

Re-run by replacing the last portion of your command: '-t Human -g -1 hsa -v' with '-t hsa'.

[Yashrajsinh Jadeja]

Did that. Still the same error. Is there any other workaround for this?

Answer 2

[Ziliang Luo]

before "precursors.fa_stack found" error came out and killed the program. it reported excision of precursor at certain iteration. e.g

#excising precursors
...
46  217072
47  210636
48  204216
49  198362
No file mirdeep_runs/run_28_08_2019_t_15_49_37/tmp/precursors.fa_stack found

What I did is set the -g option just bigger than where it stopped. For example, -g 200000. Then it can continue the precursor excision. I also tried -g -1 which worked as well. I guess it's because something wrong with the number of the precursor excision.