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  • Running MISO with paired-end reads

    Hi,

    I am running MISO with paired-end reads but it comes up the error message. Just wondering if anyone had the same problem? Tia!

    python test_miso.py
    Testing conversion of SAM to BAM...
    Executing: python ./Projects/tools/MISO/sam_to_bam.py --convert ./Projects/tools/MISO/test-data/sam-data/c2c12.Atp2b1.sam ./Projects/tools/MISO/test-output/sam-output
    Converting SAM to BAM...
    - Executing: samtools view -Sbh ./Projects/tools/MISO/test-data/sam-data/c2c12.Atp2b1.sam > ./Projects/tools/MISO/test-output/sam-output/c2c12.Atp2b1.bam
    [samopen] SAM header is present: 35 sequences.
    Sorting BAM file...
    - Executing: samtools sort ./Projects/tools/MISO/test-output/sam-output/c2c12.Atp2b1.bam ./Projects/tools/MISO/test-output/sam-output/c2c12.Atp2b1.sorted
    Indexing BAM...
    - Executing: samtools index ./Projects/tools/MISO/test-output/sam-output/c2c12.Atp2b1.sorted.bam
    Conversion took 0.00 minutes.
    .Testing single-end SE event interface...
    Executing: python ./Projects/tools/MISO/run_events_analysis.py --compute-events-psi se-sample ./Projects/tools/MISO/test-data/se-counts/se_test.counts --output-dir ./Projects/tools/MISO/test-output/SE-output --read-len 35 --overhang-len 4 --event-type SE
    Loading settings from: ./Projects/tools/MISO/settings/miso_settings.txt
    Settings:
    short_queue_name quick
    long_queue_name long
    cluster_command qsub
    filter_results True
    min_event_reads 20
    lag 10
    burn_in 500
    num_iters 5000
    Loading settings file from: ./Projects/tools/MISO/settings/miso_settings.txt
    Computing Psi for events...
    Filter events?
    - yes
    Computing Psi for events of type SE
    - samples used: ['se-sample']
    Processing sample: label=se-sample, filename=./Projects/tools/MISO/test-data/se-counts/se_test.counts
    Filtering events...
    Filtering SE events...
    Running on a total of 2 events.
    Serializing a total of 2 events by Pickle.
    Executing: python ./Projects/tools/MISO/run_miso.py --compute-two-iso-psi ./Projects/tools/MISO/test-output/SE-output/SE/se-sample/pickle/se-sample.pickle ./Projects/tools/MISO/test-output/SE-output/SE/se-sample --event-type SE --read-len 35 --overhang-len 4
    Loading settings from: ./Projects/tools/MISO/settings/miso_settings.txt
    Settings:
    short_queue_name quick
    long_queue_name long
    cluster_command qsub
    filter_results True
    min_event_reads 20
    lag 10
    burn_in 500
    num_iters 5000
    Running MISO on: ./Projects/tools/MISO/test-output/SE-output/SE/se-sample/pickle/se-sample.pickle
    - Output directory: ./Projects/tools/MISO/test-output/SE-output/SE/se-sample/
    Called on: ./Projects/tools/MISO/test-output/SE-output/SE/se-sample/pickle/se-sample.pickle
    Loading from Pickle file took 0.00 seconds.
    Parsing of events to genes took 0.00 seconds.
    Running sampler on a two-isoform event...
    - Gene label: chr15:79605327:79604789:-;chr15:79596283:79596217:-;chr15:79595451:79595246:- gene_id: chr15:79605327:79604789:-;chr15:79596283:79596217:-;chr15:79595451:79595246:-
    isoforms: [Isoform(gene = chr15:79605327:79604789:-;chr15:79596283:79596217:-;chr15:79595451:79595246:-, g_start = 0, g_end = 811, len = 812,
    parts = ['A', 'B', 'C']), Isoform(gene = chr15:79605327:79604789:-;chr15:79596283:79596217:-;chr15:79595451:79595246:-, g_start = 0, g_end = 811, len = 745,
    parts = ['A', 'C'])]
    - NI, NE, NB: 4, 42, 281
    Using default sampler parameters.
    Indexing by chromosome...
    Output filename ./Projects/tools/MISO/test-output/SE-output/SE/se-sample/chr15/chr15:79605327:79604789:-;chr15:79596283:79596217:-;chr15:79595451:79595246:-.miso exists, not running MISO.
    Parsing of events to genes took 0.00 seconds.
    Running sampler on a two-isoform event...
    - Gene label: chr1:192752368:192752637:+;chr1:192756746:192757071:+;chr1:192761323:192762671:+ gene_id: chr1:192752368:192752637:+;chr1:192756746:192757071:+;chr1:192761323:192762671:+
    isoforms: [Isoform(gene = chr1:192752368:192752637:+;chr1:192756746:192757071:+;chr1:192761323:192762671:+, g_start = 0, g_end = 1944, len = 1945,
    parts = ['A', 'B', 'C']), Isoform(gene = chr1:192752368:192752637:+;chr1:192756746:192757071:+;chr1:192761323:192762671:+, g_start = 0, g_end = 1944, len = 1619,
    parts = ['A', 'C'])]
    - NI, NE, NB: 96, 5, 148
    Using default sampler parameters.
    Indexing by chromosome...
    Output filename ./Projects/tools/MISO/test-output/SE-output/SE/se-sample/chr1/chr1:192752368:192752637:+;chr1:192756746:192757071:+;chr1:192761323:192762671:+.miso exists, not running MISO.
    Submitted total of 2 events.
    Loading events from: ./Projects/tools/MISO/test-output/SE-output/SE/se-sample/
    Writing summary to: ./Projects/tools/MISO/test-output/SE-output/SE/se-sample/summary/events_summary.miso
    /Library/Frameworks/Python.framework/Versions/2.6/lib/python2.6/site-packages/numpy/lib/npyio.py:1164: DeprecationWarning: The use of `skiprows` is deprecated.
    Please use `skip_header` instead.
    DeprecationWarning)
    - Summarized a total of 2 events.
    .Testing gene-level Psi...
    Testing GFF indexing of: ./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1.mm9.gff
    Executing: python ./Projects/tools/MISO/index_gff.py --index ./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1.mm9.gff ./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1/indexed
    Indexing GFF...
    ./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1.mm9.gff appears to already be indexed. Aborting.
    Executing: python ./Projects/tools/MISO/run_events_analysis.py --compute-genes-psi ./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1/indexed ./Projects/tools/MISO/test-output/sam-output/c2c12.Atp2b1.sorted.bam --output-dir ./Projects/tools/MISO/test-output/gene-psi-output --read-len 35 --paired-end 250 30
    Loading settings from: ./Projects/tools/MISO/settings/miso_settings.txt
    Settings:
    short_queue_name quick
    long_queue_name long
    cluster_command qsub
    filter_results True
    min_event_reads 20
    lag 10
    burn_in 500
    num_iters 5000
    Loading settings file from: ./Projects/tools/MISO/settings/miso_settings.txt
    Mapping genes to their indexed GFF representation, using ./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1/indexed
    Skipping: ./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1/indexed/genes_to_filenames.shelve
    Computing gene-level Psi for 1 genes...
    - GFF index: ./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1/indexed
    - BAM: ./Projects/tools/MISO/test-output/sam-output/c2c12.Atp2b1.sorted.bam
    - Read length: 35
    - Output directory: ./Projects/tools/MISO/test-output/gene-psi-output
    - Executing: python ./Projects/tools/MISO/run_miso.py --compute-gene-psi "ENSMUSG00000019943" "./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1/indexed/chr10/ENSMUSG00000019943.pickle" ./Projects/tools/MISO/test-output/sam-output/c2c12.Atp2b1.sorted.bam ./Projects/tools/MISO/test-output/gene-psi-output --read-len 35 --paired-end 250.0 30.0 --settings-filename ./Projects/tools/MISO/settings/miso_settings.txt
    Loading settings from: ./Projects/tools/MISO/settings/miso_settings.txt
    Settings:
    short_queue_name quick
    long_queue_name long
    cluster_command qsub
    filter_results True
    min_event_reads 20
    lag 10
    burn_in 500
    num_iters 5000
    Computing Psi for 1 genes...
    - ENSMUSG00000019943
    - GFF filename: ./Projects/tools/MISO/gff-events/mm9/genes/Atp2b1/indexed/chr10/ENSMUSG00000019943.pickle
    - BAM: ./Projects/tools/MISO/test-output/sam-output/c2c12.Atp2b1.sorted.bam
    - Outputting to: ./Projects/tools/MISO/test-output/gene-psi-output
    - Paired-end mode: (250.0, 30.0)
    Loading BAM filename from: ./Projects/tools/MISO/test-output/sam-output/c2c12.Atp2b1.sorted.bam
    Loading took 0.01 seconds
    Aligning reads gene...
    Traceback (most recent call last):
    File "./Projects/tools/MISO/run_miso.py", line 621, in <module>
    main()
    File "./Projects/tools/MISO/run_miso.py", line 601, in main
    event_type=options.event_type)
    File "./Projects/tools/MISO/run_miso.py", line 382, in compute_gene_psi
    paired_end=paired_end)
    File "./Projects/tools/MISO/sam_utils.py", line 290, in sam_reads_to_isoforms
    reads, num_reads = sam_pe_reads_to_isoforms(samfile, gene)
    File "./Projects/tools/MISO/sam_utils.py", line 236, in sam_pe_reads_to_isoforms
    paired_reads = pair_sam_reads(samfile)
    File "./Projects/tools/MISO/sam_utils.py", line 189, in pair_sam_reads
    unpaired_reads[read_name] = read
    UnboundLocalError: local variable 'read_name' referenced before assignment

    Loading settings from: ./Projects/tools/MISO/settings/miso_settings.txt
    Settings:
    short_queue_name quick
    long_queue_name long
    cluster_command qsub
    filter_results True
    min_event_reads 20
    lag 10
    burn_in 500
    num_iters 5000
    .
    ----------------------------------------------------------------------
    Ran 3 tests in 4.803s

    OK

  • #2
    Hi unidodo,

    Just noticed this post now. To answer your question, recent versions of MISO fix this bug. I recommend using the version from the "dev" branch in the GitHub repository for MISO, or download the packaged .zip version of the software from our website (see http://genes.mit.edu/burgelab/miso/software.html).

    Best, Yarden

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