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<div style="font-size: 14px; font-family: Calibri, sans-serif;">Hello,</div>
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<div style="font-size: 14px; font-family: Calibri, sans-serif;">I’ve been having some trouble getting MISO to work with my RNA-seq data. I’m relatively new to RNA-seq analysis and I’m probably just missing something relatively simple. I installed MISO using
the latest version from the GitHub repository and Python’s pip install misopy. I checked that all the packages were installed correctly and was able to Test MISO and it seemed to be working correctly. I was also able to compute the insert length distribution
for my BAM file. </div>
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<div style="font-size: 14px; font-family: Calibri, sans-serif;">When I try to run MISO with my BAM file I get the following errors:</div>
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<p style="font-size: 11px; font-family: Menlo; margin: 0px;">miso --run /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Reference_genome/indexed/ /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam --output-dir Mut_1/ --read-len 125 --paired-end
167 33</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">MISO (Mixture of Isoforms model)</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Probabilistic analysis of RNA-Seq data for detecting differential isoforms</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Use --help argument to view options.</p>
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<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Using MISO settings file: /Library/Python/2.7/site-packages/misopy/settings/miso_settings.txt</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Computing Psi values...</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - GFF index: /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Reference_genome/indexed</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - BAM: /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Read length: 125</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Output directory: /Users/bowmanlab/Mut_1</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Checking your GFF annotation and BAM for mismatches...</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Checking if BAM has mixed read lengths...</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Found reads of length 125 in BAM.</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Mapping genes to their indexed GFF representation, using /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Reference_genome/indexed</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Searching for /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Reference_genome/indexed/genes_to_filenames.shelve..</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Found shelved file.</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Preparing to run 4 batches of jobs...</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Running batch of 8434 genes..</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Executing: python /Library/Python/2.7/site-packages/misopy/run_miso.py --compute-genes-from-file "/Users/bowmanlab/Mut_1/batch-genes/batch-0_genes.txt" /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam
/Users/bowmanlab/Mut_1 --read-len 125 --paired-end 167.0 33.0 --settings-filename /Library/Python/2.7/site-packages/misopy/settings/miso_settings.txt</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Submitted thread batch-0</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Running batch of 8434 genes..</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Executing: python /Library/Python/2.7/site-packages/misopy/run_miso.py --compute-genes-from-file "/Users/bowmanlab/Mut_1/batch-genes/batch-1_genes.txt" /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam
/Users/bowmanlab/Mut_1 --read-len 125 --paired-end 167.0 33.0 --settings-filename /Library/Python/2.7/site-packages/misopy/settings/miso_settings.txt</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Submitted thread batch-1</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/Library/Python/2.7/site-packages/misopy/miso_sampler.py:306: RuntimeWarning: Long inserted alignment at pysplicing/src/solve.c:291</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> stop_cond)</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Running batch of 8434 genes..</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Executing: python /Library/Python/2.7/site-packages/misopy/run_miso.py --compute-genes-from-file "/Users/bowmanlab/Mut_1/batch-genes/batch-2_genes.txt" /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam
/Users/bowmanlab/Mut_1 --read-len 125 --paired-end 167.0 33.0 --settings-filename /Library/Python/2.7/site-packages/misopy/settings/miso_settings.txt</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Submitted thread batch-2</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/Library/Python/2.7/site-packages/misopy/miso_sampler.py:306: RuntimeWarning: Long deleted alignment at pysplicing/src/solve.c:281</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> stop_cond)</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Running batch of 8435 genes..</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Executing: python /Library/Python/2.7/site-packages/misopy/run_miso.py --compute-genes-from-file "/Users/bowmanlab/Mut_1/batch-genes/batch-3_genes.txt" /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam
/Users/bowmanlab/Mut_1 --read-len 125 --paired-end 167.0 33.0 --settings-filename /Library/Python/2.7/site-packages/misopy/settings/miso_settings.txt</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> - Submitted thread batch-3</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/Library/Python/2.7/site-packages/misopy/miso_sampler.py:306: RuntimeWarning: Long deleted alignment at pysplicing/src/solve.c:281</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> stop_cond)</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">Waiting on 4 threads...</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/Library/Python/2.7/site-packages/misopy/miso_sampler.py:306: RuntimeWarning: Long inserted alignment at pysplicing/src/solve.c:291</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> stop_cond)</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/Library/Python/2.7/site-packages/misopy/miso_sampler.py:306: RuntimeWarning: Long deleted alignment at pysplicing/src/solve.c:281</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> stop_cond)</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/Library/Python/2.7/site-packages/misopy/miso_sampler.py:306: RuntimeWarning: Long inserted alignment at pysplicing/src/solve.c:291</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> stop_cond)</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/Library/Python/2.7/site-packages/misopy/miso_sampler.py:306: RuntimeWarning: Long deleted alignment at pysplicing/src/solve.c:281</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> stop_cond)</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/Library/Python/2.7/site-packages/misopy/miso_sampler.py:306: RuntimeWarning: Long inserted alignment at pysplicing/src/solve.c:291</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;"> stop_cond)</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">python(23371,0x7fff736a4310) malloc: *** error for object 0x7faf43321c30: incorrect checksum for freed object - object was probably modified after being freed.</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">*** set a breakpoint in malloc_error_break to debug</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/bin/sh: line 1: 23371 Abort trap: 6 python /Library/Python/2.7/site-packages/misopy/run_miso.py --compute-genes-from-file "/Users/bowmanlab/Mut_1/batch-genes/batch-3_genes.txt" /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam
/Users/bowmanlab/Mut_1 --read-len 125 --paired-end 167.0 33.0 --settings-filename /Library/Python/2.7/site-packages/misopy/settings/miso_settings.txt >> "/Users/bowmanlab/Mut_1/batch-logs/batch-3-03-30-15_09:23:29.log"</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/bin/sh: line 1: 23369 Segmentation fault: 11 python /Library/Python/2.7/site-packages/misopy/run_miso.py --compute-genes-from-file "/Users/bowmanlab/Mut_1/batch-genes/batch-2_genes.txt" /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam
/Users/bowmanlab/Mut_1 --read-len 125 --paired-end 167.0 33.0 --settings-filename /Library/Python/2.7/site-packages/misopy/settings/miso_settings.txt >> "/Users/bowmanlab/Mut_1/batch-logs/batch-2-03-30-15_09:23:28.log"</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">python(23366,0x7fff736a4310) malloc: *** error for object 0x7fd52157b420: incorrect checksum for freed object - object was probably modified after being freed.</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">*** set a breakpoint in malloc_error_break to debug</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/bin/sh: line 1: 23366 Abort trap: 6 python /Library/Python/2.7/site-packages/misopy/run_miso.py --compute-genes-from-file "/Users/bowmanlab/Mut_1/batch-genes/batch-1_genes.txt" /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam
/Users/bowmanlab/Mut_1 --read-len 125 --paired-end 167.0 33.0 --settings-filename /Library/Python/2.7/site-packages/misopy/settings/miso_settings.txt >> "/Users/bowmanlab/Mut_1/batch-logs/batch-1-03-30-15_09:23:28.log"</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">python(23364,0x7fff736a4310) malloc: *** error for object 0x7ff814359150: incorrect checksum for freed object - object was probably modified after being freed.</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">*** set a breakpoint in malloc_error_break to debug</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">/bin/sh: line 1: 23364 Abort trap: 6 python /Library/Python/2.7/site-packages/misopy/run_miso.py --compute-genes-from-file "/Users/bowmanlab/Mut_1/batch-genes/batch-0_genes.txt" /Volumes/Sherlock/RNA-Seq_analysis_RCC_Jan_2015/Mut_1/Mut_1_sorted.bam
/Users/bowmanlab/Mut_1 --read-len 125 --paired-end 167.0 33.0 --settings-filename /Library/Python/2.7/site-packages/misopy/settings/miso_settings.txt >> "/Users/bowmanlab/Mut_1/batch-logs/batch-0-03-30-15_09:23:27.log"</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">03/30/2015 09:28:15 AM - miso_main - WARNING - Thread batch-0 might have failed...</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">03/30/2015 09:28:15 AM - miso_main - WARNING - Thread batch-1 might have failed...</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">03/30/2015 09:28:15 AM - miso_main - WARNING - Thread batch-2 might have failed...</p>
<p style="font-size: 11px; font-family: Menlo; margin: 0px;">03/30/2015 09:28:15 AM - miso_main - WARNING - Thread batch-3 might have failed…</p>
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<p style="margin: 0px;">I used the STAR aligner to create my SAM files and converted them to sorted indexed BAM files with samtools (outside of Miso). I’m working with zebrafish and downloaded my gtf file from Ensembl (Danio_rerio.Zv9.78.gtf) and then used
the Galaxy (GTF-to-GFF3) to convert from gtf to a gff3 file (I used Galaxy because I had some trouble with the gtf2gff3.pl script). I wanted to include a small version of my BAM file but I wasn’t sure what the best way to do this would be.</p>
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<p style="margin: 0px;">Any suggestions would be greatly appreciated!</p>
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<p style="margin: 0px;">Thank you,</p>
<p style="margin: 0px;">Rosannah </p>
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