<div dir="ltr">Dear All:
<div><br></div><div style>I am using MISO to test for differential exon usage between a control and a treatment group. I got an error <b>when computing the insert length distribution using pe_utils.py --compute-insert-len</b>. I list the steps I used below:</div>
<div style><br></div><div style>1. sort the BAM file from TopHat (by coordinate):</div><div style>samtools sort control.bam control_sorted<br></div><div style><br></div><div style>2. index the BAM file:</div><div style>samtools index control_sorted.bam control_sorted.bai<br>
</div><div style><br></div><div style>3. run pe_utils.py:</div><div style>python pe_utils.py --compute-insert-len controlam /directories/exons/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff --output-dir /directories/insert-dist/<br>
</div><div style><br></div><div style>After the command above, I got the<b> error message</b>:</div><div style><br></div><div style><div>Preparing to call bedtools 'tagBam'</div><div>tagBam -i control.bam -files /directories/exons/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff -labels gff -intervals -f 1 | samtools view - -h | egrep '^@|:gff:' | samtools view - -Shb -o /directories/insert-dist/bam2gff_Homo_sapiens.GRCh37.65.min_1000.const_exons.gff/control.bam</div>
<div>[samopen] SAM header is present: 25 sequences.</div><div>[sam_read1] reference 'ID:TopHat CL:/informatics/tools/Linux-AS5/bin/tophat -o Lane3 -g 1 --coverage-search --microexon -r 100 --phred64-quals --library-type fr-unstranded -p 4 -G gene_models/Homo_sapiens.GRCh37.72_norm.gtf --transcriptome-index=gene_models/transcripts /directories/Genomes/NCBI_Jul-09-2012/Human/bowtie/human_ref_genome Lane3_1.fq.gz Lane3_2.fq.gz VN:1.4.1</div>
<div>' is recognized as '*'.</div><div>[main_samview] truncated file.</div><div>Traceback (most recent call last):</div><div> File "/pe_utils.py", line 520, in <module></div><div> main()</div>
<div> File "pe_utils.py", line 517, in main</div><div> sd_max=sd_max)</div><div> File "pe_utils.py", line 271, in compute_insert_len</div><div> output_dir)</div><div> File "exon_utils.py", line 185, in map_bam2gff</div>
<div> raise Exception, "Error: tagBam call failed."</div><div>Exception: Error: tagBam call failed.</div><div><br></div><div style>I used <b>Homo_sapiens.GRCh37.72_norm.gtf</b> from Ensembl as the annotation file when preparing my data, but downloaded </div>
<ul class="" style="color:rgb(0,0,0);font-family:ff-meta-web-pro-1,ff-meta-web-pro-2,Arial,'Helvetica Neue',sans-serif;line-height:24px"><li><a class="" href="http://genes.mit.edu/burgelab/miso/annotations/ver2/miso_annotations_hg19_v2.zip" style="color:rgb(68,68,68);text-decoration:none;border-bottom-width:1px;border-bottom-style:solid;border-bottom-color:rgb(204,204,204)">Human genome (hg19) alternative events v2.0</a></li>
</ul><div style><font color="#000000" face="ff-meta-web-pro-1, ff-meta-web-pro-2, Arial, Helvetica Neue, sans-serif"><span style="line-height:24px">from the MISO website and unzipped. I saw it is based on Homo_sapiens.GRCh37.65. Is this the version problem? If so, could anyone provide the latest GFF3 file for use? </span></font><span style="line-height:24px;color:rgb(0,0,0);font-family:ff-meta-web-pro-1,ff-meta-web-pro-2,Arial,'Helvetica Neue',sans-serif">Thank you for your suggestions!</span></div>
<div style><span style="line-height:24px;color:rgb(0,0,0);font-family:ff-meta-web-pro-1,ff-meta-web-pro-2,Arial,'Helvetica Neue',sans-serif"><br></span></div><div style><font color="#000000" face="ff-meta-web-pro-1, ff-meta-web-pro-2, Arial, Helvetica Neue, sans-serif"><span style="line-height:24px">Best,</span></font></div>
<div style><font color="#000000" face="ff-meta-web-pro-1, ff-meta-web-pro-2, Arial, Helvetica Neue, sans-serif"><span style="line-height:24px">Gu</span></font></div></div></div>