Breakdancer output error

Question

[shumaila.sayyab]

Hi everyone,

I have 4 samples (matepair illumina) and run Breakdancer_max as follows:

bam2cfg.pl -h -g -q 10 sample1.bam sample2.bam sample3.bam sample4.bam >sample1_2_sample3_4.cfg

It worked without error msg.

breakdancer-max -o chrX -q 10 -d Fastq_SVReadsFile_X -g GBrowse_OUTFile_X.bed -l -h sample1_2_sample3_4.cfg >chrX_SV_allSample.ctx

This also worked generating outputs with last mag as Kahan error (which I ignored as read on other blogs Breakdancer Max Kahan Error:0)

The problem is that the output file chrX_SV_allSample.ctx only contains SV specific to sample4. while all other samples contains NA in their columns. I do not know where I am making mistake.

Output (first few lines)

Software: 1.4.4-unstable-7-6213d5a (commit 6213d5a)
Command: breakdancer-max -o chrX -q 10 -d Fastq_SVReadsFile_X -g GBrowse_OUTFile_X.bed -l -h sample1_2_sample3_4.cfg
Library Statistics:
sample4.bam mean:4218.95 std:552.31 uppercutoff:6393.21 lowercutoff:1975.9 readlen:50.82 library:tight reflen:111700677 seqcov:0.939753 phycov:39.008 1:8326 2:6802 3:2818 4:436 8:8414 32:20695
sample4.bam mean:3889.35 std:680.67 uppercutoff:6865.34 lowercutoff:1401.76 readlen:50.82 library:wide reflen:111700677 seqcov:0.701866 phycov:26.8576 1:6586 2:2710 3:2022 4:326 8:6506 32:15637
Chr1 Pos1 Orientation1 Chr2 Pos2 Orientation2 Type Size Score num_Reads num_Reads_lib Allele_frequency sample1.bam sample2.bam sample3.bam sample4.bam
chrX 229 312+317- chrX 292963 312+317- INV 6106 99 118 sample4.bam|118 -nan NA NA NA NA
chrX 327014 58+52- chrX 372108 58+52- INV -1620 70 20 sample4.bam|20 -nan NA NA NA NA
chrX 372154 58+52- chrX 430662 103+43- INV 10905 99 43 sample4.bam|43 0.301335 NA NA NA 1.40
chrX 491312 5+2- chrX 494412 5+2- INV -3118 34 2 sample4.bam|2 -nan NA NA NA NA

Please guide.

Any help or suggestion is valuable

Answer 1

[ernfrid]

Do your BAM files have proper readgroup information in the header and in the reads? Can you post your configuration file?

[shumaila.sayyab]

I think Yes, each of my sample has two libraries (tight and wide), and when I run the script to generate the configuration file, I received this file (attached)

readgroup:sample1_tight    platform:Illumina    map:../sample1_sample.dedup.bam    readlen:50.80    lib:tight    num:10001    lower:2298.05    upper:6428.40    mean:4663.57    std:518.01    SWnormality:-63.07    flag:0(21.58%)1(0.23%)18(77.00%)2(0.14%)32(0.65%)4(0.11%)8(0.29%)25934    exe:samtools view
readgroup:sample1_wide    platform:Illumina    map:../sample1_sample.dedup.bam    readlen:50.82    lib:wide    num:9999    lower:2137.45    upper:8151.25    mean:5085.55    std:750.93    SWnormality:-42.96    flag:1(0.37%)18(98.26%)2(0.20%)32(0.77%)4(0.12%)8(0.28%)20327    exe:samtools view
readgroup:sample2_tight    platform:Illumina    map:../sample2_sample.dedup.bam    readlen:50.79    lib:tight    num:10001    lower:1461.89    upper:6183.87    mean:4448.08    std:592.35    SWnormality:-73.76    flag:0(34.48%)1(0.94%)18(62.94%)2(0.06%)32(0.47%)4(0.25%)8(0.86%)31752    exe:samtools view
readgroup:sample2_wide    platform:Illumina    map:../sample2_sample.dedup.bam    readlen:50.82    lib:wide    num:9998    lower:1248.42    upper:7635.35    mean:4645.63    std:799.94    SWnormality:-46.68    flag:1(1.07%)18(96.56%)2(0.09%)32(0.88%)4(0.31%)8(1.09%)20704    exe:samtools view
readgroup:sample3_wide    platform:Illumina    map:../sample3_sample.dedup.bam    readlen:50.81    lib:wide    num:9998    lower:0.00    upper:9689.16    mean:3902.64    std:1278.45    SWnormality:-34.61    flag:1(0.64%)18(97.64%)2(0.10%)32(0.74%)4(0.33%)8(0.55%)20472    exe:samtools view
readgroup:sample3_tight    platform:Illumina    map:../sample3_sample.dedup.bam    readlen:50.80    lib:tight    num:9992    lower:1396.67    upper:5864.12    mean:3621.16    std:558.34    SWnormality:-46.06    flag:0(27.73%)1(0.39%)18(70.41%)2(0.19%)32(0.57%)4(0.30%)8(0.41%)28393    exe:samtools view
readgroup:sample4_wide    platform:Illumina    map:../sample4_sample.dedup.bam    readlen:50.82    lib:wide    num:9996    lower:1401.76    upper:6865.34    mean:3889.35    std:680.67    SWnormality:-32.26    flag:1(0.26%)18(98.19%)2(0.24%)32(1.05%)4(0.08%)8(0.18%)20357    exe:samtools view
readgroup:sample4_tight    platform:Illumina    map:../sample4_sample.dedup.bam    readlen:50.82    lib:tight    num:9990    lower:1975.90    upper:6393.21    mean:4218.95    std:552.31    SWnormality:-50.96    flag:0(21.69%)1(0.19%)18(76.93%)2(0.32%)32(0.62%)4(0.10%)8(0.16%)25965    exe:samtools view

[shumaila.sayyab]

Thanks ernfrid, did this answer your question? Please let me know if you have any suggestion for the conf. file?

[ernfrid]

It certainly did and I have a theory. Can you a) Check that your reads have RG:Z: entries in each BAM file tying them back to the readgroups in the header and b) attempt giving unique library names in each BAM file (as opposed to tight and wide). I believe the current code only looks at the library name so the same name across samples will result in the behavior you are seeing.

[shumaila.sayyab]

Thank you ernfrid, it worked after giving unique library names.