What's wrong with my pipeline of BreakDancer?

[shixinyi]

Hello! I am new in this research region. I try to use breakdancer to detect the SVs. I process the following steps:

1. I wrote a program to simulatively generate pair-end reads. There are two files named reads_out1.fq and reads_out2.fq.

2. I used bwa to generate sam file, the command is:
PATH_P=/home/shixy/data/human/chromx/2012_09_01

/home/shixy/tools/bwa-0.6.2/bwa index -a bwtsw $PATH_P/hs.fa

/home/shixy/tools/bwa-0.6.2/bwa aln -t 16 $PATH_P/hs.fa $PATH_P/reads_out1.fq > $PATH_P/anl_sa_1.sai

/home/shixy/tools/bwa-0.6.2/bwa aln -t 16 $PATH_P/hs.fa $PATH_P/reads_out2.fq > $PATH_P/anl_sa_2.sai

/home/shixy/tools/bwa-0.6.2/bwa sampe -r '@RG\tID:ga\tSM:hs\tLB:ga\tPL:Illumina\tPI:200' $PATH_P/hs.fa $PATH_P/anl_sa_1.sai $PATH_P/anl_sa_2.sai $PATH_P/reads_out1.fq $PATH_P/reads_out2.fq > $PATH_P/sampleC.sam

/home/shixy/tools/samtools/samtools view -bS $PATH_P/sampleC.sam > $PATH_P/sampleC.bam

/home/shixy/tools/samtools/samtools sort $PATH_P/sampleC.bam $PATH_P/sampleC_sort

/home/shixy/tools/samtools/samtools index $PATH_P/sampleC_sort.bam

3. I use command 'perl bam2cfg.pl sampleC_sort.bam > config.cfg' to generate the config file. This step ran successfully, and the result is:
'readgroup:ga platform:Illumina map:/home/shixy/data/human/chromx/2012_09_01/sampleC_sort.bam readlen:36.00 lib:ga num:9998 lower:4.54 upper:491.60 mean:205.02 std:60.46 SWnormality:-44.88 exe:samtools view'

4. Then I use command 'breakdancer_max confg.cfg > sampleC.ctx'. But I just got nothing but this:
#Software: BreakDancerMax-1.1r112
#Command: ./breakdancer_max config.cfg
#Library Statistics:
#/home/shixy/data/human/chromx/2012_09_01/sampleC_sort.bam mean:205.02 std:60.46 uppercutoff:491.6 lowercutoff:4.54 readlen:36 library:ga reflen:143733241 seqcov:23.4655 phycov:66.8181 2:85414
#Chr1 Pos1 Orientation1 Chr2 Pos2 Orientation2 Type Size Score num_Reads num_Reads_lib xxx_sort.bam

Could you give me some advice! Please help me! It's really hard to me.

Thanks a lot!

[TiborNagy]

Hi, your reference is a multi fasta file? Maybe the order of the contigs not proper. samtools sort does not order the contigs, try picard's ReorderSam if it is the problem.

[shixinyi]

There is only one chromosome in my reference file.
I found that someone wrote the command :"bam2cfg.pl -g -h tumor.bam normal.bam > BRC6.cfg".
I don't know how to get the file normal.bam. I think maybe it is the problem.
Could you tell me how to get the normal.bam?
Thanks a lot!

[Bukowski]

How did you generate the SV's in your reads just out of interest?

[Irsan_Kooi]

I found that someone wrote the command :"bam2cfg.pl -g -h tumor.bam normal.bam > BRC6.cfg".

That is definetely not the problem. With breakdancer you can specify multiple .bam files. It just piles them up and uses all the reads together and in the end for each called structural variant it reports the amount of reads that were used for that structural event per .bam file you supplied.

So nothing wrong with:

perl bam2cfg.pl sampleC_sort.bam > config.cfg

Your problem is most likely in the bam-file