Breakdancer BAM2cfg output blank

[vas72985]

Hello,

I am attempting to generate a configuration file for breakdancer using the BAM2cfg perl script, however when I run the script on my two BAM files, it generates an empty file as my output. It doesn't give any errors. Anyone have any idea what might be wrong?

[ernfrid]

Others have reported this issue when your BAM files and reads don't contain @RG tags. Which version of breakdancer are you trying to use? How is the header of your BAM file constructed? Are there @RG entries on your reads?

[vas72985]

So I came across that and added @RG tags using Picard. Now when I run it, it doesn't create the output file at all but just hangs for 1-2 minutes and then says it's done, but there is no output.

[ernfrid]

Are you running bam2cfg with any options?

[vas72985]

only -g and -h

[ernfrid]

In looking at the code, the problem is almost certainly with the formatting of your BAM file. Could you post your header and an example entry?

[vas72985]

Here's the header from one of my BAM files (which is sorted):

@HD VN:1.0 SO:unsorted
@SQ SN:chr15 LN:102531392
@SQ SN:chr1_gl000191_random LN:106433
@SQ SN:chr14 LN:107349540
@SQ SN:chr13 LN:115169878
@SQ SN:chrUn_gl000227 LN:128374
@SQ SN:chrUn_gl000228 LN:129120
@SQ SN:chr12 LN:133851895
@SQ SN:chr11 LN:135006516
@SQ SN:chr10 LN:135534747
@SQ SN:chrUn_gl000214 LN:137718
@SQ SN:chr9 LN:141213431
@SQ SN:chr8 LN:146364022
@SQ SN:chrUn_gl000226 LN:15008
@SQ SN:chrX LN:155270560
@SQ SN:chrUn_gl000221 LN:155397
@SQ SN:chr7 LN:159138663
@SQ SN:chr19_gl000209_random LN:159169
@SQ SN:chrUn_gl000218 LN:161147
@SQ SN:chrUn_gl000220 LN:161802
@SQ SN:chrUn_gl000213 LN:164239
@SQ SN:chrM LN:16571
@SQ SN:chrUn_gl000211 LN:166566
@SQ SN:chr17_ctg5_hap1 LN:1680828
@SQ SN:chr9_gl000199_random LN:169874
@SQ SN:chr6 LN:171115067
@SQ SN:chrUn_gl000217 LN:172149
@SQ SN:chrUn_gl000216 LN:172294
@SQ SN:chrUn_gl000215 LN:172545
@SQ SN:chr17_gl000205_random LN:174588
@SQ SN:chrUn_gl000219 LN:179198
@SQ SN:chrUn_gl000224 LN:179693
@SQ SN:chrUn_gl000223 LN:180455
@SQ SN:chr5 LN:180915260
@SQ SN:chr7_gl000195_random LN:182896
@SQ SN:chrUn_gl000212 LN:186858
@SQ SN:chrUn_gl000222 LN:186861
@SQ SN:chr9_gl000200_random LN:187035
@SQ SN:chr4_gl000193_random LN:189789
@SQ SN:chr4 LN:191154276
@SQ SN:chr4_gl000194_random LN:191469
@SQ SN:chr3 LN:198022430
@SQ SN:chrUn_gl000229 LN:19913
@SQ SN:chrUn_gl000225 LN:211173
@SQ SN:chr2 LN:243199373
@SQ SN:chr1 LN:249250621
@SQ SN:chrUn_gl000231 LN:27386
@SQ SN:chr21_gl000210_random LN:27682
@SQ SN:chrUn_gl000239 LN:33824
@SQ SN:chrUn_gl000235 LN:34474
@SQ SN:chr9_gl000201_random LN:36148
@SQ SN:chrUn_gl000247 LN:36422
@SQ SN:chrUn_gl000245 LN:36651
@SQ SN:chr8_gl000197_random LN:37175
@SQ SN:chr17_gl000203_random LN:37498
@SQ SN:chrUn_gl000246 LN:38154
@SQ SN:chrUn_gl000249 LN:38502
@SQ SN:chr8_gl000196_random LN:38914
@SQ SN:chrUn_gl000248 LN:39786
@SQ SN:chrUn_gl000244 LN:39929
@SQ SN:chrUn_gl000238 LN:39939
@SQ SN:chr11_gl000202_random LN:40103
@SQ SN:chrUn_gl000234 LN:40531
@SQ SN:chrUn_gl000232 LN:40652
@SQ SN:chr17_gl000206_random LN:41001
@SQ SN:chrUn_gl000240 LN:41933
@SQ SN:chrUn_gl000236 LN:41934
@SQ SN:chrUn_gl000241 LN:42152
@SQ SN:chr18_gl000207_random LN:4262
@SQ SN:chrUn_gl000243 LN:43341
@SQ SN:chrUn_gl000242 LN:43523
@SQ SN:chrUn_gl000230 LN:43691
@SQ SN:chrUn_gl000237 LN:45867
@SQ SN:chrUn_gl000233 LN:45941
@SQ SN:chr6_dbb_hap3 LN:4610396
@SQ SN:chr6_qbl_hap6 LN:4611984
@SQ SN:chr6_apd_hap1 LN:4622290
@SQ SN:chr6_mann_hap4 LN:4683263
@SQ SN:chr6_cox_hap2 LN:4795371
@SQ SN:chr21 LN:48129895
@SQ SN:chr6_mcf_hap5 LN:4833398
@SQ SN:chr6_ssto_hap7 LN:4928567
@SQ SN:chr22 LN:51304566
@SQ SN:chr1_gl000192_random LN:547496
@SQ SN:chr4_ctg9_hap1 LN:590426
@SQ SN:chr19 LN:59128983
@SQ SN:chrY LN:59373566
@SQ SN:chr20 LN:63025520
@SQ SN:chr18 LN:78077248
@SQ SN:chr17 LN:81195210
@SQ SN:chr17_gl000204_random LN:81310
@SQ SN:chr9_gl000198_random LN:90085
@SQ SN:chr16 LN:90354753
@SQ SN:chr19_gl000208_random LN:92689
@RG ID:1 PL:Illumina PU:H0W3EADXX:1 LB:TS SM:L1En
@PG ID:bowtie2 PN:bowtie2 VN:2.0.6

I'm running:

bam2cfg.pl -g -h [BAM1] [BAM2] > [output.cfg] and getting a blank output.cfg file

I've adjusted the path to samtools in the script. And I'm running the most up-to-date release of BreakDancer.

[ernfrid]

That header looks reasonable to me. Can you also show one of your alignment records? (e.g. samtools view my.bam | head -1)

[vas72985]

I appreciate your help. Here it is:

HWI-ST281:389:H0W3EADXX:1:1113:3366:13843 161 chr15 20068461 0 62M1I37M chr14 19093543 0 ACTATCCTCCCAGTTACCCAGGCATAAAACCTTGGAATTCTATCGAATTATTTTATTTCCTTAACATCTCATGTTTAATTAGTCACTGATTCCCATCAAT CCCFFFFFHHG<FGIGIIJIGJJIJJGHEIIJJGJ>DHIEFGIIIGGJJBIGDIIIIGHGIIEDGHGAFGGICHCHE?EH>CHC>BDDCFEEDEEE=>AC MD:Z:2C2T17T1G6C0A10A8C12A32 RG:Z:1 XG:i:1 NM:i:10 XM:i:9 XN:i:0 XO:i:1 AS:i:-58 YS:i:-32 YT:ZP

[vas72985]

The silly smiley face is a colon and a DP because it's a discordant pair...gotta love emoticons...

[GenoMax]

The silly smiley face is a colon and a DP because it's a discordant pair...gotta love emoticons...

You could enclose the section in a "Quote". (TIP: Click on the "Go Advanced" button as you edit to get additional formatting options)

HWI-ST281:389:H0W3EADXX:1:1113:3366:13843 161 chr15 20068461 0 62M1I37M chr14 19093543 0 ACTATCCTCCCAGTTACCCAGGCATAAAACCTTGGAATTCTATCGAATTATTTTATTTCCTTAACATCTCATGTTTAATTAGTCACTGATTCCCATCAAT CCCFFFFFHHG<FGIGIIJIGJJIJJGHEIIJJGJ>DHIEFGIIIGGJJBIGDIIIIGHGIIEDGHGAFGGICHCHE?EH>CHC>BDDCFEEDEEE=>AC MD:Z:2C2T17T1G6C0A10A8C12A32 RG:Z:1 XG:i:1 NM:i:10 XM:i:9 XN:i:0 XO:i:1 AS:i:-58 YS:i:-32 YT:ZP

[ernfrid]

I'm not seeing any malformation with your header or records. The record is not the best example because it is discordant and bam2cfg only looks at concordant read pairs, but things seem to be functioning fine. If you could get me some normal entries from your BAM then I might be able to troubleshoot better.

[vas72985]

So due to the fact that I was being dense, I didn't realize that I had filtered by BAM file for discordant pairs...this explains why the output was blank.

I've rerun it on the original (unfiltered) BAM file and now get a small output cfg file and the following error:

Use of uninitialized value in printf at /usr/local/bin/breakdancer-1.1_2011_02_21/perl/bam2cfg.pl line 222.

Line 222 reads:

printf "%d",$flagHgram{$rg}{all};

[ernfrid]

That's odd. I would be concerned about your config file. Do you see non-zero numbers for your flag distributions? It should be any numbers after the "flag:" string in the config file.