[KaiYe]

Hi KaiYe,

I used MarkDuplicates from Picard and mark duplicates but did not removed them.
I have a question does pindel need file with removed duplicates or can work with Marked ones?

Thank you in an advance.

It is better to remove duplicates. Pindel has an internal counting procedure to output total support and unique support.

In the original Pindel output file (not converted to vcf) per sample you will see something like:
NA18507 + 10 8 - 12 9
While 10 is total count from + strand, 8 is the unique count.
12 and 9 are for other strand, total or unique.

Kai

[mikhmv]

It was a quick reply!!! Thanks.

I have a several more questions now.
1. Does it mean that Pindel does not check flag that this read is duplicate?
2. If you have count it independently maybe it just doesn't matter was duplicates removed or not as a amount unique reads will be same?
3. Did pindel take in account sequencing error rate while determining amount of unique reads?

[libiyagirl]

Hi Kai,

I found that the bam_configuration method takes different number of reads for analysis, compared with the bam2pindel.pl method. Could you please explain it to me? And the new version, pindel024n processes way more reads compared with Pindel_0.2.4d. Does the two versions filter the reads differently?

Thank you so much for your help,

[KaiYe]

It was a quick reply!!! Thanks.

I have a several more questions now.
1. Does it mean that Pindel does not check flag that this read is duplicate?
2. If you have count it independently maybe it just doesn't matter was duplicates removed or not as a amount unique reads will be same?
3. Did pindel take in account sequencing error rate while determining amount of unique reads?

Pindel doesn't check flag for duplicate but count by itself. It reports both total and unique counts for both strands. It is up to the users to decide which number to use. Pindel considers 3% sequence error rate by default but you can change this parameter.

Kai

[KaiYe]

Hi Kai,

I found that the bam_configuration method takes different number of reads for analysis, compared with the bam2pindel.pl method. Could you please explain it to me? And the new version, pindel024n processes way more reads compared with Pindel_0.2.4d. Does the two versions filter the reads differently?

Thank you so much for your help,

Yes, the perl program does slightly different things than Pindel internal BAM read function, although we tried to make them consistent. We changed a few parameters in pindel024n, resulting at least 3 times more candidate reads. We are investigating this impact. So far no significant difference (except for more sensitive) was observed.

Kai

[mikhmv]

Hi KaiYe,

I have an error message during run pindel2vcf.
The executing command was:
pindel2vcf -r $REFGENOME -R hg19b37 -d 20120310 -p 5173N_TD.res -v 5173N_TD.res2.vcf
The last few lines from program:
Processing chromosome chr11
Reading region 0-300000000
Reading chromosome chr11 into memory.
Total reads: 180
Sorting completed
Removing chromosome chr11 from memory.
Processing chromosome chr11_gl000202_random
Reading region 0-300000000
Total reads: 0
Sorting completed
Segmentation fault

Can you help to resolve this problem?

Regards

[mikhmv]

KaiYe,
I think it is a bug. I got this message on different chromosome depend on file but error everytime after Total reads = 0.

Is it possible to fix?

Regards,
Max

[KaiYe]

KaiYe,
I think it is a bug. I got this message on different chromosome depend on file but error everytime after Total reads = 0.

Is it possible to fix?

Regards,
Max

Hi Max,

Yes, we also noticed this bug. Please try our latest source code 0.2.4o, updated on March 9 2012.

Please do contact us if you experience the same or other issues.

Thanks,

Kai

[mikhmv]

Hi Max,

Yes, we also noticed this bug. Please try our latest source code 0.2.4o, updated on March 9 2012.

Please do contact us if you experience the same or other issues.

Thanks,

Kai

Thanks KaiYe.
"o" is working well.

[mikhmv]

Hi KaiYe,
I have a question how can we annotate BreakPoints? pindel2vcf doesn't support it.

Regards

[mikhmv]

Hi KaiYe,
Does pindel2vcf provide quality score?
How can I get it in vcf?
Regards,

[bwubb]

I recently upgraded to pindel024o and may have found some sort of bug; or maybe Im doing something I shouldnt be...

When I execute pindel now all of my bins are empty and it produces no results. I do not get an error, I just get no results, as such:

Pindel version 0.2.4o, March 9 2012.
Looping over all chromosomes.
Processing chromosome: 1
Chromosome Size: 249250621
NumBoxes: 60001	BoxSize: 8975

Looking at chromosome 1 bases 0 to 10000000.
There are no reads for this bin.

Looking at chromosome 1 bases 10000000 to 20000000.
There are no reads for this bin.

Looking at chromosome 1 bases 20000000 to 30000000.
There are no reads for this bin.

Looking at chromosome 1 bases 30000000 to 40000000.
There are no reads for this bin.

Looking at chromosome 1 bases 40000000 to 50000000.
There are no reads for this bin.

Looking at chromosome 1 bases 50000000 to 60000000.
There are no reads for this bin.
...

This is suppose to be exome sequencing data. Now the odd thing is that I have previous pindel024m results for this sample, so everything should work as I did not change anything. The only change besides the version is where I am executing from.

I made a dir 'my_test' in my pindel024o directory (~/pindel024o/my_test/). Previously I was in a different path, something like ~/NGS/Pindel_restults/

The BAM files used have not changed, nor have the locations. If I execute pindel from my old directory it still works.

Im just confused by this.

[KaiYe]

I recently upgraded to pindel024o and may have found some sort of bug; or maybe Im doing something I shouldnt be...

When I execute pindel now all of my bins are empty and it produces no results. I do not get an error, I just get no results, as such:

Pindel version 0.2.4o, March 9 2012.
Looping over all chromosomes.
Processing chromosome: 1
Chromosome Size: 249250621
NumBoxes: 60001	BoxSize: 8975

Looking at chromosome 1 bases 0 to 10000000.
There are no reads for this bin.

Looking at chromosome 1 bases 10000000 to 20000000.
There are no reads for this bin.

Looking at chromosome 1 bases 20000000 to 30000000.
There are no reads for this bin.

Looking at chromosome 1 bases 30000000 to 40000000.
There are no reads for this bin.

Looking at chromosome 1 bases 40000000 to 50000000.
There are no reads for this bin.

Looking at chromosome 1 bases 50000000 to 60000000.
There are no reads for this bin.
...

This is suppose to be exome sequencing data. Now the odd thing is that I have previous pindel024m results for this sample, so everything should work as I did not change anything. The only change besides the version is where I am executing from.

I made a dir 'my_test' in my pindel024o directory (~/pindel024o/my_test/). Previously I was in a different path, something like ~/NGS/Pindel_restults/

The BAM files used have not changed, nor have the locations. If I execute pindel from my old directory it still works.

Im just confused by this.

This is indeed odd. Can you share a tiny BAM (first 1k reads) with me so that I can check this out. We don't see this on our data but we would like to find out. Please contact my private email address [email protected] to discuss how to share the data.

Thank you.

Kai

[bwubb]

Hi Kai,

I appreciate you offering to help with this issue, but I believe I resolved the problem though. The problem was my config file. I was not actually using the same exact config file, but had written an identical one to my 'version m' data.

Identical... except for an embarrassing mistake in that the newer config file did not have a new line character at the end.

This seemed to result in the error/results I posted above.

[EWLameijer]

Hi Kai,

I appreciate you offering to help with this issue, but I believe I resolved the problem though. The problem was my config file. I was not actually using the same exact config file, but had written an identical one to my 'version m' data.

Identical... except for an embarrassing mistake in that the newer config file did not have a new line character at the end.

This seemed to result in the error/results I posted above.

Hi Bwubb,

It's very good that you resolved the problem on your own. To save you and other people some time in the future, Kai and I have now implemented some changes in the bam-file reading parts, and submitted it as Pindel-0.2.4p.

Pindel-0.2.4p does not fundamentally change the pindel output, but will give a clear error message if an user by accident:
-uses the -i option on a bam-configuration that does not exist
-uses a bam-configuration file that is empty or truncated
-refers in the bam-configuration file to bam-files that don't exist
-refers in the bam-configuration file to bam-files that have not been indexed yet

Inspired by your report, it should now also work if the last line does not end in a newline character.

Thank you for your message and help in clearing this up,


Eric-Wubbo