[bwubb]

What is SVTYPE RPL?

[bwubb]

Can I request a max supporting samples option for the pindel2vcf program? I did not see that as an option in the --help and for my purposes, I want to filter out any SV that occurs in more then 2 samples.

Or am I missing how to do this? Thank you.

[Seq_student]

Pindel empty output

Hi Kai

I posted this on a separated thread already, but my problem is that I keep getting output like this:

*** Calling SV using Split-Read Analysis: /home/usr/apps/pindel024s/src ***
>> Running Pindel on ALL: /home/usr/apps/pindel024s/src/pindel -f /srv/gs1/projects/lab/usr/data/hg19/ucsc.hg19.fasta -i /srv/gs1/projects/lab/usr/dir.2/JS2.sra.cfg -o /srv/gs1/projects/lab/usr/dir.2/JS2.sra -c ALL
Pindel version 0.2.4s, June 18 2012.
Looping over all chromosomes.
Processing chromosome: chrM
Chromosome Size: 16571
NumBoxes: 60020 BoxSize: 667

Looking at chromosome chrM bases 0 to 10000000.
getReads chrM 20016571
Insertsize in bamreads: 195
Number of reads in current window: 0, + 0 - 0
Number of reads where the close end could be mapped: 0, + 0 - 0
Percentage of reads which could be mapped: + 0.00% - 0.00%

No currentState.Reads for chrM found in /srv/gs1/projects/lab/usr/dir.2/JS2_L1_1_pf_aa.sorted.recal.bam
BAM file index 0 0
There are no reads for this bin.


And the configuration file looks like this:

/srv/gs1/projects/lab/usr/dir.2/JS2_L1_1_pf_aa.sorted.recal.bam 195 JS2


I originally used an older version and then reinstalled the latest version of pindel (0.2.4s) and I get the same problem, the "BAM file index 0 0" "no reads in this bin" for every bin for every chromosome.

The only thing I can think of is that maybe something went wrong at the compilation stage of pindel? When i compiled pindel I saw a few errors:

/pindel024s> make SAMTOOLS=/home/usr/apps/samtools-0.1.14
Created Makefile.local, please review the configuration
make: *** No rule to make target `Makefile.local', needed by `pindel'. Stop.
scg2:~/apps/pindel024s> make SAMTOOLS=/home/usr/apps/samtools-0.1.14
make -C src pindel
make[1]: Entering directory `/home/usr/apps/pindel024s/src'
make[1]: Leaving directory `/home/usr/apps/pindel024s/src'
make[1]: Entering directory `/home/usr/apps/pindel024s/src'
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 pindel.cpp -o pindel.o
pindel.cpp: In function 'int main(int, char**)':
pindel.cpp:1196:38: warning: variable 'Time_Mine_E' set but not used [-Wunused-but-set-variable]
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 reader.cpp -o reader.o
reader.cpp: In function 'int fetch_func_RP(const bam1_t*, void*)':
reader.cpp:770:24: warning: variable 'b1_core' set but not used [-Wunused-but-set-variable]
reader.cpp:772:18: warning: variable 'b2_core' set but not used [-Wunused-but-set-variable]
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 reporter.cpp -o reporter.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 searcher.cpp -o searcher.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 parameter.cpp -o parameter.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 refreader.cpp -o refreader.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 control_state.cpp -o control_state.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 search_deletions_nt.cpp -o search_deletions_nt.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 search_inversions.cpp -o search_inversions.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 search_inversions_nt.cpp -o search_inversions_nt.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 bam2depth.cpp -o bam2depth.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 search_tandem_duplications.cpp -o search_tandem_duplications.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 search_tandem_duplications_nt.cpp -o search_tandem_duplications_nt.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 output_sorter.cpp -o output_sorter.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 farend_searcher.cpp -o farend_searcher.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 search_variant.cpp -o search_variant.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 searchshortinsertions.cpp -o searchshortinsertions.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 searchdeletions.cpp -o searchdeletions.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 output_file_data.cpp -o output_file_data.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 bddata.cpp -o bddata.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 shifted_vector.cpp -o shifted_vector.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 read_buffer.cpp -o read_buffer.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 assembly.cpp -o assembly.o
assembly.cpp: In function 'void TryLI(const std::vector<Chromosome>&, std::map<std::basic_string<char>, int>&, ControlState&, ParCollection&, const Assembly&, std::vector<SPLIT_READ>&, std::vector<SPLIT_READ>&, std:fstream&)':
assembly.cpp:606:24: warning: variable 'SecondLength' set but not used [-Wunused-but-set-variable]
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 genotyping.cpp -o genotyping.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 line_reader.cpp -o line_reader.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 ifstream_line_reader.cpp -o ifstream_line_reader.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 gz_line_reader.cpp -o gz_line_reader.o
g++ -Wall -g -c -O3 -fopenmp -I/home/usr/apps/samtools-0.1.14 pindel_read_reader.cpp -o pindel_read_reader.o
g++ pindel.o reader.o reporter.o searcher.o parameter.o refreader.o control_state.o search_deletions_nt.o search_inversions.o search_inversions_nt.o bam2depth.o search_tandem_duplications.o search_tandem_duplications_nt.o output_sorter.o farend_searcher.o search_variant.o searchshortinsertions.o searchdeletions.o output_file_data.o bddata.o shifted_vector.o read_buffer.o assembly.o genotyping.o line_reader.o ifstream_line_reader.o gz_line_reader.o pindel_read_reader.o -O3 -fopenmp -lm -lz -L/home/usr/apps/samtools-0.1.14 -lbam -o pindel
make[1]: Leaving directory `/home/usr/apps/pindel024s/src'

(the reason I'm using samtools 0.1.14 is because the pipeline I'm installing was set up with this version and the indexed bam was created using this version. I also tried installing pindel and compiling with samtools 0.1.18 and then running it, but it did not solve my problems and I ended up with the same output...)

Any help/solutions you can offer would be greatly appreciated!

[mikhmv]

KaiYe: Does pindel accept breakdancer files?

I tried to use this command: pindel --fasta "human_g1k_v37_decoy_optimized.fasta" \
--config-file "config_1" \
--output-prefix "$PREFIX-$CHROM" \
--chromosome $CHROM \
--number_of_threads 10 \
--max_range_index 5 \
--report_inversions --report_duplications --report_long_insertions --report_breakpoints --report_close_mapped_reads \
--min_NT_size 50 \
--min_inversion_size 50 \
--min_num_matched_bases 30 \
--additional_mismatch 1 \
--min_perfect_match_around_BP 3 \
--sequencing_error_rate 0.03 \
--maximum_allowed_mismatch_rate 0.1 \
--anchor_quality 20 \
--balance_cutoff 100 \
--window_size 300 \
--minimum_support_for_event 3 \
--genotyping \
--breakdancer "file_1190575.txt" \
--output_of_breakdancer_events "breakdancer-events-$CHROM.txt" \
--name_of_logfile $CHROM.log

But I didn't get neither breakdancer-events neither log file. Does this options work?

[d12ajd]

Thanks for your responses! I will run with the realigned files, Alex

[mikhmv]

d12ajd: I think after realignment will be better. I have seen following deletions marked as independent with the same starting position in pindel:
A-TTTTTC and
ATTT-TTC
So, you will loose this deletions as they will have low score hits/total.

[KaiYe]

Hi Kai,

I have incorporated pindel into my pipeline, but I have a query about where best to place it.

Can I run pindel with the realigned.bam files from GATK? Or is it better to run it before this step?

Thanks,

Alex

Both are fine but do it after realignment may be better.

Kai

[d12ajd]

Hi Kai,

I have incorporated pindel into my pipeline, but I have a query about where best to place it.

Can I run pindel with the realigned.bam files from GATK? Or is it better to run it before this step?

Thanks,

Alex

[Yong-Biao Zhang]

As Pindel ignores reads supporting the reference allele to speed up, we only report the number of variant supporting reads per sample with strand and uniqueness information. You may use the two score S1 and SUM_MS to do filterings.

Hi Kai

Thanks for you reply.
Another question, in the output file of large insertion, it seems that there were no columns of S1 and SUM_MS, as welll as the length of large insertion. Did I make a mistake in manipulating pindel?
By the way, to get a proper confidence of SV, what cutoff value shoud I set to filter the results?

Thanks

[KaiYe]

Hi Kai

Thanks for you help.

One more question, there is a column named "The number of reads supporting the SV", but I can not find the total number of reads cover the SV per sample, unless I check this by samtools -tview. I just want to know the confidence of the SVs in the output files. Is there a parameter to reflect this?

Thanks.

As Pindel ignores reads supporting the reference allele to speed up, we only report the number of variant supporting reads per sample with strand and uniqueness information. You may use the two score S1 and SUM_MS to do filterings.

[Yong-Biao Zhang]

Please use "-c ALL", not "-c all". Also make sure that you use the same reference file here you used for mapping. "chr1" is different than "1".

Hi Kai

Thanks for you help.

One more question, there is a column named "The number of reads supporting the SV", but I can not find the total number of reads cover the SV per sample, unless I check this by samtools -tview. I just want to know the confidence of the SVs in the output files. Is there a parameter to reflect this?

Thanks.

[bwubb]

Hi Kai,

I had a few questions I was hoping you could address. Could you provide a bit of information about how incorporating BreakDancer output affects Pindel. I am trying to compare results of Pindel with and without using BreakDancer output as an option. Does BreakDancer make Pindel results more reliable somehow?


Additionally, I am curious about the 'insert size' for each sample included in the pindel config file. Previously I was just putting 200 as I have 100bp PE data, but this seems incorrect. A more accurate number might be the average size of my library prep (minus the adapter size), which is a bit different for each sample. Is fragment size recommended instead? How will that improve the results?

Thank you very much.

[KaiYe]

Hi kai,

I met a problem when I run pindel.

this is the command line:
pindel -f ../ref/hs_ref_GRCh37.p2.all.fa -i config.txt -c all -o pindeloutput -T 8

this is the configure file:
A1.sort.bam 300 A1
A2.sort.bam 300 A2
A3.sort.bam 300 A3

the bam files were generated by BWA, I also try the unsorted bam file, not work too.

this is the prompt:
Pindel version 0.2.4o, March 9 2012.
Processing chromosome: gi|224589800|ref|NC_000001.10|
Skipping chromosome: gi|224589800|ref|NC_000001.10|
Processing chromosome: gi|224589811|ref|NC_000002.11|
Skipping chromosome: gi|224589811|ref|NC_000002.11|
Processing chromosome: gi|224589815|ref|NC_000003.11|
Skipping chromosome: gi|224589815|ref|NC_000003.11|
.............
Processing chromosome: gi|224514683|ref|NT_167243.1|
Skipping chromosome: gi|224514683|ref|NT_167243.1|
Processing chromosome: gi|251831106|ref|NC_012920.1|
Skipping chromosome: gi|251831106|ref|NC_012920.1|
Loading genome sequences and reads: 0 seconds.
Mining, Sorting and output results: 0 seconds.

could you help me to solve this problem?

Regards

Yongbiao

Please use "-c ALL", not "-c all". Also make sure that you use the same reference file here you used for mapping. "chr1" is different than "1".

[Yong-Biao Zhang]

Hi kai,

I met a problem when I run pindel.

this is the command line:
pindel -f ../ref/hs_ref_GRCh37.p2.all.fa -i config.txt -c all -o pindeloutput -T 8

this is the configure file:
A1.sort.bam 300 A1
A2.sort.bam 300 A2
A3.sort.bam 300 A3

the bam files were generated by BWA, I also try the unsorted bam file, not work too.

this is the prompt:
Pindel version 0.2.4o, March 9 2012.
Processing chromosome: gi|224589800|ref|NC_000001.10|
Skipping chromosome: gi|224589800|ref|NC_000001.10|
Processing chromosome: gi|224589811|ref|NC_000002.11|
Skipping chromosome: gi|224589811|ref|NC_000002.11|
Processing chromosome: gi|224589815|ref|NC_000003.11|
Skipping chromosome: gi|224589815|ref|NC_000003.11|
.............
Processing chromosome: gi|224514683|ref|NT_167243.1|
Skipping chromosome: gi|224514683|ref|NT_167243.1|
Processing chromosome: gi|251831106|ref|NC_012920.1|
Skipping chromosome: gi|251831106|ref|NC_012920.1|
Loading genome sequences and reads: 0 seconds.
Mining, Sorting and output results: 0 seconds.

could you help me to solve this problem?

Regards

Yongbiao

[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