GMAP gsnap input error

[Irina Pulyakhina]

Hi everyone,

I'm trying to run gsnap programm with paired-end Illumina data. This is the command:

gsnap -D /data/database -d hg19 s_5.1.fastq .s_5.2.fastq -A sam >> s_5__hg19.sam

After two hours of running and 1,000,000 lines in the resulting sam file I get the following error:

========================
Signal received: Segmentation fault
Problem sequence: DD7DT8Q1:4:1101:20988:58254#ACACAA (100 bp)
>DD7DT8Q1:4:1101:20988:58254#ACACAA
GATGAAGGGAGGGATAGAGGGAGAATGGATGAAGGGAGAGTGGATGGATGGATGGGTGTAGGGTGGATGTGTGGAGGGCAGGTCGGAAGAGCACACGCCT
TCCATCCATACATGCACCCTTCATCCATCCATTCATCCACTCTCCCTTCATCCATTCTCCCTCTATCCCTCCCTTCATCAGATCGGAAGAGCGTCGTGTA
/usr/local/gridengine/default/spool/makoshark/job_scripts/225677: line 12: 30062 Aborted (core dumped)
========================

This sequence looks like this:
========================
@DD7DT8Q1:4:1101:20988:58254#ACACAA/1
GATGAAGGGAGGGATAGAGGGAGAATGGATGAAGGGAGAGTGGATGGATGGATGGGTGTAGGGTGGATGTGTGGAGGGCAGGTCGGAAGAGCACACGCCT
+DD7DT8Q1:4:1101:20988:58254#ACACAA/1
c\_b`[ddd`ddd`_`dXddZQcccf^^TeYTTV[cT]`^`a`_BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
========================

I don't see the problem with this sequence, but anyway gsnap doesn't accept it. Does anyone know what the problem might be?

Thanks in advance,
Irina

[twu]

Developer of GMAP and GSNAP

Hi Irina,

I have just created an account on SEQanswers to help answer questions about GMAP and GSNAP (and GSTRUCT when it is finally released).

To answer your issue, there is a known bug in version 2011-09-14 for reads of 100 bp or longer. This bug has been fixed in the latest release, 2011-10-01, available at the distribution Web site at http://research-pub.gene.com/gmap. Sorry for any inconvenience. If you have any further issues, you can send email directly to me at [email protected].

Regards,

Thomas D. Wu, M.D., Ph.D.
Senior Scientist
Bioinformatics and Computational Biology
Genentech, Inc.
South San Francisco, CA 94080, USA

[uma.maheswari]

Hi Thomas,

I am getting similar error as Irina with the current version 2012-07-20 (http://research-pub.gene.com/gmap/sr...2-07-20.tar.gz)

thanks
Uma

[twu]

Error

Hi Uma,

Please feel free to send more details to me at [email protected], and I can take a closer look. I have had no other bug reports from others so far on the 2012-07-20 release, so I would be interested to know what you are seeing.

Regards,

Tom

[simonwhite1971]

Error

I am seeing a similar error with version 2012-11-09

[afkoeppel]

I am trying to align paired end reads with GSNAP (version 2013-03-27) but I am getting a similar error.

My input fastq file is in the format described in the readme:

=================================
>@HWI-ST273:279:C05D8ACXX:7:1101:12952:5966
CTTTTAAGAGGATTCTTTCTGAGCAAATTTCAGTTATGAGGTAGATTCTAATTGTTCAATGAGTAACATTCTCAGGATTTCTAACTCAAGTGATCAGATC
GATCACTTGAGTTAGAAATCCTGAGAATGTTACTCATTGAACAATTAGAATCTACCTCATAACTGAAATTTGCTCAGAAAGAATCCTCTTAAAAGAGATC
+
CCCFFFFFHHHHHJJJJJJJJGIJJJJJJJJJJIJJJJJJJFHIJJJJJJIJJJIJJJJJJJJIJJIJJJJJJJJJJJJIJJJJJHHHHHEEFFFFEFEC
CCCFFFFFHHHHHJJJJJJJJJJJJJJJJJJJJJJJJJIJJJJJJJJJJJIJJJJJJJJJJJJJJJJJJJJJJJJIJJJJJJHHHHHFFFFFFFEDEEED
>@HWI-ST273:279:C05D8ACXX:7:1101:19991:5793
CCCGACTCCTTCAACCCACCAATGCCAAGCTGAGCCCTGCTTTCTCCCACAAGTCCCATATCTGGTGCCAAAAGTATTATTCCCAAGCTTGATCCATAAC
GGCCTTCCAAGGTGAAACCTGAGTTATGGATCAAGCTTGGGAATAATACTTTTGGCACCAGATATGGGACTTGTGGGAGAAAGCAGGGCTCAGCTTGGCA
+
CCCFFFFFHHHHHJJJJJJJJIJJJJJJJJJJJJJJJJJJJJJJJJJJJJIJJJJJJJJJJJJHHHHHFFFEDE6;CEEEFEEDDCDDDDDDDDCDCCD@
CCCFFFFFHHHHFHJJJJJIJHIHIIIJIHIJJIIJJGIJJHIJJJIJJJGIIJJJJJJJJIJIIJJJIIHHHHHFFFFDEEECDDDDDDDDDDDDDDD@
============================

etc.

I called GSNAP as follows:
gsnap -t 4 -A sam -m 4 -d mm9 -s mm9.introns.iit -A sam CRF0003_GsnapForm.fastq > test.sam

and got:

290272 unique splicesites...
290272 splicesites are valid...splicetrie_obs has 290272 entries...done
GMAP modes: pairsearch, indel_knownsplice, terminal, improvement
Starting alignment
Unexpected pairtype 0
Aborted

It did create a partial alignment with (test.sam had about 4k lines in what looked to be good SAM format), but it seems to be quitting partway through.

Any ideas as to what I might be doing wrong?

[afkoeppel]

I am trying to align paired end reads with GSNAP (version 2013-03-27) but I am getting a similar error.

My input fastq file is in the format described in the readme:

=================================
>@HWI-ST273:279:C05D8ACXX:7:1101:12952:5966
CTTTTAAGAGGATTCTTTCTGAGCAAATTTCAGTTATGAGGTAGATTCTAATTGTTCAATGAGTAACATTCTCAGGATTTCTAACTCAAGTGATCAGATC
GATCACTTGAGTTAGAAATCCTGAGAATGTTACTCATTGAACAATTAGAATCTACCTCATAACTGAAATTTGCTCAGAAAGAATCCTCTTAAAAGAGATC
+
CCCFFFFFHHHHHJJJJJJJJGIJJJJJJJJJJIJJJJJJJFHIJJJJJJIJJJIJJJJJJJJIJJIJJJJJJJJJJJJIJJJJJHHHHHEEFFFFEFEC
CCCFFFFFHHHHHJJJJJJJJJJJJJJJJJJJJJJJJJIJJJJJJJJJJJIJJJJJJJJJJJJJJJJJJJJJJJJIJJJJJJHHHHHFFFFFFFEDEEED
>@HWI-ST273:279:C05D8ACXX:7:1101:19991:5793
CCCGACTCCTTCAACCCACCAATGCCAAGCTGAGCCCTGCTTTCTCCCACAAGTCCCATATCTGGTGCCAAAAGTATTATTCCCAAGCTTGATCCATAAC
GGCCTTCCAAGGTGAAACCTGAGTTATGGATCAAGCTTGGGAATAATACTTTTGGCACCAGATATGGGACTTGTGGGAGAAAGCAGGGCTCAGCTTGGCA
+
CCCFFFFFHHHHHJJJJJJJJIJJJJJJJJJJJJJJJJJJJJJJJJJJJJIJJJJJJJJJJJJHHHHHFFFEDE6;CEEEFEEDDCDDDDDDDDCDCCD@
CCCFFFFFHHHHFHJJJJJIJHIHIIIJIHIJJIIJJGIJJHIJJJIJJJGIIJJJJJJJJIJIIJJJIIHHHHHFFFFDEEECDDDDDDDDDDDDDDD@
============================

etc.

I called GSNAP as follows:
gsnap -t 4 -A sam -m 4 -d mm9 -s mm9.introns.iit -A sam CRF0003_GsnapForm.fastq > test.sam

and got:

290272 unique splicesites...
290272 splicesites are valid...splicetrie_obs has 290272 entries...done
GMAP modes: pairsearch, indel_knownsplice, terminal, improvement
Starting alignment
Unexpected pairtype 0
Aborted

It did create a partial alignment with (test.sam had about 4k lines in what looked to be good SAM format), but it seems to be quitting partway through.

Any ideas as to what I might be doing wrong?

Quick Update: When I limited my input file to 1000 reads it worked just fine. There's something it's not liking about some (but not all) of my sequences.

One of the offending sequences is:
>@HWI-ST273:279:C05D8ACXX:7:1201:18203:90202
GTATAGTAATGCCTGCGGCTAGCACTGGTAGTGATAATAGGAGCAGTACGGCTGTAATAAGTACGGATCAGACAAATAGTGGAGTTTGATACTAGATCGG
AGTATCAAACTCCACTATTTGTCTGATCCGTACTTATTACAGCCGTACTGCTCCTATTATCACTACCAGTGCTAGCCGCAGGCATTACTATACAGATCGG
+
BB@FFFEDHHHHHJJJJIGJJJJIJIII?DEHGGIJJJGIJIJIGH@F@HIIGI@ECHEGIEHHEDFFDEECE=C?CDDDDADB:ACC3@ACD@>@C@BD
CCCF<DEFHFHHGJJIJIJJJHIJJIIIIJHHIJIJJIIJJJIGGGFHIHHHHHIJJJIIIJJJJJIIJGHJIIHHHHFDDDDDDDDCDEEDDEDDDDDD

...but I'm not seeing any obvious difference between it and the others.

[twu]

Please use version 2013-03-31

That particular bug was fixed in version 2013-03-31. Sorry for the inconvenience.

Regards,

Tom

[zuotian]

Hi,

I still run into the segmentation fault in the 2014-05-15 version. Here is the error message.

=====================================
Looking for index files in directory /references/H.Sapiens/hg20/gmap/reference
Pointers file is reference.ref153offsets64meta
Offsets file is reference.ref153offsets64strm
Positions file is reference.ref153positions
Offsets compression type: bitpack64
Allocating memory for ref offset pointers, kmer 15, interval 3...done (134,217,744 bytes, 1.32 sec)
Allocating memory for ref offsets, kmer 15, interval 3...done (470,734,416 bytes, 4.19 sec)
Allocating memory for ref positions, kmer 15, interval 3...done (3,913,147,304 bytes, 34.40 sec)
GMAP modes: pairsearch, indel_knownsplice, terminal, improvement
Starting alignment
Signal received: Segmentation fault
Problem sequence: FCC2EJEACXX:8:1214:12079:6463#ATCACGAT (90 bp)
>FCC2EJEACXX:8:1214:12079:6463#ATCACGAT
ACGCATACACACAGAGACATACATACACATGTGCATACACACATACAAACACACGCATACACACATACACACGCATACACACAGAGACAT
TGTGTATGCATGTGTGTGTATGCATATGTGTATGTGTGTATGCGTGTGTATGTCTCTGTGTGTATGCATGTGTATGTGTATATGTGTGTG
/bin/sh: line 1: 41324 Aborted (core dumped) SGE_RREQ="-q all.q -pe BWA 4 -l h_vmem=8G" /usr/local/gmap/gmap-2014-05-15/bin/gsnap --format sam --nthreads 4 --db reference --novelsplicing 1 --npaths 1 --batch 4 --quiet-if-excessive --dir /references/H.Sapiens/hg20/gmap read1.qc.fq read2.qc.fq > output.hg20.sam
=====================================

I used the hg20 analysis set as the reference. The gmap index worked fine for some samples, but not this one.

Thanks,
Zuotian

[hohosharon]

[GSNAP] Signal received: Segmentation fault

I am trying to align single end reads with GSNAP (version 2014-09-22) but I am getting a similar error.

My input fastq file is in the format described in the readme:

============[Sample 1]===============
@HWUSI-EAS1759R:40:FC:6:30:3714:19225 1:N:0:AGTCAA
CGGGGTTTCACCATCTTAACCAGGCTGGTCTCAAACTCCTGACCTCAGGTG
+
GGG4GIIIIIIHIIIIIGIIHIIIIHIIIIIIIIIIIIIIDHIIIIIIICG
==================================

============[Sample 2]===============
@HWUSI-EAS1759R:52:FC:2:20:5324:12059 1:N:0:TGACCA
CTCAGGTGATCCAACCGTCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGT
+
IIIIHGFGGGIEIHIIGHIHIIGIIIIHIIB>EAEIFIIGHBHEGIIHIIC
==================================

etc.

I called GSNAP as follows:
gsnap -d hg19 -s hg19.splicesites --query-unk-mismatch=1 -N 1 -B 4 -t 20 -O -A sam --force-xs-dir --force-single-end sample1.fastq >output.sam


and got:
[Sample 1]
Signal received: Segmentation fault
Problem sequence: HWUSI-EAS1759R:40:FC:6:30:3714:19225 (51 bp)
>HWUSI-EAS1759R:40:FC:6:30:3714:19225
CGGGGTTTCACCATCTTAACCAGGCTGGTCTCAAACTCCTGACCTCAGGTG

[Sample 2]
Signal received: Segmentation fault
Problem sequence: HWUSI-EAS1759R:52:FC:2:20:5324:12059 (51 bp)
>HWUSI-EAS1759R:52:FC:2:20:5324:12059
CTCAGGTGATCCAACCGTCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGT


I wanna to see where is alternative splicing, or fusion gene...
Any ideas as to what I might be doing wrong?

thank you,
Sharon

[hohosharon]

solve my Segmentation fault problem

I think I figure out the problem....

My command line should "-B 2" instead of "-B 4" for single-end reads.

and when I change the optional....the "Segmentation fault" is disappear