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MaSuRCA 3.2.8: sometimes problems with Illumina+ONT assembly #58
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I had run a few more assemblies (now with more ONT data) and I keep running into problems in later stages of the assembly: buffer overflow during the correct overlaps step (as of now 6 of such overflow error were detected) [Do 13. Sep 22:57:55 CEST 2018] Processing pe library reads |
tried different parameters and subsets of the input ONT data without success. I think the biggest issues is at the Gap consensus step with ufasta (see below). I am running out of ideas what to change/try. Any advice would be greatly appreciated! /opt/MaSuRCA-3.2.8/bin/mega_reads_assemble_cluster.sh: line 620: 52887 Aborted (core dumped) ufasta split -i refs.renamed.fa ${ref_names[@]} [So 23. Sep 22:05:12 CEST 2018] Gap consensus |
It seems MaSuRCA have some issues at consensus calling stage. I got the same error with Illumina PE+ONT reads.
|
Hello, I have the same problem when running MaSuRCA with Illumina+ONT reads. Genome size should be 150Mb. Looking at CA.mr.41.15.15.0.02.log I find:
Here is the log file: CA.mr.41.15.15.0.02.log AH |
Dear @alekseyzimin, We are also having the same problem when using MaSuRCA 3.2.8 for illumina PE + ONT data in the gap consensus step. ** Warning! Some or all gap consensus jobs failed, see files in mr.41.15.15.0.02.join_consensus.tmp We tested two different sets of data for different species and we got the same error log. |
Dear @alekseyzimin I have been checking the code and I would guess that the problem might be in line 504 from file mega_reads_assemble_cluster.sh
I wonder if other users also experimenting this issue could tell us if they are seeing the same problem. Thanks, |
Hi,
The most likely cause of this problem is incomplete/failed MaSuRCA
installation. Please re-run install.sh, and capture the output and look
for errors. I guess Mummer did not compile properly. Please let me know
what you find out.
…--Aleksey
On Mon, Nov 12, 2018 at 5:13 AM Jose Francisco Sanchez-Herrero < ***@***.***> wrote:
Dear @alekseyzimin <https://github.com/alekseyzimin>
I have found that during the Refining of alignments, files
*$COORDS.matches*.all.txt.tmp* are empty, only containing ">". That
generates that the following steps result in errors leading to the final
error in gap consensus step that we have reported here.
I have been checking the code and I would guess that the problem might be
in line 504 from file *mega_reads_assemble_cluster.sh*
cat <(ufasta extract -f $COORDS.single.txt $COORDS.txt) <(ufasta extract
-v -f $COORDS.single.txt $COORDS.mr.txt)| awk
'{if($0~/^>/){pb=substr($1,2);print $0} else { print $3" "$4" "$5" "$6"
"$10" "pb" "$11" "$9}}' | add_pb_seq.pl $LONGREADS1 |
split_matches_file.pl $NUM_LONGREADS_READS_PER_BATCH .matches && ls
.matches.* | xargs -P $NUM_THREADS -I % refine.sh $COORDS % $KMER && cat
$COORDS.matches*.all.txt.tmp > $COORDS.all.txt && rm .matches.* && rm
$COORDS.matches*.all.txt.tmp
I wonder if other users also experimenting this issue could tell us if
they are seeing the same problem.
Thanks,
P.D. We really appreciate your work on MaSurCa and we are all waiting to
resolve this problem and obtain a draft assembly for our data.
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Johns Hopkins University,
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(301)-437-6260
http://www.genome.umd.edu
http://masurca.blogspot.com
|
Hello, |
Yep, that is the problem. Can you see why? Is there an error during
compilation of libumdmummer?
…On Thu, Nov 15, 2018 at 1:10 PM Antoine Houtain ***@***.***> wrote:
Hello,
After a quick check I find in the logs:
libtool: install: warning: 'libumdmummer' has not been installed in lib
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Dr. Alexey V. Zimin
Associate Research Scientist
Department of Biomedical Engineering,
Johns Hopkins University,
Baltimore, MD, USA
(301)-437-6260
http://www.genome.umd.edu
http://masurca.blogspot.com
|
I checked the lib directory and find the libumdmummer.la file in it. In the bin directory mummer seems to be working. I rapidly checked the install.sh log but couldn't find anything here are the log files: installsh.cout.log Thanks for your help! |
Yes, another installation of mummer (especially from miniconda) will cause
exactly that problem. Mummer is installed along with MaSuRCA. Can you
remove mummer from the path and try to reinstall?
…On Thu, Nov 15, 2018 at 2:52 PM Antoine Houtain ***@***.***> wrote:
I checked the lib directory and find the libumdmummer.la file in it. In
the bin directory mummer seems to be working.
Is it possible that another mummer in $PATH causes troubles?
I rapidly checked the install.sh log but couldn't find anything here are
the log files:
installsh.cout.log
<https://github.com/alekseyzimin/masurca/files/2586790/installsh.cout.log>
install.log
<https://github.com/alekseyzimin/masurca/files/2586791/install.log>
Thanks for your help!
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Associate Research Scientist
Department of Biomedical Engineering,
Johns Hopkins University,
Baltimore, MD, USA
(301)-437-6260
http://www.genome.umd.edu
http://masurca.blogspot.com
|
Hi there,
I wonder if other users have experienced the same after reinstalling or if you guys did solve the problem and got the assembler running succesfully. |
No, I have not hear that this problem is common. Can you delete the
MaSuRCA and re-untar/unzip and re-run install.sh on a clean configuration
and see if there are any errors?
…--Aleksey
On Mon, Nov 19, 2018 at 10:58 AM Jose Francisco Sanchez-Herrero < ***@***.***> wrote:
Hi there,
I discarded from my path and environment all mummer libs I found and I
downloaded and reinstalled MaSuRCA 3.2.8 but I still got the same problem
as I stated before:
Dear @alekseyzimin <https://github.com/alekseyzimin>
I have found that during the Refining of alignments, files
*$COORDS.matches.all.txt.tmp** are empty, only containing ">". That
generates that the following steps result in errors leading to the final
error in gap consensus step that we have reported here.
I have been checking the code and I would guess that the problem might be
in line 504 from file *mega_reads_assemble_cluster.sh*
cat <(ufasta extract -f $COORDS.single.txt $COORDS.txt) <(ufasta extract
-v -f $COORDS.single.txt $COORDS.mr.txt)| awk
'{if($0~/^>/){pb=substr($1,2);print $0} else { print $3" "$4" "$5" "$6"
"$10" "pb" "$11" "$9}}' | add_pb_seq.pl $LONGREADS1 |
split_matches_file.pl $NUM_LONGREADS_READS_PER_BATCH .matches && ls
.matches.* | xargs -P $NUM_THREADS -I % refine.sh $COORDS % $KMER && cat
$COORDS.matches*.all.txt.tmp > $COORDS.all.txt && rm .matches.* && rm
$COORDS.matches*.all.txt.tmp
I wonder if other users have experienced the same after reinstalling or if
you guys did solve the problem and got the assembler running succesfully.
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Associate Research Scientist
Department of Biomedical Engineering,
Johns Hopkins University,
Baltimore, MD, USA
(301)-437-6260
http://www.genome.umd.edu
http://masurca.blogspot.com
|
Yes I did. |
The thing is that I got to finish the first test I did with illumina and Nanopore data a couple of months ago with no errors. |
Hello, I am assembling a genome of approximately 150 Mbs using 25x Pacbio data and ~70x Illumina data. And I believe that I am running into the same problem as mentioned here before, I have been following this thread for a while. [Mon 19 Nov 12:15:37 GMT 2018] Processing pe library reads Thanks for helping and providing this assembler! |
Hi,
Likely not the same problem. Can you send me the output of "ls -lth" on
your assembly folder to aleksey.zimin@gmail.com
…On Tue, Nov 20, 2018 at 10:43 AM LironShv ***@***.***> wrote:
Hello,
I am assembling a genome of approximately 150 Mbs using 25x Pacbio data
and ~70x Illumina data. And I believe that I am running into the same
problem as mentioned here before, I have been following this thread for a
while.
I have run the assembly a couple of times, and even after reinstalling
masurca I get the following issues:
[Mon 19 Nov 12:15:37 GMT 2018] Processing pe library reads
[Mon 19 Nov 12:23:24 GMT 2018] Average PE read length 150
[Mon 19 Nov 12:23:24 GMT 2018] Using kmer size of 99 for the graph
[Mon 19 Nov 12:23:25 GMT 2018] MIN_Q_CHAR: 33
[Mon 19 Nov 12:23:25 GMT 2018] Creating mer database for Quorum
[Mon 19 Nov 12:35:57 GMT 2018] Error correct PE
[Mon 19 Nov 13:12:00 GMT 2018] Estimating genome size
[Mon 19 Nov 13:17:56 GMT 2018] Estimated genome size: 160727761
[Mon 19 Nov 13:17:56 GMT 2018] Creating k-unitigs with k=99
[Mon 19 Nov 13:29:51 GMT 2018] Computing super reads from PE
[Mon 19 Nov 14:37:22 GMT 2018] Using CABOG from
/home/ls001/genomes/software/MaSuRCA-3.2.8/bin/../CA8/Linux-amd64/bin
[Mon 19 Nov 14:37:22 GMT 2018] Running mega-reads correction/assembly
[Mon 19 Nov 14:37:22 GMT 2018] Using mer size 15 for mapping, B=17, d=0.029
[Mon 19 Nov 14:37:22 GMT 2018] Estimated Genome Size 160727761
[Mon 19 Nov 14:37:22 GMT 2018] Estimated Ploidy 1
[Mon 19 Nov 14:37:22 GMT 2018] Using 48 threads
[Mon 19 Nov 14:37:23 GMT 2018] Output prefix mr.41.15.17.0.029
[Mon 19 Nov 14:37:23 GMT 2018] Pacbio coverage >25x, using 25x of the
longest reads
[Mon 19 Nov 14:38:18 GMT 2018] Reducing super-read k-mer size
[Mon 19 Nov 14:44:41 GMT 2018] Mega-reads pass 1
[Mon 19 Nov 14:44:41 GMT 2018] Running locally in 1 batch
[Tue 20 Nov 09:09:30 GMT 2018] Mega-reads pass 2
[Tue 20 Nov 09:09:30 GMT 2018] Running locally in 1 batch
[Tue 20 Nov 11:08:27 GMT 2018] Refining alignments
[Tue 20 Nov 11:17:44 GMT 2018] Joining
[Tue 20 Nov 11:22:27 GMT 2018] Gap consensus
[Tue 20 Nov 11:22:30 GMT 2018] Warning! Some or all gap consensus jobs
failed, see files in mr.41.15.17.0.029.join_consensus.tmp, proceeding
anyway, to rerun gap consensus erase mr.41.15.17.0.029.1.fa and re-run
assemble.sh
[Tue 20 Nov 11:22:49 GMT 2018] Generating assembly input files
[Tue 20 Nov 11:54:47 GMT 2018] Coverage threshold for splitting unitigs is
35 minimum ovl 250
[Tue 20 Nov 11:54:47 GMT 2018] Running assembly
Processed 500000 super reads, irreducible 399967, processing 1043 super
reads per second
*** buffer overflow detected ***: ufasta terminated
======= Backtrace: =========
/lib/x86_64-linux-gnu/libc.so.6(+0x777e5)[0x7f253f2d37e5]
/lib/x86_64-linux-gnu/libc.so.6(__fortify_fail+0x5c)[0x7f253f37515c]
/lib/x86_64-linux-gnu/libc.so.6(+0x117160)[0x7f253f373160]
/lib/x86_64-linux-gnu/libc.so.6(+0x1190a7)[0x7f253f3750a7]
ufasta[0x41bd6e]
ufasta[0x403d96]
/lib/x86_64-linux-gnu/libc.so.6(__libc_start_main+0xf0)[0x7f253f27c830]
ufasta[0x404199]
======= Memory map: ========
00400000-00429000 r-xp 00000000 00:2c 425594311
/home/ls001/genomes/software/MaSuRCA-3.2.8/bin/ufasta
00628000-00629000 r--p 00028000 00:2c 425594311
/home/ls001/genomes/software/MaSuRCA-3.2.8/bin/ufasta
00629000-0062b000 rw-p 00029000 00:2c 425594311
/home/ls001/genomes/software/MaSuRCA-3.2.8/bin/ufasta
00bd6000-00c7f000 rw-p 00000000 00:00 0 [heap]
7f253f25c000-7f253f41c000 r-xp 00000000 08:02 23075723
/lib/x86_64-linux-gnu/libc-2.23.so
7f253f41c000-7f253f61c000 ---p 001c0000 08:02 23075723
/lib/x86_64-linux-gnu/libc-2.23.so
7f253f61c000-7f253f620000 r--p 001c0000 08:02 23075723
/lib/x86_64-linux-gnu/libc-2.23.so
7f253f620000-7f253f622000 rw-p 001c4000 08:02 23075723
/lib/x86_64-linux-gnu/libc-2.23.so
ect.
ffffffffff600000-ffffffffff601000 r-xp 00000000 00:00 0 [vsyscall]
/home/ls001/genomes/software/MaSuRCA-3.2.8/bin/mega_reads_assemble_cluster.sh:
line 620: 33945
Aborted (core dumped) ufasta split -i refs.renamed.fa ${ref_names[@]}
xargs: invalid number "-I" for -P option
Usage: xargs [OPTION]... COMMAND [INITIAL-ARGS]...
Run COMMAND with arguments INITIAL-ARGS and more arguments read from input.
Thanks for helping and providing this assembler!
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Johns Hopkins University,
Baltimore, MD, USA
(301)-437-6260
http://www.genome.umd.edu
http://masurca.blogspot.com
|
Dear @alekseyzimin, I have downloaded it and installed it from scratch (taking into account for mummer dependencies again). But I got the same error in the Refining of Alignments.
As stated before, I got thousands of error messages saying:
I have noticed that now join_consensus folder contains some files, although most of them are empty.
I have also attached a txt file with "ls -lth" of my assembly folder. I am still thinking that the error is coming from Refining alignments step where all files $COORDS.matches.all.txt.tmp contain only ">" and no sequence at all. Thank you very much |
I think my problem is also different. the mummer library was present in my previous install (which made problems). After installing the new 2.3.9 version, the was not ufasta related gap-consensus error anymore, so thats good. In the assembly step I still get Buffer overflow errors: Running assembly With the previous install I sometimes had error-free assembly runs with the same Illumina input data and settings but with a much smaller subset of ONT reads. Thatswhy I initially suspected the ONT input is the problem. I, however, can't any obvious problem with it (re-basecalling or check s for proper formatting etc did not give any problematic results). |
I get the different error. The work stop at the 4-unitigger.
Fragment correction job 1089 failed.
Fragment correction job 1090 failed.
================================================================================
runCA failed.
----------------------------------------
Stack trace:
at /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin/runCA
line 1613.
main::caFailure('1090 overlap jobs failed; remove
/scratch/snyder/f/fu115/Geno...', undef) called at
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin/runCA
line 4372
main::overlapCorrection() called at
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin/runCA
line 6526
----------------------------------------
Failure message:
1090 overlap jobs failed; remove
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/3-overlapcorrection/frgcorr.sh
to try again
----------------------------------------START Tue Nov 20 17:31:47 2018
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart -O
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.ovlStore
-G
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.gkpStore
-T
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.tigStore
-B 1702554 -eg 0.03 -Eg 1000 -em 0.03 -Em 1000 -repeatdetect 76 76 76
-el 63 -RS -o
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/genome
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/unitigger.err
2>&1
sh: line 1: 27889 Segmentation fault
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart -O
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.ovlStore
-G
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.gkpStore
-T
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.tigStore
-B 1702554 -eg 0.03 -Eg 1000 -em 0.03 -Em 1000 -repeatdetect 76 76 76 -el
63 -RS -o
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/genome
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/unitigger.err
2>&1
----------------------------------------END Tue Nov 20 18:10:42 2018 (2335
seconds)
ERROR: Failed with signal SEGV (11)
================================================================================
runCA failed.
----------------------------------------
Stack trace:
at /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin/runCA
line 1613.
main::caFailure('failed to unitig',
'/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.m...') called
at /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin/runCA
line 4795
main::unitigger() called at
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin/runCA
line 6528
----------------------------------------
Last few lines of the relevant log file
(/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/unitigger.err):
WARNING: bogus overlap found for A=653754368 B=570347520
WARNING: A len=74 hang=14 ovl=-42671
WARNING: B len=72 hang=-42731 ovl=72
WARNING: bogus overlap found for A=1257537536 B=530341888
WARNING: A len=-389244560 hang=2713 ovl=-389275920
WARNING: B len=71 hang=-28647 ovl=71
WARNING: bogus overlap found for A=1257701376 B=526004224
WARNING: A len=-1933301984 hang=3180 ovl=-1933305164
WARNING: B len=71 hang=11298 ovl=-11227
WARNING: bogus overlap found for A=1257734144 B=526061568
WARNING: A len=-1393298272 hang=2744 ovl=-1393301016
WARNING: B len=71 hang=21029 ovl=-20958
WARNING: bogus overlap found for A=1257734144 B=526077952
WARNING: A len=-1393298272 hang=5968 ovl=-1393363583
WARNING: B len=71 hang=-59343 ovl=71
WARNING: bogus overlap found for A=1257799680 B=525946880
WARNING: A len=-296162976 hang=283 ovl=-296163259
WARNING: B len=71 hang=30471 ovl=-30400
WARNING: bogus overlap found for A=1257799680 B=526090240
WARNING: A len=-296162976 hang=291 ovl=-296163267
WARNING: B len=71 hang=46379 ovl=-46308
WARNING: bogus overlap found for A=1257799680 B=531431424
WARNING: A len=-296162976 hang=4821 ovl=-296230469
WARNING: B len=71 hang=-62672 ovl=71
Failed with 'Segmentation fault'
Backtrace (mangled):
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x477426]
/usr/lib64/libpthread.so.0(+0xf5e0)[0x2b58994d45e0]
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x45d756]
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x45e0f4]
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x4031b7]
/usr/lib64/libc.so.6(__libc_start_main+0xf5)[0x2b589a1cac05]
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x404724]
Backtrace (demangled):
[0] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x477426]
[1] /usr/lib64/libpthread.so.0::(null) + 0xf5e0 [0x2b58994d45e0]
[2] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x45d756]
[3] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x45e0f4]
[4] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x4031b7]
[5] /usr/lib64/libc.so.6::(null) + 0xf5 [0x2b589a1cac05]
[6] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x404724]
GDB:
----------------------------------------
Failure message:
failed to unitig
----------------------------------------START Tue Nov 20 18:10:43 2018
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart -O
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.ovlStore
-G
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.gkpStore
-T
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.tigStore
-B 1702554 -eg 0.03 -Eg 1000 -em 0.03 -Em 1000 -repeatdetect 76 76 76
-el 63 -RS -o
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/genome
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/unitigger.err
2>&1
sh: line 1: 35180 Segmentation fault
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart -O
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.ovlStore
-G
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.gkpStore
-T
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/genome.tigStore
-B 1702554 -eg 0.03 -Eg 1000 -em 0.03 -Em 1000 -repeatdetect 76 76 76 -el
63 -RS -o
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/genome
/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/unitigger.err
2>&1
----------------------------------------END Tue Nov 20 18:46:32 2018 (2149
seconds)
ERROR: Failed with signal SEGV (11)
================================================================================
runCA failed.
…----------------------------------------
Stack trace:
at /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin/runCA
line 1613.
main::caFailure('failed to unitig',
'/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.m...') called
at /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin/runCA
line 4795
main::unitigger() called at
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin/runCA
line 6528
----------------------------------------
Last few lines of the relevant log file
(/scratch/snyder/f/fu115/Genome_assembly/masurca/Assembly/CA.mr.41.15.17.0.029/4-unitigger/unitigger.err):
WARNING: bogus overlap found for A=653754368 B=570347520
WARNING: A len=74 hang=14 ovl=-42671
WARNING: B len=72 hang=-42731 ovl=72
WARNING: bogus overlap found for A=1257635840 B=525258752
WARNING: A len=-1775698480 hang=4303 ovl=-1775707105
WARNING: B len=71 hang=-4322 ovl=71
WARNING: bogus overlap found for A=1257668608 B=526073856
WARNING: A len=-1238679648 hang=495 ovl=-1238680143
WARNING: B len=71 hang=0 ovl=71
WARNING: bogus overlap found for A=1257701376 B=526004224
WARNING: A len=-687012064 hang=3180 ovl=-687015244
WARNING: B len=71 hang=11298 ovl=-11227
WARNING: bogus overlap found for A=1257734144 B=526061568
WARNING: A len=-147008352 hang=2744 ovl=-147011096
WARNING: B len=71 hang=21029 ovl=-20958
WARNING: bogus overlap found for A=1257734144 B=526077952
WARNING: A len=-147008352 hang=5968 ovl=-147073663
WARNING: B len=71 hang=-59343 ovl=71
WARNING: bogus overlap found for A=1257799680 B=525946880
WARNING: A len=950126944 hang=283 ovl=950126661
WARNING: B len=71 hang=30471 ovl=-30400
WARNING: bogus overlap found for A=1257799680 B=526090240
WARNING: A len=950126944 hang=291 ovl=950126653
WARNING: B len=71 hang=46379 ovl=-46308
Failed with 'Segmentation fault'
Backtrace (mangled):
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x477426]
/usr/lib64/libpthread.so.0(+0xf5e0)[0x2afde39625e0]
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x45d756]
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x45e0f4]
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x4031b7]
/usr/lib64/libc.so.6(__libc_start_main+0xf5)[0x2afde4658c05]
/depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart[0x404724]
Backtrace (demangled):
[0] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x477426]
[1] /usr/lib64/libpthread.so.0::(null) + 0xf5e0 [0x2afde39625e0]
[2] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x45d756]
[3] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x45e0f4]
[4] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x4031b7]
[5] /usr/lib64/libc.so.6::(null) + 0xf5 [0x2afde4658c05]
[6] /depot/bioinfo/apps/apps/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/bogart()
[0x404724]
GDB:
----------------------------------------
Failure message:
failed to unitig
The runSpec is
batOptions=-repeatdetect 76 76 76 -el 63 -RS
useGrid=0
cnsOnGrid=0
gridSubmitCommand=qsub
cnsConcurrency=20
cnsMinFrags=10000
obtMerSize=22
ovlMerSize=22
unitigger=bogart
merylMemory=65536
ovlStoreMemory=65536
utgGraphErrorLimit=1000
utgMergeErrorLimit=1000
utgGraphErrorRate=0.03
utgMergeErrorRate=0.03
ovlCorrBatchSize=100000
ovlCorrConcurrency=8
frgCorrThreads=20
frgCorrConcurrency=6
mbtThreads=20
ovlThreads=2
ovlHashBlockLength=10000000
ovlRefBlockSize=197194010
ovlConcurrency=20
doOverlapBasedTrimming=1
doUnitigSplitting=0
doChimeraDetection=normal
merylThreads=20
stoneLevel=0
doExtendClearRanges=0
computeInsertSize=0
maxRepeatLength=12000
ovlErrorRate=0.1
cnsErrorRate=0.1
cgwErrorRate=0.1
cgwMergeMissingThreshold=-1
cgwMergeFilterLevel=1
cgwDemoteRBP=0
cnsReuseUnitigs=1
doFragmentCorrection=0
On Wed, Nov 21, 2018 at 6:37 AM stollec ***@***.***> wrote:
I think my problem is also different. the mummer library was present in my
previous install (which made problems). After installing the new 2.3.9
version, the was not ufasta related gap-consensus error anymore, so thats
good. In the assembly step I still get Buffer overflow errors:
Running assembly
*** buffer overflow detected ***:
/opt/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/correct-olaps terminated
======= Backtrace: =========
/lib/x86_64-linux-gnu/libc.so.6(+0x777e5)[0x7ff08df2a7e5]
/lib/x86_64-linux-gnu/libc.so.6(__fortify_fail+0x5c)[0x7ff08dfcc15c]
/lib/x86_64-linux-gnu/libc.so.6(+0x117160)[0x7ff08dfca160]
/lib/x86_64-linux-gnu/libc.so.6(+0x1164b2)[0x7ff08dfc94b2]
/opt/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/correct-olaps[0x403b12]
/opt/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/correct-olaps[0x4026d4]
/lib/x86_64-linux-gnu/libc.so.6(__libc_start_main+0xf0)[0x7ff08ded3830]
/opt/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/correct-olaps[0x402c09]
======= Memory map: ========
00400000-00424000 r-xp 00000000 08:12 3935483
/opt/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/correct-olaps
00623000-00624000 r--p 00023000 08:12 3935483
/opt/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/correct-olaps
00624000-00625000 rw-p 00024000 08:12 3935483
/opt/MaSuRCA-3.2.9/CA8/Linux-amd64/bin/correct-olaps
With the previous install I sometimes had error-free assembly runs with
the same Illumina input data and settings but with a much smaller subset of
ONT reads. Thatswhy I initially suspected the ONT input is the problem. I,
however, can't any obvious problem with it (re-basecalling or check s for
proper formatting etc did not give any problematic results).
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Hello, I get the same error after upgrading to Masurca 3.2.9
Further on in the log I also find:
When I go to the 3-overlapcorrection folder and look in the 000 (int) .err files I find :
When I look into .err files in the 8-consensus folder, I cannot find anything causing an error, all files genome_00X.err seem fine saying everything is already computed and skipped. Any idea? |
Dear @alekseyzimin, After some debugging and installing problems we finally sorted it out and get to install Masurca with no dependencies to other library previously installed in the server. The UFASTA error due to Mummer libraries problem was solved and Masurca successfully finished and generated a nice an long assembly! Thanks!
Thank you very much |
Hello, I have a similar problem with the gap consensus step with my assembly (80X Illumina PE 250 + 35X ONT). First I tried to assemble with v 3.2.8 and I got following errors:
The folder mr.41.15.15.0.02.join_consensus.tmp contained two emtpy files:
I then tried with v 3.2.9 (from bioconda) and got following errors:
Now the folder mr.41.15.15.0.02.join_consensus.tmp contains more files, but I couldn't find any error there:
The assembly is still running, I don't know whether it will succeed and if so, whether the result will be influenced by these errors. |
Excellent! Can you tell me what was the problem and how you've addressed
it?
…On Mon, Dec 3, 2018 at 4:18 AM Jose Francisco Sanchez-Herrero < ***@***.***> wrote:
Dear @alekseyzimin <https://github.com/alekseyzimin>,
After some debugging and installing problems we finally sorted it out and
get to install Masurca with no dependencies to other library previously
installed in the server.
The UFASTA error due to Mummer libraries problem was solved and Masurca
successfully finished and generated a nice an long assembly! Thanks!
I have found that during the Refining of alignments, files
*$COORDS.matches.all.txt.tmp** are empty, only containing ">". That
generates that the following steps result in errors leading to the final
error in gap consensus step that we have reported here.
As stated before, I got thousands of error messages saying:
ERROR: Could not parse delta file, /dev/stdin
error no: 402
I have noticed that now join_consensus folder contains some files,
although most of them are empty.
I am still thinking that the error is coming from Refining alignments step
where all files *$COORDS.matches.all.txt.tmp* contain only ">" and no
sequence at all.
Thank you very much
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Was it for me this comment?
I am afraid the problems was derived from UFASTA during the refining alingments although the error was coming later in the consensus step. What I had to do in order to avoid problems with MUmmer or any other libraries and successfully install masurca was:
Using this approach I avoided installing masurca in the whole server but only in the node where I was going to do the job but also I avoided mis-configuration of third party libraries such as Mummer discarding environment variables before installation. Thanks! |
Thank you for letting me know! Having clean environment is essential for
MaSuRCA installation.
All the Best,
Aleksey
…On Tue, Dec 4, 2018 at 10:40 AM Jose Francisco Sanchez-Herrero < ***@***.***> wrote:
Was it for me this comment?
Excellent! Can you tell me what was the problem and how you've addressed
it?
I am afraid the problems was derived from UFASTA during the refining
alingments although the error was coming later in the consensus step.
What I had to do in order to avoid problems with MUmmer or any other
libraries and successfully install masurca was:
1. I download from the github web, release 3.2.9 version
2. tar -zxvf into a folder
3. Modify install.sh. I added at the beginning of the lines to remove
PATH variable from my environment and only use basic variables. To do so i
added:
export
PATH=/usr/local/bin:/usr/bin:/bin:/usr/local/games:/usr/games:/opt/openmpi-184/bin:/usr/lib/jvm/java-8-oracle/bin:/usr/lib/jvm/java-8-oracle/db/bin:/usr/lib/jvm/java-8-oracle/jre/bin
1.
Because of the specific configuration of my server, I had to connect
to node within my server where I will be sending the command and install it
(sh install.sh).
2.
Generate the assemble.sh using the configuration file.
3.
Exit to the master node and send via grid engine queues the call for
masurca to the node where I had installed it.
Using this approach I avoided installing masurca in the whole server but
only in the node where I was going to do the job but also I avoided
mis-configuration of third party libraries such as Mummer discarding
environment variables before installation.
Thanks!
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http://masurca.blogspot.com
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Hello,
I think the error comes from something around line 582 of mega_reads_assemble_cluster.sh. Since MaSuRCA cannot find to_join.fa because of a buffer overflow in ufasta split Thanks! |
Hi,
Can you test ufasta split separately? That is run
ufasta split -i file.fasta file1 file2 file3 file4 -- this should split the
fasta file into 4 files
I have not yet seen buffer overflow error in ufasta split. But I can
change the code if it causes problems.
…--Aleksey
On Fri, Dec 7, 2018 at 12:14 PM Antoine Houtain ***@***.***> wrote:
Hello,
I installed with the added line export PATH=... I could not find errors in
the log of installation so I went on.
Then during the assembly I have the following issue:
[Fri Dec 7 00:06:00 CET 2018] Processing pe library reads
[Fri Dec 7 01:17:57 CET 2018] Average PE read length 250
[Fri Dec 7 01:17:57 CET 2018] Using kmer size of 127 for the graph
[Fri Dec 7 01:17:57 CET 2018] MIN_Q_CHAR: 33
[Fri Dec 7 01:17:57 CET 2018] Creating mer database for Quorum
[Fri Dec 7 01:37:04 CET 2018] Error correct PE
[Fri Dec 7 05:07:24 CET 2018] Estimating genome size
[Fri Dec 7 05:23:34 CET 2018] Estimated genome size: 187883011
[Fri Dec 7 05:23:34 CET 2018] Creating k-unitigs with k=127
[Fri Dec 7 06:44:43 CET 2018] Computing super reads from PE
[Fri Dec 7 08:40:48 CET 2018] Using CABOG from /home/lege/tools/MaSuRCA-3.2.9/bin/../CA8/Linux-amd64/bin
[Fri Dec 7 08:40:48 CET 2018] Running mega-reads correction/assembly
[Fri Dec 7 08:40:48 CET 2018] Using mer size 15 for mapping, B=15, d=0.02
[Fri Dec 7 08:40:48 CET 2018] Estimated Genome Size 187883011
[Fri Dec 7 08:40:48 CET 2018] Estimated Ploidy 1
[Fri Dec 7 08:40:48 CET 2018] Using 44 threads
[Fri Dec 7 08:40:48 CET 2018] Output prefix mr.41.15.15.0.02
[Fri Dec 7 08:40:48 CET 2018] Using 25x of the longest ONT reads
[Fri Dec 7 08:51:43 CET 2018] Reducing super-read k-mer size
[Fri Dec 7 09:05:35 CET 2018] Mega-reads pass 1
[Fri Dec 7 09:05:35 CET 2018] Running locally in 1 batch
[Fri Dec 7 11:31:42 CET 2018] Mega-reads pass 2
[Fri Dec 7 11:31:42 CET 2018] Running locally in 1 batch
[Fri Dec 7 14:31:26 CET 2018] Refining alignments
[Fri Dec 7 14:44:17 CET 2018] Joining
[Fri Dec 7 14:53:43 CET 2018] Gap consensus
*** buffer overflow detected ***: /home/lege/tools/MaSuRCA-3.2.9/bin/ufasta terminated
/home/lege/tools/MaSuRCA-3.2.9/bin/mega_reads_assemble_cluster.sh: line 624: 1742 Aborted (core dumped) $MYPATH/ufasta split -i refs.renamed.fa ${ref_names[@]}
Error: Failed to open reference file 'to_join.1.fa'
Usage: nucmer [options] ref:path qry:path+
Use --help for more information
ERROR: Could not parse delta file, /dev/stdin
error no: 402
Error: Failed to open reference file 'to_join.2.fa'
Usage: nucmer [options] ref:path qry:path+
Use --help for more information
ERROR: Could not parse delta file, /dev/stdin
error no: 402
Error: Failed to open reference file 'to_join.3.fa'
Usage: nucmer [options] ref:path qry:path+
Use --help for more information
ERROR: Could not parse delta file, /dev/stdin
error no: 402
Error: Failed to open reference file 'to_join.5.fa'
Usage: nucmer [options] ref:path qry:path+
Use --help for more information
Error: Failed to open reference file 'to_join.4.fa'
Usage: nucmer [options] ref:path qry:path+
Use --help for more information
ERROR: Could not parse delta file, /dev/stdin
error no: 402
ERROR: Could not parse delta file, /dev/stdin
error no: 402
Error: Failed to open reference file 'to_join.6.fa'
Usage: nucmer [options] ref:path qry:path+
Use --help for more information
ERROR: Could not parse delta file, /dev/stdin
error no: 402
xargs: ./do_consensus.sh: exited with status 255; aborting
xargs: ./do_consensus.sh: exited with status 255; aborting
Error: Failed to open reference file 'to_join.1.fa'
Usage: nucmer [options] ref:path qry:path+
Use --help for more information
ERROR: Could not parse delta file, /dev/stdin
error no: 402
Error: Failed to open reference file 'to_join.2.fa'
Usage: nucmer [options] ref:path qry:path+
Use --help for more information
ERROR: Could not parse delta file, /dev/stdin
error no: 402
xargs: ./do_consensus.sh: exited with status 255; aborting
xargs: ./do_consensus.sh: exited with status 255; aborting
cat: 'merges.[0-9]*.txt': No such file or directory
[Fri Dec 7 14:53:49 CET 2018] Warning! Some or all gap consensus jobs failed, see files in mr.41.15.15.0.02.join_consensus.tmp, proceeding anyway, to rerun gap consensus erase mr.41.15.15.0.02.1.fa and re-run assemble.sh
[Fri Dec 7 14:53:55 CET 2018] Generating assembly input files
[Fri Dec 7 15:15:02 CET 2018] Coverage threshold for splitting unitigs is 20 minimum ovl 250
[Fri Dec 7 15:15:02 CET 2018] Running assembly
I think the error comes from something around line 582 of
mega_reads_assemble_cluster.sh. Since MaSuRCA cannot find to_join.fa
because of a buffer overflow in ufasta split
Then it calls nucmer that seem to be well compiled
Any ideas?
Thanks!
AH
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I have tried to do it, it did not fail so I am searching now what caused the buffer overflow error. Join consensus files in the directory are empty and coords.1, coords.2 etc are also empty. However, ref.1.fa ref.2.fa, etc are not. |
you have to execute that command inside the mr......join_consensus.tmp
folder.
…On Fri, Dec 7, 2018 at 5:31 PM Antoine Houtain ***@***.***> wrote:
I found that when I do:
perl -ane '{if($F[0] =~ /^>/){$rn=$F[0];}else{$seq=$F[0]; $seq=~
tr/a-zA-Z//s; print "$rn\n$F[0]\n" if(length($seq)>length($F[0])*0.1);}}'
../mr.41.15.0.0.02.1.to_join.fa.tmp | ~/tools/MaSuRCA-3.2.9/bin/
split_reads_to_join.pl qrys.txt to_join (list of ref.fa files)
I get: Can't open ../mr.41.15.0.0.02.1.to_join.fa.tmp: No such file or
directory.
I find a mr.41.15.0.0.02.1.to_join.fa.tmp file of 4Gb in the directory
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http://masurca.blogspot.com
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Hi there
I was running into some issues recently when I tried to assemble a small insect (250Mb) genome with Illumina (120x, PE 150bp) with ca 25x ONT 1D data. I am getting these buffer overflow issues and errors at the Refining alignments step, but the script proceeds into the assembly and eventually finishes. With another individuals Illumina data as input I get sometimes problems at the gapfilling step, yet the process finishes eventually. Any ideas what could cause this or how it might impact the final result?
[Sa 8. Sep 12:49:07 CEST 2018] Processing pe library reads
[Sa 8. Sep 12:59:41 CEST 2018] Average PE read length 149
[Sa 8. Sep 12:59:41 CEST 2018] Using kmer size of 99 for the graph
[Sa 8. Sep 12:59:42 CEST 2018] MIN_Q_CHAR: 33
WARNING: JF_SIZE set too low, increasing JF_SIZE to at least 1314280647, this automatic increase may be not enough!
[Sa 8. Sep 12:59:42 CEST 2018] Creating mer database for Quorum
[Sa 8. Sep 13:10:32 CEST 2018] Error correct PE
[Sa 8. Sep 13:36:07 CEST 2018] Estimating genome size
[Sa 8. Sep 13:41:08 CEST 2018] Estimated genome size: 273786976
[Sa 8. Sep 13:41:08 CEST 2018] Creating k-unitigs with k=99
[Sa 8. Sep 13:56:11 CEST 2018] Computing super reads from PE
[Sa 8. Sep 14:36:19 CEST 2018] Using linking mates
[Sa 8. Sep 14:36:19 CEST 2018] Using CABOG from /opt/MaSuRCA-3.2.8/bin/../CA8/Linux-amd64/bin
[Sa 8. Sep 14:36:19 CEST 2018] Running mega-reads correction/assembly
[Sa 8. Sep 14:36:19 CEST 2018] Using mer size 15 for mapping, B=15, d=0.02
[Sa 8. Sep 14:36:19 CEST 2018] Estimated Genome Size 273786976
[Sa 8. Sep 14:36:19 CEST 2018] Estimated Ploidy 1
[Sa 8. Sep 14:36:19 CEST 2018] Using 100 threads
[Sa 8. Sep 14:36:19 CEST 2018] Output prefix mr.41.15.15.0.02
[Sa 8. Sep 14:36:19 CEST 2018] Using 25x of the longest ONT reads
[Sa 8. Sep 14:40:22 CEST 2018] Reducing super-read k-mer size
[Sa 8. Sep 14:47:19 CEST 2018] Mega-reads pass 1
[Sa 8. Sep 14:47:19 CEST 2018] Running locally in 1 batch
[Sa 8. Sep 19:05:51 CEST 2018] Mega-reads pass 2
[Sa 8. Sep 19:05:51 CEST 2018] Running locally in 1 batch
[Sa 8. Sep 20:02:50 CEST 2018] Refining alignments
ERROR: failed to merge alignments at position 273
Please file a bug report
ERROR: Could not parse delta file, /dev/stdin
error no: 402
ERROR: Could not parse delta file, /dev/stdin
error no: 402
[Sa 8. Sep 20:10:48 CEST 2018] Joining
[Sa 8. Sep 20:16:07 CEST 2018] Gap consensus
*** buffer overflow detected ***: ufasta terminated
======= Backtrace: =========
/lib/x86_64-linux-gnu/libc.so.6(+0x777e5)[0x7f72f58d77e5]
/lib/x86_64-linux-gnu/libc.so.6(__fortify_fail+0x5c)[0x7f72f597915c]
/lib/x86_64-linux-gnu/libc.so.6(+0x117160)[0x7f72f5977160]
/lib/x86_64-linux-gnu/libc.so.6(+0x1190a7)[0x7f72f59790a7]
ufasta[0x41bd6e]
ufasta[0x403d96]
/lib/x86_64-linux-gnu/libc.so.6(__libc_start_main+0xf0)[0x7f72f5880830]
ufasta[0x404199]
======= Memory map: ========
00400000-00429000 r-xp 00000000 08:12 3539149 /opt/MaSuRCA-3.2.8/bin/ufasta
00628000-00629000 r--p 00028000 08:12 3539149 /opt/MaSuRCA-3.2.8/bin/ufasta
00629000-0062b000 rw-p 00029000 08:12 3539149 /opt/MaSuRCA-3.2.8/bin/ufasta
01739000-01901000 rw-p 00000000 00:00 0 [heap]
...... [more entries here]
/opt/MaSuRCA-3.2.8/bin/mega_reads_assemble_cluster.sh: line 620: 49949 Aborted (core dumped) ufasta split -i refs.renamed.fa ${ref_names[@]}
xargs: invalid number "-I" for -P option
[more text of input parameters for the script here]
xargs: ./do_consensus.sh: No such file or directory
cat: 'merges.[0-9]*.txt': No such file or directory
[Sa 8. Sep 20:16:12 CEST 2018] Warning! Some or all gap consensus jobs failed, see files in mr.41.15.15.0.02.join_consensus.tmp, proceeding anyway, to rerun gap consensus erase mr.41.15.15.0.02.1.fa and re-run assemble.sh
[Sa 8. Sep 20:16:15 CEST 2018] Generating assembly input files
[Sa 8. Sep 20:30:35 CEST 2018] Coverage threshold for splitting unitigs is 27 minimum ovl 98
[Sa 8. Sep 20:30:35 CEST 2018] Running assembly
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