Installation issues

[ShevchenkoAlla]

Hi,
I have tried to run opera-ms and again, got error

After some digging in error files I see this -
"
/beegfs/shevch_alla/shevch_alla/OPERA-MS//tools_opera_ms//samtools: error while loading shared libraries: libncurses.so.5: cannot open shared object file: No such file or directory
"

So It didn't installed libncurses.so.5: in samtools,

So I have checked - dependencies -

"
python OPERA-MS-UTILS.py check-dependency
/beegfs/shevch_alla/shevch_alla/OPERA-MS/src_utils/checkm_analysis.py:51: SyntaxWarning: invalid escape sequence '\s'
line_list = re.split(" \s+", line)
MetaBAT2 functioning
Kraken2 functioning
/bin/sh: /beegfs/shevch_alla/shevch_alla/OPERA-MS/src_utils/../tools_opera_ms//checkm: No such file or directory
CheckM *** NOT FUNCTIONING ***
Can't open perl script "/beegfs/shevch_alla/shevch_alla/OPERA-MS/src_utils/../tools_opera_ms//maxbin2/run_MaxBin.pl": No such file or directory
MaxBin2 *** NOT FUNCTIONING ***
"

Part of them are missing,
Your suggestion is to create a conda env and install them there, but how opera-ms will work, if I haven't installed it in this particular environment?

conda create -n opera-ms python=3.6
conda activate opera-ms
conda install -c bioconda maxbin2
ln -sf $(dirname $(readlink -f which run_MaxBin.pl)) tools_opera_ms/maxbin2
conda install -c bioconda checkm-genome
ln -sf $(readlink -f which checkm) tools_opera_ms/
pip install pandas==1.0.3
pip install scikit-learn==0.22.2

It's confusing, I am sorry.

Could you please provide full instructions from the begining, to get opera-ms installation properly?

Thanks,
Alla

[jsgounot]

Please check my answer in a separated issue here.

[ShevchenkoAlla]

Please check my answer in a separated issue here.

It didn't work
I still have - samtools: error while loading shared libraries: libcrypto.so.1.0.0: cannot open shared object file: No such file or directory

Althought,

perl OPERA-MS.pl check-dependency
*** OPERA-LG functional
*** sigma functional

samtools found in /beegfs/shevch_alla/shevch_alla/OPERA-MS//tools_opera_ms/ is not functional. Checking path for samtools.

samtools found in path.

mv /beegfs/shevch_alla/shevch_alla/OPERA-MS//tools_opera_ms//samtools /beegfs/shevch_alla/shevch_alla/OPERA-MS//tools_opera_ms//samtools.bk

ln -s /home/shevch_alla/beegfs/shevch_alla/miniconda3/envs/operams/bin/samtools /beegfs/shevch_alla/shevch_alla/OPERA-MS//tools_opera_ms/

*** samtools functional
*** blasr functional
*** bwa functional
*** minimap2 functional
*** mash functional
*** mummer functional
*** megahit functional
*** spades functional
*** racon functional
*** pilon functional

*** All compiled software are functional.
*** Please try to run OPERA-MS on the test dataset.

[jsgounot]

This can probably be resolved following this answer.

[ShevchenkoAlla]

This can probably be resolved following this answer.

Now, I have Gapfilling issue, -

rm -r /home/intermediate_files/opera_long_read/GAPFILLING/TILLING/QUERY
rm: cannot remove '/home/intermediate_files/opera_long_read/GAPFILLING/TILLING/QUERY': No such file or directory
rm: cannot remove '/home/intermediate_files/opera_long_read/GAPFILLING/TILLING/REF': No such file or directory
mkdir /home/intermediate_files/opera_long_read/GAPFILLING/TILLING/REF
*** split /home/intermediate_files/opera_long_read/GAPFILLING/TILLING/consensus.fa
Use of uninitialized value $genome in print at //OPERA-LG/bin//run_mummer_large_ref.pl line 152.
mkdir /home/intermediate_files/opera_long_read/GAPFILLING/TILLING/QUERY
*** split /home/intermediate_files/megahit_assembly/final.contigs.fa
*** run the mummer mapping
rm -r /home/intermediate_files/opera_long_read/GAPFILLING/mummer
rm: cannot remove '/home/intermediate_files/opera_long_read/GAPFILLING/mummer': No such file or directory
mkdir /home/intermediate_files/opera_long_read/GAPFILLING/mummer
cat /home/intermediate_files/opera_long_read/GAPFILLING/mummer/cmd_delta.txt | xargs -I cmd --max-procs=20 bash -c cmd > /dev/null

Error in during nucmer.

[jsgounot]

Can you check that you linked a samtools version <= 0.1.19 as indicated in my last post?

[ShevchenkoAlla]

Can you check that you linked a samtools version <= 0.1.19 as indicated in my last post?

Thanks,
You are right samtools were 1.21 version, after I downgrated it looks ok.

perl OPERA-MS.pl check-dependency
*** OPERA-LG functional
*** sigma functional
*** samtools functional
*** blasr functional
*** bwa functional
*** minimap2 functional
*** mash functional
*** mummer functional
*** megahit functional
*** spades functional
*** racon functional
*** pilon functional

*** All compiled software are functional.
*** Please try to run OPERA-MS on the test dataset.

Thank you for your quick responces.
Best

[ShevchenkoAlla]

No,
Unfortinatly it crashed again showing - Error in during nucmer
I see a few opened issues with the same problem, but there are no solution.
How can I find the reason for that?

[jsgounot]

Hi. I don't know which opened issues you're mentioning ... Have you looked at the several closed issues related to this error? Please take the time to look at the different solutions offered in the past and try those first. If those don't work, please provide a clear description of your problem. Thank you.

[ShevchenkoAlla]

Hi. I don't know which opened issues you're mentioning ... Have you looked at the several closed issues related to this error? Please take the time to look at the different solutions offered in the past and try those first. If those don't work, please provide a clear description of your problem. Thank you.

Thank you for your quick resoinse
I have tried your suggestion from #85

I run - intermediate_files/opera_long_read/GAPFILLING/mummer/cmd_delta.txt

I have got:

1: PREPARING DATA
2,3: RUNNING mummer AND CREATING CLUSTERS

reading input file "split_1.fa_split_2.fa.ntref" /home/OPERA-MS/tools_opera_ms/MUMmer3.23/mummer: empty sequence in multiple fasta file

ERROR: mummer and/or mgaps returned non-zero

[jsgounot]

This is once again covered by another issue here. Please follow what was discussed in the whole thread and report the different results here if this still don't work.

[ShevchenkoAlla]

This is really strange...

From another sample I got another issue

Exception in thread "main" java.lang.reflect.InvocationTargetException
at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
at java.base/jdk.internal.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
at java.base/java.lang.reflect.Method.invoke(Method.java:566)
at com.simontuffs.onejar.Boot.run(Boot.java:340)
at com.simontuffs.onejar.Boot.main(Boot.java:166)
Caused by: java.lang.OutOfMemoryError: Java heap space
at org.broadinstitute.pilon.GenomeRegion.(GenomeRegion.scala:56)
at org.broadinstitute.pilon.GenomeFile$$anonfun$contigRegions$1.apply(GenomeFile.scala:72)
at org.broadinstitute.pilon.GenomeFile$$anonfun$contigRegions$1.apply(GenomeFile.scala:72)
at scala.collection.TraversableLike$$anonfun$map$1.apply(TraversableLike.scala:234)
at scala.collection.TraversableLike$$anonfun$map$1.apply(TraversableLike.scala:234)
at scala.collection.immutable.Range.foreach(Range.scala:160)
at scala.collection.TraversableLike$class.map(TraversableLike.scala:234)
at scala.collection.AbstractTraversable.map(Traversable.scala:104)
at org.broadinstitute.pilon.GenomeFile.contigRegions(GenomeFile.scala:72)
at org.broadinstitute.pilon.GenomeFile$$anonfun$2.apply(GenomeFile.scala:52)
at org.broadinstitute.pilon.GenomeFile$$anonfun$2.apply(GenomeFile.scala:52)
at scala.collection.immutable.List.map(List.scala:277)
at org.broadinstitute.pilon.GenomeFile.(GenomeFile.scala:52)
at org.broadinstitute.pilon.Pilon$.main(Pilon.scala:98)
at org.broadinstitute.pilon.Pilon.main(Pilon.scala)
... 6 more

Although, I put 50Gb of memory for this job.

[jsgounot]

What is the size of your dataset? Pilon can request extremely high amount of memory for some samples. If you have a very high amount of reads, I'd suggest to first run OMS without Pilon (check options) and then run Pilon on a subsample of your reads.