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I can't make FastaAlternateReferenceMaker works

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Luca Stefanucci

Hi All, 

I want to retrieve FASTA sequences with the alternative variants. but I don't manage to have FastaAlternateReferenceMaker working. I don't get any output from this script and I can't see any obvious error message. Could you please help me?

Can you please provide
a) GATK version used

GATK v4.1.0.0


b) Exact GATK commands used

gatk FastaAlternateReferenceMaker \

-O outputproof_Script \

-R /rds/project/who1000-1/rds-who1000-cbrc/ref/b37/human_g1k_v37.fasta \

-V proof.vcf


c) The entire error log if applicable.

Using GATK jar /usr/local/Cluster-Apps/gatk/4.1.0.0/gatk-package-4.1.0.0-local.jar

Running:

    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /usr/local/Cluster-Apps/gatk/4.1.0.0/gatk-package-4.1.0.0-local.jar FastaAlternateReferenceMaker -O outputproof_Script -R /rds/project/who1000-1/rds-who1000-cbrc/ref/b37/human_g1k_v37.fasta -V proof.vcf

14:56:09.449 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/usr/local/Cluster-Apps/gatk/4.1.0.0/gatk-package-4.1.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so

Jul 09, 2020 2:56:09 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine

WARNING: Failed to detect whether we are running on Google Compute Engine.

shaded.cloud_nio.com.google.api.client.http.HttpResponseException: 404 Not Found

<html>

<head>

  <title>404 Not Found</title>

</head>

<body>

  <h1>404 Not Found</h1>

  The resource could not be found.<br /><br />

 

 

 

</body>

</html>

at shaded.cloud_nio.com.google.api.client.http.HttpRequest.execute(HttpRequest.java:1072)

at shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials.runningOnComputeEngine(ComputeEngineCredentials.java:210)

at shaded.cloud_nio.com.google.auth.oauth2.DefaultCredentialsProvider.tryGetComputeCredentials(DefaultCredentialsProvider.java:290)

at shaded.cloud_nio.com.google.auth.oauth2.DefaultCredentialsProvider.getDefaultCredentialsUnsynchronized(DefaultCredentialsProvider.java:207)

at shaded.cloud_nio.com.google.auth.oauth2.DefaultCredentialsProvider.getDefaultCredentials(DefaultCredentialsProvider.java:124)

at shaded.cloud_nio.com.google.auth.oauth2.GoogleCredentials.getApplicationDefault(GoogleCredentials.java:127)

at shaded.cloud_nio.com.google.auth.oauth2.GoogleCredentials.getApplicationDefault(GoogleCredentials.java:100)

at com.google.cloud.ServiceOptions.defaultCredentials(ServiceOptions.java:304)

at com.google.cloud.ServiceOptions.<init>(ServiceOptions.java:278)

at com.google.cloud.storage.StorageOptions.<init>(StorageOptions.java:83)

at com.google.cloud.storage.StorageOptions.<init>(StorageOptions.java:31)

at com.google.cloud.storage.StorageOptions$Builder.build(StorageOptions.java:78)

at org.broadinstitute.hellbender.utils.gcs.BucketUtils.setGlobalNIODefaultOptions(BucketUtils.java:353)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:182)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:210)

at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:162)

at org.broadinstitute.hellbender.Main.mainEntry(Main.java:205)

at org.broadinstitute.hellbender.Main.main(Main.java:291)

 

Jul 09, 2020 2:56:09 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine

WARNING: Failed to detect whether we are running on Google Compute Engine.

shaded.cloud_nio.com.google.api.client.http.HttpResponseException: 404 Not Found

<html>

<head>

  <title>404 Not Found</title>

</head>

<body>

  <h1>404 Not Found</h1>

  The resource could not be found.<br /><br />

 

 

 

</body>

</html>

at shaded.cloud_nio.com.google.api.client.http.HttpRequest.execute(HttpRequest.java:1072)

at shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials.runningOnComputeEngine(ComputeEngineCredentials.java:210)

at shaded.cloud_nio.com.google.auth.oauth2.DefaultCredentialsProvider.tryGetComputeCredentials(DefaultCredentialsProvider.java:290)

at shaded.cloud_nio.com.google.auth.oauth2.DefaultCredentialsProvider.getDefaultCredentialsUnsynchronized(DefaultCredentialsProvider.java:207)

at shaded.cloud_nio.com.google.auth.oauth2.DefaultCredentialsProvider.getDefaultCredentials(DefaultCredentialsProvider.java:124)

at shaded.cloud_nio.com.google.auth.oauth2.GoogleCredentials.getApplicationDefault(GoogleCredentials.java:127)

at shaded.cloud_nio.com.google.auth.oauth2.GoogleCredentials.getApplicationDefault(GoogleCredentials.java:100)

at com.google.cloud.ServiceOptions.defaultCredentials(ServiceOptions.java:304)

at com.google.cloud.ServiceOptions.<init>(ServiceOptions.java:278)

at com.google.cloud.storage.StorageOptions.<init>(StorageOptions.java:83)

at com.google.cloud.storage.StorageOptions.<init>(StorageOptions.java:31)

at com.google.cloud.storage.StorageOptions$Builder.build(StorageOptions.java:78)

at org.broadinstitute.hellbender.utils.gcs.BucketUtils.setGlobalNIODefaultOptions(BucketUtils.java:353)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:182)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:210)

at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:162)

at org.broadinstitute.hellbender.Main.mainEntry(Main.java:205)

at org.broadinstitute.hellbender.Main.main(Main.java:291)

 

Jul 09, 2020 2:56:09 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine

WARNING: Failed to detect whether we are running on Google Compute Engine.

shaded.cloud_nio.com.google.api.client.http.HttpResponseException: 404 Not Found

<html>

<head>

  <title>404 Not Found</title>

</head>

<body>

  <h1>404 Not Found</h1>

  The resource could not be found.<br /><br />

 

 

 

</body>

</html>

at shaded.cloud_nio.com.google.api.client.http.HttpRequest.execute(HttpRequest.java:1072)

at shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials.runningOnComputeEngine(ComputeEngineCredentials.java:210)

at shaded.cloud_nio.com.google.auth.oauth2.DefaultCredentialsProvider.tryGetComputeCredentials(DefaultCredentialsProvider.java:290)

at shaded.cloud_nio.com.google.auth.oauth2.DefaultCredentialsProvider.getDefaultCredentialsUnsynchronized(DefaultCredentialsProvider.java:207)

at shaded.cloud_nio.com.google.auth.oauth2.DefaultCredentialsProvider.getDefaultCredentials(DefaultCredentialsProvider.java:124)

at shaded.cloud_nio.com.google.auth.oauth2.GoogleCredentials.getApplicationDefault(GoogleCredentials.java:127)

at shaded.cloud_nio.com.google.auth.oauth2.GoogleCredentials.getApplicationDefault(GoogleCredentials.java:100)

at com.google.cloud.ServiceOptions.defaultCredentials(ServiceOptions.java:304)

at com.google.cloud.ServiceOptions.<init>(ServiceOptions.java:278)

at com.google.cloud.storage.StorageOptions.<init>(StorageOptions.java:83)

at com.google.cloud.storage.StorageOptions.<init>(StorageOptions.java:31)

at com.google.cloud.storage.StorageOptions$Builder.build(StorageOptions.java:78)

at org.broadinstitute.hellbender.utils.gcs.BucketUtils.setGlobalNIODefaultOptions(BucketUtils.java:353)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:182)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:210)

at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:162)

at org.broadinstitute.hellbender.Main.mainEntry(Main.java:205)

at org.broadinstitute.hellbender.Main.main(Main.java:291)

 

14:56:09.630 INFO  FastaAlternateReferenceMaker - ------------------------------------------------------------

14:56:09.630 INFO  FastaAlternateReferenceMaker - The Genome Analysis Toolkit (GATK) v4.1.0.0

14:56:09.630 INFO  FastaAlternateReferenceMaker - For support and documentation go to https://software.broadinstitute.org/gatk/

14:56:09.631 INFO  FastaAlternateReferenceMaker - Executing as ls760@login-e-11.data.cluster on Linux v3.10.0-957.48.1.el7.csd3.x86_64 amd64

14:56:09.631 INFO  FastaAlternateReferenceMaker - Java runtime: Java HotSpot(TM) 64-Bit Server VM v1.8.0_141-b15

14:56:09.631 INFO  FastaAlternateReferenceMaker - Start Date/Time: 09 July 2020 14:56:09 BST

14:56:09.631 INFO  FastaAlternateReferenceMaker - ------------------------------------------------------------

14:56:09.632 INFO  FastaAlternateReferenceMaker - ------------------------------------------------------------

14:56:09.632 INFO  FastaAlternateReferenceMaker - HTSJDK Version: 2.18.2

14:56:09.632 INFO  FastaAlternateReferenceMaker - Picard Version: 2.18.25

14:56:09.632 INFO  FastaAlternateReferenceMaker - HTSJDK Defaults.COMPRESSION_LEVEL : 2

14:56:09.632 INFO  FastaAlternateReferenceMaker - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false

14:56:09.632 INFO  FastaAlternateReferenceMaker - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true

14:56:09.632 INFO  FastaAlternateReferenceMaker - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false

14:56:09.632 INFO  FastaAlternateReferenceMaker - Deflater: IntelDeflater

14:56:09.632 INFO  FastaAlternateReferenceMaker - Inflater: IntelInflater

14:56:09.632 INFO  FastaAlternateReferenceMaker - GCS max retries/reopens: 20

14:56:09.632 INFO  FastaAlternateReferenceMaker - Requester pays: disabled

14:56:09.632 INFO  FastaAlternateReferenceMaker - Initializing engine

14:56:09.936 INFO  FeatureManager - Using codec VCFCodec to read file file:///rds/project/who1000-1/rds-who1000-cbrc/analysis/bp_epigenomes/ls760/Luca_Hi-C/TG_project_scripts/filter_bcf_with_TG_regions/retrieve_info_from_wgs/proof.vcf

14:56:09.975 INFO  FastaAlternateReferenceMaker - Done initializing engine

14:56:09.976 INFO  FastaAlternateReferenceMaker - Shutting down engine

[09 July 2020 14:56:09 BST] org.broadinstitute.hellbender.tools.walkers.fasta.FastaAlternateReferenceMaker done. Elapsed time: 0.01 minutes.

Runtime.totalMemory()=2156396544

java.lang.NullPointerException

at org.broadinstitute.hellbender.tools.walkers.fasta.FastaReferenceMaker.closeTool(FastaReferenceMaker.java:137)

at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:970)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:138)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:191)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:210)

at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:162)

at org.broadinstitute.hellbender.Main.mainEntry(Main.java:205)

at org.broadinstitute.hellbender.Main.main(Main.java:291)

0

4 comments

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  • Avatar
    Genevieve Brandt (she/her)

    Hi Luca Stefanucci, can you try running this with your output ending with .fasta? 

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  • Avatar
    Louis Bergelson

    Hi Luca Stefanucci,

    This was a known bug in version 4.1.0.0.  It's been fixed in recent versions.  Please upgrade and try with 4.1.8.0.

    0
    Comment actions Permalink
  • Avatar
    Luca Stefanucci

    Hi Louis Bergelson,

    Thanks for your answer,

    I am working on HPC and I'd like to use one of the versions which is already installed. Do you know if any of the following works ok fo FastaAlternateReferenceMaker:

    gatk/3.1        gatk/3.3        gatk/3.7.0      gatk/4.1.0.0

    gatk/3.1-1      gatk/3.5        gatk/4.0.alpha 

    gatk/3.2        gatk/3.7        gatk/4.0.beta 

    Thanks,
    Luca

    0
    Comment actions Permalink
  • Avatar
    Louis Bergelson

    I would really recommend you install the newest version of GATK if you can.  In general 4.1.0.0 should be ok, but it has a bug in FastaAlternateReferenceMaker which wasn't fixed until 4.1.5.0.  (I believe the bug only manifests when there is another error condition that occurs.  I would check that your file paths are right and your vcf looks ok.)  

    If you need to run this particular tool and you can't upgrade to a newer version you'll have to try it in 3.7 I think. 

    I do recommend you try to move to a newer version though since we've made a lot of improvements in the past year and a half.

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