Biowulf High Performance Computing at the NIH
GATK on Biowulf
Description

The Genome Analysis Toolkit (GATK) is a software package developed at the Broad Institute to analyze high-throughput sequencing data. The toolkit includes a wide variety of tools, with a focus on variant discovery and genotyping as well as emphasis on data quality assurance.


Online Tutorial: A practical introduction to GATK 4 on Biowulf

New in May 2021: A self-paced, online tutorial to work through a GATK example on Biowulf. Developed by the Biowulf staff, this tutorial includes a case study of germline variant discovery with WGS data from a trio, and benchmarks for each step. By working through the tutorial, you will learn NGS data preprocessing and how to optimize your Biowulf batch jobs.


Notes

There are multiple versions of GATK available. An easy way of selecting the version is to use modules. To see the modules available, type

    module avail GATK 

To select a module use

    module load GATK/[version]

where [version] is the version of choice.

Documentation

Release notes

References

Resource bundles

GATK Resource bundles contain reference genome assembles (.fa, .fai, .dict), variation data (dbSNP, HapMap, 1000 genomes, ...), and some genotype calls for a gold standard genome.

Resource bundles are available at

/fdb/GATK_resource_bundle

  • GATK 3
  • GATK 4
Overview

Environment module

The environment module for GATK (module load GATK or module load GATK/version)

  • loads R as a dependency
  • sets the environment variables
    • $GATK_HOME
    • $GATK_JAR
    • $GATK_JARPATH
    • $GATK_KEY
    • $GATK_QUEUE_JAR
    • $GATK_TEST_DATA
  • adds a GATK wrapper script developed in house and utilities for lifting over VFC files between genome builds

Wrapper

The GATK wrapper script provides a simplified interface to the GATK tools:

usage: GATK [options] [-m MEM] tool [gatk_tool_args]

This GATK wrapper script invokes GATK tools with a more friendly interface.
For documentation on each of the tools and their arguments see
https://www.broadinstitute.org/gatk/guide/tooldocs/

positional arguments:
  tool               GATK tool. Use 'LIST' or 'list' to get the current list
                     of available tools

optional arguments:
  -h, --help         show this help message and exit
  -m MEM, --mem MEM  maximal heap memory allowed for the Java process.
                     *Format*: <number>[kmg]. *Examples*: 1024k, 1000m, 2g,
                     32g. Passed to Java with -Xmx (default: 2g)
  -p N, --pgct N     maximal number of parallel GC threads allowed for Java
                     VM. Defaults to 1 less than the number of allocated CPUs.
                     (default: -1)
  -n, --dry-run      show command that would have been executed and set up
                     temp dir (default: False)

It takes care of finding the GATK jar and writing temporary files to either

  • /lscratch/${SLURM_JOBID}: if running on a compute node with allocated local disk space
  • ./unique_tmp_dir: if running on a compute node without allocated local disk space

Whenever possible, please use lscratch for temporary files. It will be more performant and reduce strain on the shared filesystems.

Calling GATK directly

When not using the GATK wrapper script, the basic GATK command should be

java -Xmx####g -Djava.io.tmpdir=/lscratch/${SLURM_JOBID} \
  -XX:ParallelGCThreads=## -jar $GATK_JAR -T <Toolname> [options]

which requires requesting lscratch space at job submission.

Note that /lscratch is the node-local scratch space. See the user guide for more information about allocating local scratch space.

Some tools may perform better with non-default garbage collectors.

Memory

The -Xmx option to Java specifies the maximal size of the memory allocation pool. It accepts suffixes k, m, and g. This is the option used by the wrapper script to limit memory usage.

Note that you should request additional memory (1-2GB) for batch/interactive jobs on top of the memory given to java to ensure that the whole java process stays below the allocated memory limit.

Redirect temporary files to /lscratch/${SLURM_JOBID}

GATK writes some temporary files during its run. By default, Java writes temp files to /tmp. However this is not advisable because /tmp is used by the operating system and is very limited in space. The tmp files should be redirected to /lscratch/${SLURM_JOBID} (if allocating local disk space)

-Djava.io.tmpdir=/lscratch/${SLURM_JOBID}

to the java command as shown above. The wrapper script takes care of this automatically. Note that the temp dir must already exist if specifying it manually. The wrapper script will automatically create it if necessary.

Limit the number of parallel garbage collection threads

The Java VM has a tendency to start many threads including parallel threads for garbage collection since it automatically detects all CPUs on a compute node irrespective of the allocation. This can be aggravated when there is not sufficient memory allocated to the JVM. Use -XX:ParallelGCThreads=## to limit the number of parallel garbage collection threads to the number of allocated CPUs up to 8 CPUs plus 5 for every 8 beyond that. The GATK wrapper by default sets the number of GC threads correctly one less than the number of allocated CPUs.

GATK 3.8-0 made the intel de/infaltor the default. While slightly faster than their jdk equivalents, this did introduce a bug that can lead to segfaults when allocating large heaps. Adding --use_jdk_inflater --use_jdk_deflater to the GATK tool options avoids this. The GATK wrapper takes care of this automatically.

Phone home

Note that GATK has a phone home feature which will send some generic information about each GATK run to the developers at the Broad Institute. This feature will not work on the Biowulf computational nodes, which are on a private network. This will not affect your runs in most cases. If you're getting an error related to 'phone-home', add -et NO_ET -K $GATK_KEY to your GATK command line to disable the phone-home feature.

Open files

GATK opens many files simultaneously. It can happen that you hit the open-file limit on Biowulf, and see errors like:

##### ERROR MESSAGE: Couldn't read file .... (Too many open files)

The most important change you can make is to reduce the multithreading. If your GATK command has any multithreading flags turned on (e.g. '-nt' and 'nct'), reduce the number of threads to 1.

If that happens, the limit on the number of open files has to be increased from its default soft limit of 4096. This can be done by adding the following command to your batch script: ulimit -n 16384.

Here are some other possibilities (thanks to Ryan Neff, NHGRI):

  • Reduce the number of open files. When doing variant calling, first create gVCF files for each sample and then combine them together in batches of 100-200, and then do the final variant calls on the remaining 5-20 files (GATK's HaplotypeCaller and GenotypeGVCFs). If you are using the UG or a different variant caller, try first discovering variant calls on a per-sample basis (-gt mode DISCOVERY), combining all of those files together, and then genotyping each sample on it's own from the list of all variants (-gt mode GENOTYPE_GIVEN_ALLELES).
  • Reduce the number of open file pointers. This can be done by increasing RAM, reducing the number of temporary files open at one time. Sometimes, in the GATK, setting --disableAutoIndexCreationAndLockingWhenReadingRods helps with performance and other I/O issues.

Relevant discussions from the GATK mailing list:

GATK Parallelization

Some GATK tools can make use of more than one CPU using option -nt or -nct. See the GATK documentation for more detail.

Running a single GATK batch job on Biowulf

Set up a batch script along the following lines:

#! /bin/bash
# gatk.bat
set -e

module load GATK/3.8-0 || exit 1

cd /data/$USER/test_data/GATK

# use 8 GB memory
java -Xmx8g -Djava.io.tmpdir=/lscratch/$SLURM_JOBID -jar $GATK_JAR \
  -T CountReads \
  -R exampleFASTA.fasta \
  -I exampleBAM.bam \
  -et NO_ET -K $GATK_KEY

Submit to the queue with sbatch:

biowulf$ sbatch --mem=9g --gres=lscratch:10 gatk.bat

Since the simple CountReads tool does not support multithreading, only a single core (the default) is requested.

Running a swarm of GATK batch jobs on Biowulf

The following swarm commend file would run the RealignerTargetCreator ,which determines which genomic intervals require realignment, for a number of samples:

# this file is gatk-swarm
GATK -m 7g RealignerTargetCreator \
  -R ref.fasta
  -I input1.bam \
  -o output1.intervals \
  --known /fdb/GATK_resource_bundle/hg19/1000G_phase1.indels.hg19.vcf.gz
GATK -m 7g RealignerTargetCreator \
  -R ref.fasta
  -I input2.bam \
  -o output2.intervals \
  --known /fdb/GATK_resource_bundle/hg19/1000G_phase1.indels.hg19.vcf.gz
[...]

These command lines specify that the process will use 7 GB of memory (-Xmx7g). Thus, this swarm job should be submitted with the '-g 7' flag, to tell swarm that each process will require 7 GB of memory.

biowulf$ swarm -g 7 -f gatk-swarm --module GATK/3.8-1
Interactive job on Biowulf

Allocate an interactive session with sinteractive and use as shown below

[user@biowulf]$ sinteractive --gres=lscratch:50 --cpus-per-task=2 --mem=6g
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn3144 are ready for job

[user@cn3144]$ cd /lscratch/$SLURM_JOB_ID
[user@cn3144]$ module load GATK/3.8-1    
[user@cn3144]$ cp ${GATK_TEST_DATA:-none}/NA12878.ba[im]* .
[user@cn3144]$ ls -lh
total 18M
-rw-r--r-- 1 user group 2.2M Jun 30 13:45 NA12878.bai
-rw-r--r-- 1 user group  15M Jun 30 13:45 NA12878.bam
[user@cn3144]$ GATK CountReads \
                    -R /fdb/GATK_resource_bundle/hg38/Homo_sapiens_assembly38.fasta \
                    -I NA12878.bam
directory for temp files: /lscratch/60475214
Executing ' java -Djava.io.tmpdir=/lscratch/60475214 -Xmx2g -XX:ParallelGCThreads=2 -jar /usr/local/apps/GATK/3.8-1/GenomeAnalysisTK.jar -T CountReads -R /fdb/GATK_resource_bundle/hg38/Homo_sapiens_assembly38.fasta -I NA12878.bam --use_jdk_inflater --use_jdk_deflater '
INFO  13:50:21,043 HelpFormatter - ----------------------------------------------------------------
INFO  13:50:21,047 HelpFormatter - The Genome Analysis Toolkit (GATK) v3.8-1-0-gf15c1c3ef, Compiled...
INFO  13:50:21,048 HelpFormatter - Copyright (c) 2010-2016 The Broad Institute
INFO  13:50:21,048 HelpFormatter - For support and documentation go to https://software.broadinstit...
INFO  13:50:21,049 HelpFormatter - [Tue Jun 30 13:50:21 EDT 2020] Executing on Linux 3.10.0-862....
INFO  13:50:21,049 HelpFormatter - OpenJDK 64-Bit Server VM 1.8.0_181-b13
INFO  13:50:21,056 HelpFormatter - Program Args: -T CountReads -R /fdb/GATK_resource_bundle/hg38/Ho...
INFO  13:50:21,064 HelpFormatter - Executing as user@cn3293 on Linux 3.10.0-862.14.4.el7.x86_64 a...
INFO  13:50:21,065 HelpFormatter - Date/Time: 2020/06/30 13:50:21
INFO  13:50:21,065 HelpFormatter - ----------------------------------------------------------------
INFO  13:50:21,066 HelpFormatter - ----------------------------------------------------------------
INFO  13:50:21,072 GenomeAnalysisEngine - Deflater: JdkDeflater
INFO  13:50:21,073 GenomeAnalysisEngine - Inflater: JdkInflater
INFO  13:50:21,074 GenomeAnalysisEngine - Strictness is SILENT
INFO  13:50:23,245 GenomeAnalysisEngine - Downsampling Settings: No downsampling
INFO  13:50:23,258 SAMDataSource$SAMReaders - Initializing SAMRecords in serial
INFO  13:50:23,540 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.28
INFO  13:50:24,955 GenomeAnalysisEngine - Preparing for traversal over 1 BAM files
INFO  13:50:24,961 GenomeAnalysisEngine - Done preparing for traversal
INFO  13:50:24,962 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING]
INFO  13:50:24,962 ProgressMeter -                 | processed |    time |    per 1M |           |   total | remaining
INFO  13:50:24,962 ProgressMeter -        Location |     reads | elapsed |     reads | completed | runtime |   runtime
INFO  13:50:24,964 ReadShardBalancer$1 - Loading BAM index data
INFO  13:50:24,966 ReadShardBalancer$1 - Done loading BAM index data
INFO  13:50:26,502 CountReads - CountReads counted 61614 reads in the traversal
INFO  13:50:26,507 ProgressMeter -            done     61614.0     1.0 s      25.0 s       99.9%     1.0 s       0.0 s
INFO  13:50:26,508 ProgressMeter - Total runtime 1.55 secs, 0.03 min, 0.00 hours
INFO  13:50:26,512 MicroScheduler - 0 reads were filtered out during the traversal out of approximately 61614 total reads (0.00%)
INFO  13:50:26,514 MicroScheduler -   -> 0 reads (0.00% of total) failing BadCigarFilter
INFO  13:50:26,515 MicroScheduler -   -> 0 reads (0.00% of total) failing MalformedReadFilter
------------------------------------------------------------------------------------------
Done. There were no warn messages.
------------------------------------------------------------------------------------------
GATK finished (exit code 0)
[user@cn3144]$ exit

        
LiftOverVCF -- converts a VCF file from one reference build to another

LiftOverVCF has been deprecated with release 3.5-0. Use picard's LiftoverVCF instead.

The procedure for lifting over VCF file from one genome build to different build in GATK is a three step process - (1) LiftoverVCF (2) sort the VCF and (3) FilterLiftedVCF. This process is automated by liftOverVCF

Usage: liftOverVCF
   -vcf            <input vcf>
   -gatk           <path to gatk trunk>
   -chain          <chain file>
   -newRef         <path to new reference prefix;
                    we will need newRef.dict, .fasta, and .fasta.fai>
   -oldRef         <path to old reference prefix; we will need oldRef.fasta>
   -out            <output vcf>
   -recordOriginalLocation
                   <Should we record what the
                    original location was in the INFO field?; defaults to false>

This procedure is to use liftOverVCF to switch from, say, hg18 to hg19.

Required data:

  • Chain files from the Broad ftp site are mirrored in fdb/GATK_resource_bundle/liftover_chains. In addtion a hg18 to hg19 liftover file from UCSC is included as well.
  • Reference sequences and .dict, .fai files for references can also be found in the resource bundles.

Example:

liftOverVCF  \
    -vcf test.hg18.vcf.gz \
    -chain /fdb/GATK_resource_bundle/liftover_chains/hg18ToHg19.over.chain \
    -newRef /fdb/GATK_resource_bundle/hg19/ucsc.hg19 \
    -oldRef /fdb/GATK_resource_bundle/hg18/Homo_sapiens_assembly18 \
    -out test.hg19.vzf
Overview

Environment module

The environment module for GATK (module load GATK or module load GATK/version)

  • loads R as a dependency
  • sets the environment variables
    • $GATK_TEST_DATA

Whenever possible, please use lscratch for temporary files. It will be more performant and reduce strain on the shared filesystems.

Calling GATK

GATK now includes its own wrapper script (gatk) and therefore the Biowulf-supplied wrapper for GATK 3 is not available for GATK 4. The basic GATK 4 command is

gatk --java-options "[java options]" <Toolname> [tool options]

For detailed GATK help use

gatk --help

 Usage template for all tools (uses --spark-runner LOCAL when used with a Spark tool)
    gatk AnyTool toolArgs

 Usage template for Spark tools (will NOT work on non-Spark tools)
    gatk SparkTool toolArgs  [ -- --spark-runner  sparkArgs ]

 Getting help
    gatk --list       Print the list of available tools

    gatk Tool --help  Print help on a particular tool

 Configuration File Specification
     --gatk-config-file                PATH/TO/GATK/PROPERTIES/FILE

 gatk forwards commands to GATK and adds some sugar for submitting spark jobs

   --spark-runner     controls how spark tools are run
     valid targets are:
     LOCAL:      run using the in-memory spark runner
     SPARK:      run using spark-submit on an existing cluster
                 --spark-master must be specified
                 --spark-submit-command may be specified to control the Spark submit command
                 arguments to spark-submit may optionally be specified after --
     GCS:        run using Google cloud dataproc
                 commands after the -- will be passed to dataproc
                 --cluster  must be specified after the --
                 spark properties and some common spark-submit parameters will be translated
                 to dataproc equivalents

   --dry-run      may be specified to output the generated command line without running it
   --java-options 'OPTION1[ OPTION2=Y ... ]'   optional - pass the given string of options to the
                 java JVM at runtime.
                 Java options MUST be passed inside a single string with space-separated values.

Memory

The -Xmx option to Java specifies the maximal size of the heap memory. It accepts suffixes k, m, and g. This is passed to gatk in the --java-otions string. For example

gatk --java-options "-Xmx6g" <Toolname> [tool options]

Note that you should request additional memory (1-2GB) for batch/interactive jobs on top of the memory given to java to ensure that the whole java process stays below the allocated memory limit.

Note that some tools may perform better with non-default garbage collectors. For example here are some possible options for a large heap allocation using ParallelGCThreads:

gatk --java-options "-Djava.io.tmpdir=/lscratch/$SLURM_JOBID -Xms48G -Xmx48G \
     -XX:ParallelGCThreads=8" ...

Redirect temporary files to /lscratch/${SLURM_JOBID}

GATK writes some temporary files during its run. By default, Java writes temp files to /tmp. However this is not advisable because /tmp is used by the operating system and is very limited in space. The tmp files should be redirected to /lscratch/${SLURM_JOBID} by adding

-Djava.io.tmpdir=/lscratch/${SLURM_JOBID}

to the java options.

For more details on the node-local storage under /lscratch see the user guide

Parallel garbage collection threads

If you find that your GATK runs have more active threads than you were expecting you may have to limit the number of prallel garbage collection threads. The JVM options -XX:ParallelGCThreads and -XX:ConcGCThreads can be used to tune the number of threads dedicated to garbage collection.

Open files

GATK may open many files simultaneously. It can happen that you hit the open-file limit on Biowulf, and see errors like:

##### ERROR MESSAGE: Couldn't read file .... (Too many open files)

If that happens, the limit on the number of open files has to be increased from its default soft limit of 4096. This can be done by adding the following command to your batch script: ulimit -n 16384.

Here are some other possibilities (thanks to Ryan Neff, NHGRI):

  • Reduce the number of open files. When doing variant calling, first create gVCF files for each sample and then combine them together in batches of 100-200, and then do the final variant calls on the remaining 5-20 files (GATK's HaplotypeCaller and GenotypeGVCFs).
GATK Parallelization

The paradigm for parallelization for GATK 4 is completely different from GATK 3. Spark is used to parallelize workloads and the tools capable of using this form of parallelism include 'Spark' in their toolname. Some of these tools are still in beta and outputs may not be compatible with non-parallel variants. See the GATK documentation for more detail.

Running a single GATK batch job on Biowulf

Set up a batch script along the following lines:

#! /bin/bash
# gatk.bat
set -e

module load GATK/4.1.8.0 || exit 1
cd /lscratch/$SLURM_JOB_ID || exit 1

ref=/fdb/GATK_resource_bundle/hg38/Homo_sapiens_assembly38.fasta
bam=${GATK_TEST_DATA:-none}/HG00133.alt_bwamem_GRCh38DH.20150826.GBR.exome.bam
# use 8 GB memory
gatk --java-options "-Xmx8g -Djava.io.tmpdir=/lscratch/$SLURM_JOBID" \
  CountReads \
  -R $ref \
  -I $bam \

Submit to the queue with sbatch:

[user@biowulf]$ sbatch --mem=9g --gres=lscratch:10 gatk.bat

Since the simple CountReads tool does not support multithreading, only a single core (the default) is requested.

Running a swarm of GATK batch jobs on Biowulf

The following swarm commend file would run the MarkDuplicatesSpark. It will also sort and index the bam file. This is a Spark implementation of Picard MarkDuplicates that allows the tool to be run in parallel on multiple cores:

# this file is gatk-swarm
gatk --java-options "-Xmx8g" MarkDuplicatesSpark \
  -I input1.bam \
  -O input1_markdup.bam \
  --spark-runner LOCAL \
  --spark-master local[$SLURM_CPUS_PER_TASK] \
gatk --java-options "-Xmx8g" MarkDuplicatesSpark \
  -I input2.bam \
  -O input2_markdup.bam \
  --spark-runner LOCAL \
  --spark-master local[$SLURM_CPUS_PER_TASK] \
[...]

These command lines specify that the process will use 8 GB of memory (-Xmx8g). Thus, this swarm job should be submitted with the '-g 9' flag, to tell swarm that each process will require 9 GB (8GB plus some buffer) of memory.

biowulf$ swarm -g 9 -f gatk-swarm -t 16 --module GATK/4.1.8.0
Running an interactive job on Biowulf

It may be useful for debugging purposes to run GATK jobs interactively. Such jobs should not be run on the Biowulf login node. Instead allocate an interactive node as described below, and run the interactive job there.

[user@biowulf]$ sinteractive --gres=lscratch:50 --cpus-per-task=2 --mem=6g
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn3144 are ready for job

[user@cn3144]$ cd /lscratch/$SLURM_JOB_ID
[user@cn3144]$ module load GATK/4.1.8.0    
[user@cn3144]$ cp ${GATK_TEST_DATA:-none}/NA12878.ba[im]* .
[user@cn3144]$ ls -lh
total 18M
-rw-r--r-- 1 user group 2.2M Jun 30 13:45 NA12878.bai
-rw-r--r-- 1 user group  15M Jun 30 13:45 NA12878.bam
[user@cn3144]$ gatk --java-options "-Xmx5g -Xms5g -Djava.io.tmpdir=/lscratch/$SLURM_JOBID" \
                   CountReads \
                   -R /fdb/igenomes/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa \
                   -I NA12878.bam
Using GATK jar /usr/local/apps/GATK/4.1.8.0/gatk-package-4.1.8.0-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 -Xmx5g -Xms5g 
    -Djava.io.tmpdir=/lscratch/60475214 
    -jar /usr/local/apps/GATK/4.1.8.0/gatk-package-4.1.8.0-local.jar CountReads -R 
     /fdb/igenomes/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa -I NA12878.bam
17:06:09.007 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/usr/local/apps/GAT
K/4.1.8.0/gatk-package-4.1.8.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
17:06:09.513 INFO  CountReads - ------------------------------------------------------------
17:06:09.514 INFO  CountReads - The Genome Analysis Toolkit (GATK) v4.1.8.0
17:06:09.514 INFO  CountReads - For support and documentation go to https://software.broadinstitute.org/
gatk/
17:06:09.515 INFO  CountReads - Executing as user@cn3293 on Linux v3.10.0-862.14.4.el7.x86_64 amd64
17:06:09.515 INFO  CountReads - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_181-b13
17:06:09.516 INFO  CountReads - Start Date/Time: June 30, 2020 5:06:08 PM EDT
17:06:09.517 INFO  CountReads - ------------------------------------------------------------
17:06:09.517 INFO  CountReads - ------------------------------------------------------------
17:06:09.519 INFO  CountReads - HTSJDK Version: 2.22.0
17:06:09.519 INFO  CountReads - Picard Version: 2.22.8
17:06:09.519 INFO  CountReads - HTSJDK Defaults.COMPRESSION_LEVEL : 2
17:06:09.520 INFO  CountReads - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
17:06:09.520 INFO  CountReads - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
17:06:09.521 INFO  CountReads - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
17:06:09.521 INFO  CountReads - Deflater: IntelDeflater
17:06:09.521 INFO  CountReads - Inflater: IntelInflater
17:06:09.521 INFO  CountReads - GCS max retries/reopens: 20
17:06:09.521 INFO  CountReads - Requester pays: disabled
17:06:09.521 INFO  CountReads - Initializing engine
WARNING: BAM index file /lscratch/60475214/NA12878.bai is older than BAM /lscratch/60475214/NA12878.bam
17:06:10.212 INFO  CountReads - Done initializing engine
17:06:10.212 INFO  ProgressMeter - Starting traversal
17:06:10.212 INFO  ProgressMeter -        Current Locus  Elapsed Minutes       Reads Processed     Reads
/Minute
17:06:11.192 INFO  CountReads - 0 read(s) filtered by: WellformedReadFilter

17:06:11.194 INFO  ProgressMeter - chrUn_KN707963v1_decoy:19294              0.0                 61614
      3768440.4
17:06:11.195 INFO  ProgressMeter - Traversal complete. Processed 61614 total reads in 0.0 minutes.
17:06:11.195 INFO  CountReads - CountReads counted 61614 total reads
17:06:11.195 INFO  CountReads - Shutting down engine
[June 30, 2020 5:06:11 PM EDT] org.broadinstitute.hellbender.tools.CountReads done. Elapsed time: 0.04 m
inutes.
Runtime.totalMemory()=5145362432
Tool returned:
61614