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#cloudbreak

Cloudbreak is a Hadoop-based structural variation (SV) caller for Illumina paired-end DNA sequencing data. Currently Cloudbreak calls genomic insertions and deletions; we are working on adding support for other types of SVs.

Cloudbreak contains a full pipeline for aligning your data in the form of FASTQ files using alignment pipelines that generate many possible mappings for every read, in the Hadoop framework. It then contains Hadoop jobs for computing genomic features from the alignments, and for calling insertion and deletion variants from those features.

You can get Cloudbreak by downloading a pre-packaged release from the "releases" tab in the GitHub repository, or by building from source as described below.

You can read more about how Cloudbreak works in our paper, which is currently available as a preprint at http://arxiv.org/abs/1307.2331.

##Building From Source

To build the latest version of Cloudbreak, clone the GitHub repository. You'll need to install Maven to build the executables. (http://maven.apache.org/) Enter the top level directory of the Cloudbreak repository and type the command:

mvn package

This should compile the code, execute tests, and create a distribution file, cloudbreak-$VERSION-dist.tar.gz in the target/ directory. You can then copy that distribution file to somewhere else on your system, unpack it with tar -xzvf cloudbreak-$VERSION-dist.tar.gz and access the Cloudbreak jar file and related scripts and properties files.

##Dependencies

Cloudbreak requires a cluster Hadoop 0.20.2 or Cloudera CDH3 to run (the older mapreduce API). If you don't have a Hadoop cluster, Cloudbreak can also use the Apache Whirr API to automatically provision a cluster on the Amazon Elastic Compute Cloud (EC2). See the section on using WHIRR below.

Currently Cloudbreak requires that the Hadoop cluster have the native libraries to support Snappy compression installed. This comes standard with many Hadoop distributions, including Cloudera's distribution and AMIs for use on EC2. However, if you are running an older installation of Apache's Hadoop distribution, you may not have snappy installed. If so, you will have to install snappy to run Cloudbreak, either by reinstalling from the distribution package or as an add-on using the hadoop-snappy package:

https://code.google.com/p/hadoop-snappy/

We are working to fix this dependency on snappy so that those without snappy support can run Cloudbreak (without the benefits of its fast compression algorithms, of course).

If you wish to run alignments using Cloudbreak, you will need one of the following supported aligners:

##User Guide

You can use Cloudbreak in several different ways, depending on whether you want to start with FASTQ files and use Hadoop to help parallelize your alignments, or if you already have an aligned BAM file and just want to use Cloudbreak to call variants. In addition, the workflow is slightly different depending on whether you want to run on a local Hadoop cluster or want to run using a cloud provider like Amazon EC2. Later in this file, we've listed a set of scenarios to describe options for running the Cloudbreak pipeline. Find the scenario that best fits your use case for more details on how to run that workflow. For each scenario, we have created a template script that contains all of the steps and parameters you need, which you can modify for your particular data set.

##Running on a cloud provider like Amazon EC2 with Whirr

Cloudbreak has support for automatically deploying a Hadoop cluster on cloud providers such as Amazon EC2, transferring your data there, running the Cloudbreak algorithm, and downloading the results.

Of course, renting compute time on EC2 or other clouds costs money, so please be familiar with the appropriate usage and billing policies of your cloud provider before attempting this.

WE ARE NOT RESPONSIBLE FOR UNEXPECTED CHARGES THAT YOU MIGHT INCUR ON EC2 OR OTHER CLOUD PROVIDERS.

Many properties that affect the cluster created can be set in the file cloudbreak-whirr.properties in this distribution. You will need to edit this file to set your AWS access key and secret access key (or your credentials for other cloud provider services), and to tell it the location of the public and private SSH keys to use to access the cluster. You can also control the number and type of nodes to include in the cluster. The default settings in the file create 15 nodes of type m1.xlarge, which is sufficient to fully process a 30X simulation of human chromosome 2, including read alignment and data transfer time, in under an hour. We have only tested this capability using EC2; other cloud providers may not work as well. You can also direct Whirr to use Amazon EC2's spot instances, which are dramatically cheaper than on-demand instances, although they carry the risk of being terminated if your price is out-bid. Using recent spot pricing, it cost us about $5 to run the aforementioned chromosome 2 simulation. We recommend setting your spot bid price to be the on demand price for the instance type you are using to minimize the chance of having your instances terminated.

Please consult Amazon's EC2 documentation and the documentation for Whirr for more information on how to configure and deploy clusters in the cloud.

##Running on a Small Example Data Set

To facilitate testing of Cloudbreak, we have publicly hosted the reads from the simulated data example described in the Cloudbreak manuscript on a bucket in Amazon's S3 storage service at s3://cloudbreak-example/. We have also provided an example script that creates a cluster in Amazon EC2, copies the data to the cluster, runs the full Cloudbreak workflow including alignments with BWA, and copies the variant calls back to the local machine before destroying the cluster. The script, called Cloudbreak-EC2-whirr-example.sh is in the scripts directory of the Cloudbreak distribution. Of course, you will still need to edit the cloudbreak-whirr.properties file with your EC2 credentials, and verify that the cluster size, instance types, and spot price are to your liking before executing the example.

###Scenario 1: Compute alignments in Hadoop, using a local Hadoop cluster

To install aligner dependencies for use by Cloudbreak, first generate the index for the genome reference you would like to run against. Then, copy all of the required index files, and the executable files for the aligner into HDFS using the hadoop dfs -copyFromLocal command. For BWA you will need all of the index files created by running bwa index. You will also need an 'fai' file for the reference, containing chromosome names and lengths, generated by samtools faidx.

If your reference file is reference.fa, and bwa aln has created the files

reference.fa.amb
reference.fa.ann
reference.fa.bwt
reference.fa.pac
reference.fa.sa

and reference.fa.fai as described above, issue the following commands to load the necessary files into HDFS:

hdfs -mkdir indices/
hdfs -mkdir executables/
hdfs -copyFromLocal reference.fa.amb indices/
hdfs -copyFromLocal reference.fa.ann indices/
hdfs -copyFromLocal reference.fa.bwt indices/
hdfs -copyFromLocal reference.fa.pac indices/
hdfs -copyFromLocal reference.fa.sa indices/
hdfs -copyFromLocal reference.fa.fai indices/
hdfs -copyFromLocal /path/to/bwa/executables/bwa executables/
hdfs -copyFromLocal /path/to/bwa/executables/xa2multi.pl executables/

The basic workflow is:

  1. Load the FASTQ files into HDFS
  2. Run one of the Cloudbreak alignment commands to align your reads
  3. Create a readGroup file to describe the location and insert size characteristics of your reads, and copy it into HDFS.
  4. Run the GMM fitting feature generation step of the Cloudbreak process.
  5. Extract deletion calls from the features created in step 4.
  6. Copy the deletion calls from HDFS to a local directory.
  7. Extract insertion calls from the features created in step 4.
  8. Copy the insertion calls from HDFS to a local directory.
  9. Optionally, export the alignments back into a BAM file in your local filesystem.

We have created a script to run through the full process of executing the Cloudbreak pipeline from FASTQ files to insertion and deletion calls. The script is named Cloudbreak-full.sh and can be found in the scripts directory of the Cloudbreak distribution. To customize the script for your needs, copy it to a new location and edit the variables in the first three sections: "EXPERIMENT DETAILS", "LOCAL FILES AND DIRECTORIES", and "HDFS FILES AND DIRECTORIES".

###Scenario 2: Call variants on existing alignments, using a local Hadoop cluster

For this scenario you don't need to worry about having an aligner executable or aligner-generated reference in HDFS. You will however, need a chromosome length 'fai' file, which you can generate by running samtools faidx on your reference FASTA files and then copying to HDFS:

hdfs -copyFromLocal reference.fa.fai indices/

After that, the workflow is:

  1. Load your BAM file into HDFS and prepare it for Cloudbreak
  2. Create a readGroup file to describe the location and insert size characteristics of your reads.
  3. Run the GMM fitting feature generation step of the Cloudbreak process.
  4. Extract deletion calls from the features created in step 3.
  5. Copy the deletion calls from HDFS to a local directory.
  6. Extract insertion calls from the features created in step 3.
  7. Copy the insertion calls from HDFS to a local directory.

To prepare alignments for Cloudbreak, they must be sorted by read name. You can then use the readSAMFileIntoHDFS Cloudbreak command.

A templates for this scenario is available in the script Cloudbreak-variants-only.sh located in the scripts directory of the Cloudbreak distribution.

###Scenario 3: Compute alignments in Hadoop, using a cloud provider like EC2

First, see the section "Running on a Cloud Provider like Amazon EC2 with Whirr" above, and modify the file cloudbreak-whirr.properties to include your access credentials and the appropriate cluster specifications. After that, the workflow is similar to the workflow described for scenario #1 above, with the additional first steps of copying your reads and dependency files to the cloud and creating a cluster before processing begins, and then destroying the cluster after processing has completed.

You can see an example workflow involving EC2 by examining the script Cloudbreak-EC2-whirr.sh. This begins by transferring your reads to Amazon S3. It then uses Apache Whirr to launch an EC2 Hadoop cluster, copies the necessary executable files to EC2, and runs the algorithm.

###Scenario 4: Call variants on existing alignments, using a cloud provider like EC2

Again, please read the section "Running on a Cloud Provider like Amazon EC2 with Whirr" above to learn how to update the cloudbreak-whirr.properties file with your credentials and cluster specifications. After that, follow the template in the script Cloudbreak-EC2-whirr-variants-only.sh to create a workflow involving calling variants in the cloud.

##Output Files

The output from running Cloudbreak using one of the scripts above will be found in the files named

READ_GROUP_LIBRARY_dels_genotyped.bed
READ_GROUP_LIBRARY_ins_genotyped.bed

where READ_GROUP and LIBRARY are the names of the reads in your experiment. The format of the files is tab-delimited with the following columns:

  • CHROMOSOME: The chromosome of the deletion call
  • START: The start coordinate of the deletion call
  • END: The end coordinate of the deletion call
  • NUMBER: The cloudbreak identifier of the deletion call
  • LR: The likelihood ratio of the deletion (higher indicates a call more likely to be true)
  • TYPE: Either "INS" or "DEL"
  • W: The average weight of the estimated GMM mixing parameter alpha, used in genotyping
  • GENOTYPE: The predicted genotype of the call

##Contact information

Please contact cwhelan at gmail.com with any questions on running cloudbreak.

##Reference Guide

All of Cloudbreak's functionality is contained in the executable jar file in the directory where you unpacked the Cloudbreak distribution. Use the 'hadoop' command to run the jar file to ensure that the necessary Hadoop dependencies are available to Cloudbreak.

To invoke any Cloudbreak command, use a command line in this format:

hadoop cloudbreak-${project.version}.jar [options] [command] [command options]

Where command is the name of the command, command options are the arguments specific to that command, and options are general options, including options for how to run Hadoop jobs. For example, if you'd like to specify 50 reduce tasks for one of your commands, pass in -Dmapred.reduce.tasks=50 as one of the general options.

Each command is detailed below and its options are listed below. You can view this information by typing hadoop jar cloudbreak-${project.version}.jar without any additional parameters.

    readPairedEndFilesIntoHDFS      Load paired FASTQ files into HDFS
      Usage: readPairedEndFilesIntoHDFS [options]
  Options:
        *     --HDFSDataDir                  HDFS directory to load reads into
              --clipReadIdsAtWhitespace      Whether to clip all readnames at
                                             the first whitespace (prevents trouble
                                             with some aligners)
                                             Default: true
              --compress                     Compression codec to use on the
                                             reads stored in HDFS
                                             Default: snappy
        *     --fastqFile1                   File containing the first read in
                                             each pair
        *     --fastqFile2                   File containing the second read in
                                             each pair
              --filesInHDFS                  Use this flag if the BAM file has
                                             already been copied into HDFS
                                             Default: false
              --filterBasedOnCasava18Flags   Use the CASAVA 1.8 QC filter to
                                             filter out read pairs
                                             Default: false
              --outFileName                  Filename of the prepped reads in
                                             HDFS
                                             Default: reads
              --trigramEntropyFilter         Filter out read pairs where at
                                             least one read has a trigram entropy less
                                             than this value. -1 = no filter
                                             Default: -1.0

    readSAMFileIntoHDFS      Load a SAM/BAM file into HDFS
      Usage: readSAMFileIntoHDFS [options]
  Options:
        *     --HDFSDataDir   HDFS Directory to hold the alignment data
              --compress      Compression codec to use for the data
                              Default: snappy
              --outFileName   Filename to give the file in HDFS
                              Default: alignments
        *     --samFile       Path to the SAM/BAM file on the local filesystem

    bwaPairedEnds      Run a BWA paired-end alignment
      Usage: bwaPairedEnds [options]
  Options:
        *     --HDFSAlignmentsDir    HDFS directory to hold the alignment data
        *     --HDFSDataDir          HDFS directory that holds the read data
        *     --HDFSPathToBWA        HDFS path to the bwa executable
              --HDFSPathToXA2multi   HDFS path to the bwa xa2multi.pl executable
        *     --maxProcessesOnNode   Ensure that only a max of this many BWA
                                     processes are running on each node at once.
                                     Default: 6
              --numExtraReports      If > 0, set -n and -N params to bwa sampe,
                                     and use xa2multi.pl to report multiple hits
                                     Default: 0
        *     --referenceBasename    HDFS path of the FASTA file from which the
                                     BWA index files were generated.

    novoalignSingleEnds      Run a Novoalign alignment in single ended mode
      Usage: novoalignSingleEnds [options]
  Options:
        *     --HDFSAlignmentsDir            HDFS directory to hold the
                                             alignment data
        *     --HDFSDataDir                  HDFS directory that holds the read
                                             data
        *     --HDFSPathToNovoalign          HDFS path to the Novoalign
                                             executable
              --HDFSPathToNovoalignLicense   HDFS path to the Novoalign license
                                             filez
              --qualityFormat                Quality score format of the FASTQ
                                             files
                                             Default: ILMFQ
        *     --reference                    HDFS path to the Novoalign
                                             reference index file
        *     --threshold                    Quality threshold to use for the -t
                                             parameter

    bowtie2SingleEnds      Run a bowtie2 alignment in single ended mode
      Usage: bowtie2SingleEnds [options]
  Options:
        *     --HDFSAlignmentsDir       HDFS directory to hold the alignment
                                        data
        *     --HDFSDataDir             HDFS directory that holds the read data
        *     --HDFSPathToBowtieAlign   HDFS path to the bowtie2 executable
        *     --numReports              Max number of alignment hits to report
                                        with the -k option
        *     --reference               HDFS path to the bowtie 2 fasta
                                        reference file

    gemSingleEnds      Run a GEM alignment
      Usage: gemSingleEnds [options]
  Options:
        *     --HDFSAlignmentsDir     HDFS directory to hold the alignment data
        *     --HDFSDataDir           HDFS directory that holds the read data
        *     --HDFSPathToGEM2SAM     HDFS path to the gem-2-sam executable
        *     --HDFSPathToGEMMapper   HDFS path to the gem-mapper executable
        *     --editDistance          Edit distance parameter (-e) to use in the
                                      GEM mapping
                                      Default: 0
        *     --maxProcessesOnNode    Maximum number of GEM mapping processes to
                                      run on one node simultaneously
                                      Default: 6
        *     --numReports            Max number of hits to report from GEM
        *     --reference             HDFS path to the GEM reference file
              --strata                Strata parameter (-s) to use in the GEM
                                      mapping
                                      Default: all

    razerS3SingleEnds      Run a razerS3 alignment
      Usage: razerS3SingleEnds [options]
  Options:
        *     --HDFSAlignmentsDir   HDFS directory to hold the alignment data
        *     --HDFSDataDir         HDFS directory that holds the read data
        *     --HDFSPathToRazerS3   HDFS path to the razers3 executable file
        *     --numReports          Max number of alignments to report for each
                                    read
        *     --pctIdentity         RazerS 3 percent identity parameter (-i)
                                    Default: 0
        *     --reference           HDFS path to the reference (FASTA) file for
                                    the RazerS 3 mapper
        *     --sensitivity         RazerS 3 sensitivity parameter (-rr)
                                    Default: 0

    mrfastSingleEnds      Run a novoalign mate pair alignment
      Usage: mrfastSingleEnds [options]
  Options:
        *     --HDFSAlignmentsDir   HDFS directory to hold the alignment data
        *     --HDFSDataDir         HDFS directory that holds the read data
        *     --HDFSPathToMrfast    HDFS path to the mrfast executable file
        *     --reference           HDFS path to the mrfast reference index file
              --threshold           MrFAST threshold parameter (-e)
                                    Default: -1

    exportAlignmentsFromHDFS      Export alignments in SAM format
      Usage: exportAlignmentsFromHDFS [options]
  Options:
              --aligner        Format of the alignment records
                               (sam|mrfast|novoalign)
                               Default: sam
        *     --inputHDFSDir   HDFS path to the directory holding the alignment
                               reccords

    GMMFitSingleEndInsertSizes      Compute GMM features in each bin across the genome
      Usage: GMMFitSingleEndInsertSizes [options]
  Options:
              --aligner                            Format of the alignment
                                                   records (sam|mrfast|novoalign)
                                                   Default: sam
              --chrFilter                          If filter params are used,
                                                   only consider alignments in the
                                                   region
                                                   chrFilter:startFilter-endFilter
              --endFilter                          See chrFilter
              --excludePairsMappingIn              HDFS path to a BED file. Any
                                                   reads mapped within those intervals
                                                   will be excluded from the
                                                   processing
        *     --faidx                              HDFS path to the chromosome
                                                   length file for the reference genome
        *     --inputFileDescriptor                HDFS path to the directory
                                                   that holds the alignment records
              --legacyAlignments                   Use data generated with an
                                                   older version of Cloudbreak
                                                   Default: false
              --mapabilityWeighting                HDFS path to a BigWig file
                                                   containing genome uniqness scores. If
                                                   specified, Cloudbreak will weight reads
                                                   by the uniqueness of the regions
                                                   they mapped to
              --maxInsertSize                      Maximum insert size to
                                                   consider (= max size of deletion
                                                   detectable)
                                                   Default: 25000
              --maxLogMapqDiff                     Adaptive quality score cutoff
                                                   Default: 5.0
              --maxMismatches                      Max number of mismatches
                                                   allowed in an alignment; all other
                                                   will be ignored
                                                   Default: -1
              --minCleanCoverage                   Minimum number of spanning
                                                   read pairs for a bin to run the
                                                   GMM fitting procedure
                                                   Default: 3
              --minScore                           Minimum alignment score (SAM
                                                   tag AS); all reads with lower AS
                                                   will be ignored
                                                   Default: -1
        *     --outputHDFSDir                      HDFS path to the directory
                                                   that will hold the output of the
                                                   GMM procedure
              --resolution                         Size of the bins to tile the
                                                   genome with
                                                   Default: 25
              --startFilter                        See chrFilter
              --stripChromosomeNamesAtWhitespace   Clip chromosome names from
                                                   the reference at the first
                                                   whitespace so they match with alignment
                                                   fields
                                                   Default: false

    extractDeletionCalls      Extract deletion calls into a BED file
      Usage: extractDeletionCalls [options]
  Options:
        *     --faidx                Chromosome length file for the reference
        *     --inputHDFSDir         HDFS path to the GMM fit feature results
              --medianFilterWindow   Use a median filter of this size to clean
                                     up the results
                                     Default: 5
        *     --outputHDFSDir        HDFS Directory to store the variant calls
                                     in
              --resolution           Size of the bins to tile the genome with
                                     Default: 25
        *     --targetIsize          Mean insert size of the library
                                     Default: 0
        *     --targetIsizeSD        Standard deviation of the insert size of
                                     the library
                                     Default: 0
              --threshold            Likelihood ratio threshold to call a
                                     variant
                                     Default: 1.68

    extractInsertionCalls      Extract insertion calls into a BED file
      Usage: extractInsertionCalls [options]
  Options:
        *     --faidx                Chromosome length file for the reference
        *     --inputHDFSDir         HDFS path to the GMM fit feature results
              --medianFilterWindow   Use a median filter of this size to clean
                                     up the results
                                     Default: 5
              --noCovFilter          filter out calls next to a bin with no
                                     coverage - recommend on for BWA alignments, off for
                                     other aligners
                                     Default: true
        *     --outputHDFSDir        HDFS Directory to store the variant calls
                                     in
              --resolution           Size of the bins to tile the genome with
                                     Default: 25
        *     --targetIsize          Mean insert size of the library
                                     Default: 0
        *     --targetIsizeSD        Standard deviation of the insert size of
                                     the library
                                     Default: 0
              --threshold            Likelihood ratio threshold to call a
                                     variant
                                     Default: 1.68

    copyToS3      Upload a file to Amazon S3 using multi-part upload
      Usage: copyToS3 [options]
  Options:
        *     --S3Bucket   S3 Bucket to upload to
        *     --fileName   Path to the file to be uploaded on the local
                           filesystem

    launchCluster      Use whirr to create a new cluster in the cloud using whirr/cloudbreak-whirr.properties
      Usage: launchCluster [options]
    runScriptOnCluster      Execute a script on one node of the currently running cloud cluster
      Usage: runScriptOnCluster [options]
  Options:
        *     --fileName   Path on the local filesystem of the script to run

    destroyCluster      Destroy the currently running whirr cluster
      Usage: destroyCluster [options]
    summarizeAlignments      Gather statistics about a set of alignments: number of reads, number of mappings, and total number of mismatches
      Usage: summarizeAlignments [options]
  Options:
              --aligner        Format of the alignment records
                               (sam|mrfast|novoalign)
                               Default: sam
        *     --inputHDFSDir   HDFS path of the directory that holds the
                               alignments

    exportGMMResults      Export wig files that contain the GMM features across the entire genome
      Usage: exportGMMResults [options]
  Options:
        *     --faidx          Local path to the chromosome length file
        *     --inputHDFSDir   HDFS path to the directory holding the GMM
                               features
        *     --outputPrefix   Prefix of the names of the files to create
              --resolution     Bin size that the GMM features were computed for
                               Default: 25

    dumpReadsWithScores      Dump all read pairs that span the given region with their deletion scores to BED format (debugging)
      Usage: dumpReadsWithScores [options]
  Options:
              --aligner                            Format of the alignment
                                                   records (sam|mrfast|novoalign)
                                                   Default: sam
        *     --inputFileDescriptor                HDFS path to the directory
                                                   that holds the alignment records
              --maxInsertSize                      Maximum possible insert size
                                                   to consider
                                                   Default: 500000
              --minScore                           Minimum alignment score (SAM
                                                   tag AS); all reads with lower AS
                                                   will be ignored
                                                   Default: -1
        *     --outputHDFSDir                      HDFS path to the directory
                                                   that will hold the output
        *     --region                             region to find read pairs
                                                   for, in chr:start-end format
              --stripChromosomeNamesAtWhitespace   Clip chromosome names from
                                                   the reference at the first
                                                   whitespace so they match with alignment
                                                   fields
                                                   Default: false

    debugReadPairInfo      Compute the raw data that goes into the GMM fit procedure for each bin (use with filter to debug a particular locus)
      Usage: debugReadPairInfo [options]
  Options:
              --aligner                 Format of the alignment records
                                        (sam|mrfast|novoalign)
                                        Default: sam
        *     --chrFilter               Print info for alignments in the region
                                        chrFilter:startFilter-endFilter
        *     --endFilter               see chrFilter
              --excludePairsMappingIn   HDFS path to a BED file. Any reads
                                        mapped within those intervals will be excluded
                                        from the processing
        *     --faidx                   HDFS path to the chromosome length file
                                        for the reference genome
        *     --inputFileDescriptor     HDFS path to the directory that holds
                                        the alignment records
              --mapabilityWeighting     HDFS path to a BigWig file containing
                                        genome uniqness scores. If specified,
                                        Cloudbreak will weight reads by the uniqueness of
                                        the regions they mapped to
              --maxInsertSize           Maximum insert size to consider (= max
                                        size of deletion detectable)
                                        Default: 500000
              --minScore                Minimum alignment score (SAM tag AS);
                                        all reads with lower AS will be ignored
                                        Default: -1
        *     --outputHDFSDir           HDFS directory to hold the output
              --resolution              Size of the bins to tile the genome with
                                        Default: 25
        *     --startFilter             see chrFilter

    findAlignment      Find an alignment record that matches the input string
      Usage: findAlignment [options]
  Options:
        *     --HDFSAlignmentsDir   HDFS path to the directory that stores the
                                    alignment data
        *     --outputHDFSDir       HDFS path to the directory in which to put
                                    the results
        *     --read                Read name or portion of the read name to
                                    search for

    sortGMMResults      Sort and merge GMM Results (use with one reducer to get all GMM feature results into a single file
      Usage: sortGMMResults [options]
  Options:
        *     --inputHDFSDir    HDFS path to the directory holding the GMM
                                features
        *     --outputHDFSDir   Directory in which to put the results