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GotCloud: Alignment Pipeline (view source)

Revision as of 18:39, 28 January 2016

4,398 bytes added ,  18:39, 28 January 2016
→‎Optional Configurable Settings
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= Mapping Pipeline =
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Back to parent: [[GotCloud]]
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Back to parent: [[GotCloud]]  
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The Mapping Pipeline takes FASTQ files and generates recalibrated BAM files from them.
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== Running the GotCloud Mapping Pipeline ==
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== Overview of Alignment Pipeline Steps ==  
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The Alignment/Mapping Pipeline takes [http://en.wikipedia.org/wiki/FASTQ_format FASTQ files] and generates recalibrated [[BAM|BAM (Binary Sequence Alignment/Map format) files]] from them.
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The mapping pipeline is run using the <code>gen_biopipeline.pl</code> script found in the <code>bin/</code> directory under the <code>gotcloud/</code> installation.
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[[File:MappingSteps.png]]
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===Running the Automatic Test===
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== Running the GotCloud Alignment Pipeline ==  
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The automatic test runs the mapping pipeline on a small testset and checks the results against expected results validating that GotCloud is installed correctly.
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The alignment pipeline is run using the <code>align</code> option of the <code>gotcloud</code> script.  This option calls <code>align.pl</code> found in the <code>bin/</code> directory under the <code>gotcloud</code> installation.  
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*Run alignment pipeline test:
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Use the <code>--conf</code> parameter followed by the configuration file to specify the configuration to use for this run of the alignment pipeline.
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gen_biopipeline.pl --test OUTPUT_DIR
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where OUTPUT_DIR is the directory where you want to store the test results
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If you see "Successfully ran the test case, congratulations!", then you are ready to align samples.
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You must specify the input list of FASTQs mapped to sample id to tell the alignment pipeline what files to process.  You can do this by setting either:
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* <code>FASTQ_LIST</code> in the configuration file
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* <code>--list</code> on the command-line
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You must specify an output directory to tell the alignment pipeline where to write its output by either setting:
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* <code>OUT_DIR</code> in the configuration file
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* <code>--outdir</code> on the command-line
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'''Example of a Basic Alignment Command'''
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gotcloud align --conf myAlignTest.conf --outdir ~/gotcloudOutput/align/
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 +
 
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===Running the Automated Test===
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The automated test runs the alignment pipeline on a small set of test data and checks that the results against expected results validating that GotCloud is installed correctly.
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== Overview of Mapping Pipeline Steps ==
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*Run alignment pipeline test:
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Here is an overview of the Mapping Pipeline:
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gotcloud align --test OUTPUT_DIR
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where OUTPUT_DIR is the directory where you want to store the test results
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[[File:MappingSteps.png]]
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If you see "Successfully ran the test case, congratulations!", then you are ready to align samples.
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== Input Data:==
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== Input Data:==  
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*Raw Sequence (FASTQ) files
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*Raw Sequence (FASTQ) files  
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*Sequence Index file containing fastqs & RG info
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*FASTQ List file mapping fastq pairs to sample (optional: Read Group information)
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*Reference files
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*Reference files  
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*(Optional) Configuration file to override default options
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*(Optional) Configuration file to override default options  
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=== Raw Sequence (FASTQ) files ===
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=== Raw Sequence (FASTQ) files ===  
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These are the FASTQ files that need to be mapped to BAM files.
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These are the FASTQ files that need to be mapped to BAM files.  
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These files are specified in the [[#Sequence Index File|Sequence Index File]].
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These files are specified in the [[#FASTQ List File|FASTQ List File]].  
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=== Sequence Index File ===
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=== FASTQ List File ===  
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This file specifies the FASTQ files that need to be processed and the Read Group information for them.
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This file specifies the FASTQ files that need to be processed.  It maps the FASTQ pairs to the associated Sample ID.  Optionally Read Group information for the FASTQ pairs can be specified.  If the Read Group information is not specified, it is inferred.  
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This file is specified either via the command line parameter <code>--index_file</code> or via the configuration file setting <code>INDEX_FILE</code>.   
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This file is specified either via the command line parameter <code>--list</code> or via the configuration file setting <code>FASTQ_LIST</code>.   
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The command-line setting takes precedence over the configuration file setting.
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The command-line setting takes precedence over the configuration file setting.  
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The Sequence Index is a tab delimited file that starts with a header line.  The columns may be in any order.
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The FASTQ list is a tab delimited file that starts with a header line.  The columns may be in any order.  
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Following the header line, there is one line per single-end read and one line per paired-end read (only 1 line per pair).
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Following the header line, there is one line per single-end read and one line per paired-end read (only 1 line per pair).  
    
Required Column Names:  
 
Required Column Names:  
 
* MERGE_NAME - base name for the resulting BAM file for the sample (used to group multiple fastqs or fastq pairs into a single BAM)
 
* MERGE_NAME - base name for the resulting BAM file for the sample (used to group multiple fastqs or fastq pairs into a single BAM)
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* FASTQ1 - name of the fastq or the first in the pair if paired-end.  (Only 1 line per pair)
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** The SAMPLE column can be specified instead of MERGE_NAME.  SAMPLE will be used for both the sample and the base name.
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* FASTQ1 - name of the fastq or the first in the pair if paired-end.  (Only 1 line per pair)  
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Optional Column Names:
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Optional Column Names:  
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* FASTQ2 - name of the 2nd fastq in paired-end reads.  Specify '.' if the column exists, but this line is single-ended.
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* FASTQ2 - name of the 2nd fastq in paired-end reads.  Specify '.' if the column exists, but this line is single-ended.  
 
* RGID - Read Group ID for this entry
 
* RGID - Read Group ID for this entry
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* SAMPLE - Sample Name for this entry
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** If this field is not specified, the first line of the fastq will be used to determine the RG.
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* LIBRARY - Library for this entry
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*** If the first line does not match the expected format for determining RG, incrementing numbers per fastq file will be used.
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* CENTER - Center Name for this entry
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* SAMPLE - Sample Name for this entry  
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* PLATFORM - Platform for this entry
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** If SAMPLE is not specified, MERGE_NAME will be used for the sample name
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* LIBRARY - Library for this entry  
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** If LIBRARY is not specified, the sample name will be used
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* CENTER - Center Name for this entry  
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** If CENTER is not specified, it will default to "unknown"
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* PLATFORM - Platform for this entry  
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** If PLATFORM is not specified, it will default to ILLUMINA
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The RGID, SAMPLE, LIBRARY, CENTER, and PLATFORM are used to populate the Read Group information for this entry.  These fields are optional.  Either leave the column header out of the file or specify '.' if the column header exists, but the data is N/A.  As long as the RGID field is specified non-N/A fields are added to the BAM file.
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The RGID, SAMPLE, LIBRARY, CENTER, and PLATFORM are used to populate the Read Group information for this entry.   
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  MERGE_NAME FASTQ1 FASTQ2 RGID SAMPLE LIBRARY CENTER PLATFORM
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  MERGE_NAME FASTQ1 FASTQ2 RGID SAMPLE LIBRARY CENTER PLATFORM  
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  Sample1 fastq/S1/F1_R1.fastq.gz fastq/S1/F1_R2.fastq.gz RGID1 SampleID1 Lib1 UM ILLUMINA
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  Sample1 fastq/S1/F1_R1.fastq.gz fastq/S1/F1_R2.fastq.gz RGID1 SampleID1 Lib1 UM ILLUMINA  
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  Sample1 fastq/S1/F2_R1.fastq.gz fastq/S1/F2_R2.fastq.gz RGID1a SampleID1 Lib1 UM ILLUMINA
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  Sample1 fastq/S1/F2_R1.fastq.gz fastq/S1/F2_R2.fastq.gz RGID1a SampleID1 Lib1 UM ILLUMINA  
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  Sample2 fastq/S2/F1_R1.fastq.gz fastq/S2/F1_R2.fastq.gz RGID2 SampleID2 Lib2 UM ILLUMINA
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  Sample2 fastq/S2/F1_R1.fastq.gz fastq/S2/F1_R2.fastq.gz RGID2 SampleID2 Lib2 UM ILLUMINA  
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  Sample2 fastq/S2/F2.fastq.gz . RGID2 SampleID2 Lib2 UM ILLUMINA
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  Sample2 fastq/S2/F2.fastq.gz . RGID2 SampleID2 Lib2 UM ILLUMINA  
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The <code>--fastq</code>/<code>FASTQ</code> setting can be used to specify a prefix to the FASTQ1/FASTQ2 file paths that should be applied before using the files.
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The <code>--fastq_prefix</code>/<code>FASTQ_PREFIX</code> setting can be used to specify a prefix to the FASTQ1/FASTQ2 file paths that should be applied before using the files.
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=== Reference Files ===
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=== Reference Files ===  
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See [[GotCloud: Genetic Reference and Resource Files]] for detailed information about the multiple required reference files for the alignment pipeline, including:
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* How to obtain default references
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* Configuration keys & default values
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* How to generate your own references
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* How to point GotCloud to your reference files
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The following Reference Files are required:
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Required Reference File Types:
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* Reference File fasta files
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* [[GotCloud: Genetic Reference and Resource Files#Reference fasta Files|Reference fasta Files]]
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** Files required: .fa, -bs.umfa, .GCContent, .amb, .ann, .bwt, .pac, .rbwt, .rpac, .rsa, .sa
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* [[GotCloud: Genetic Reference and Resource Files#DBSNP VCF Files|DBSNP VCF Files]]
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*** If you don't have the -bs.umfa file, the software will try to create it in the same directory as the reference fasta.
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* [[GotCloud: Genetic Reference and Resource Files#HapMap3 VCF Files|HapMap3 VCF Files]]
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*** .GCContent can be generated using qplot, see: [[QPLOT#Input_files| QPLOT: Input Files: --gccontent]] and name the resulting file as <code>.fa.GCcontent</code>
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*** Use <code>bin/bwa index ref.fa</code> if you need to generate the bwa reference files (.amb, .ann, .bwt, .pac, .rbwt, .rpac, .rsa, .sa)
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** Configuration Name: FA_REF - specify the ref.fa/ref.fa.gz name
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* DBSNP File
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** tab delimited file/VCF, can be compressed
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*** 1st column -> chromosome
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*** 2nd column -> 1-based position
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** Configuration Name: DBSNP_VCF
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* PLINK-compatible binary genotype files
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** Files required: .bed, .bin, .fam
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** Configuration Name: PLINK
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=== Configuration File ===
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=== Configuration File ===  
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Configuration file contains the run-time options including the software binaries and command line arguments.  A default configuration file is automatically loaded.  Users may specify their own configuration file specifying just the values different than the defaults.  The configuration file is not required if there are no values to override.
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{{:GotCloud: Configuration}}
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Comments begin with a <code>#</code>
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==== Recommended Settings ====
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Format: KEY = value
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As of GotCloud version 1.16, the alignment pipeline uses <code>bwa mem</code> by default.  Prior to version 1.16, the default aligner was <code>bwa aln</code>. 
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Where KEY is the item being set and value is its new value
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You can override the defaults by setting in your configuration file:
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* to use <code>bwa mem</code> (you do not need to set this in version 1.16 and later since it is the default)
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MAP_TYPE = BWA_MEM
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* to use <code>bwa aln</code> (you do not need to set this prior to version 1.16 since it is the default)
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MAP_TYPE = BWA
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See [[#Command-Line Options|Command-Line Options]] for values that can be set either via command line or via configuration.
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==== Additional Required Settings ====
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Note: Command-line options take priority over configuration file settings
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See [[#FASTQ List File|FASTQ List File]] for how to set the index file either via command line options or via configuration.
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==== Required Settings ====
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==== Turning Off Optional Steps====  
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See [[#Reference Files|Reference Files]] for the required reference file settings.
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Quality Control steps can be disabled.  
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See [[#Sequence Index File|Sequence Index File]] for how to set the index file either via command line options or via configuration.
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To Disable QPLOT, remove qplot from the PER_MERGE_STEPS configuration by setting:
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PER_MERGE_STEPS = verifyBamID index recab
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==== Turning Off Optional Steps====
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Quality Control steps can be disabled.
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To Disable QPLOT, set:
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To Disable VerifyBamID, remove qplot from the PER_MERGE_STEPS configuration by setting:  
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  RUN_QPLOT = 0
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  PER_MERGE_STEPS = qplot index recab
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To Disable VerifyBamID, set:
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==== Optional Configurable Settings ====
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RUN_VERIFY_BAM_ID = 0
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You may want to adjust the amount of memory/threads that are used:  
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==== Optional Configurable Settings ====
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There are additional configurable settings, but these are the ones most likely to be adjusted.
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You may want to adjust the amount of memory/threads that are used:
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There are additional configurable settings, but these are the ones most likely to be adjusted.
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* BWA_THREADS = -t N  
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* BWA_THREADS = -t N
   
** Fill in the N with the number of threads you want BWA to run with, default is 1
 
** Fill in the N with the number of threads you want BWA to run with, default is 1
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* BWA_MAX_MEM = 2000000000
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* BWA_QUAL = -q N
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** Fill in the N with the trim quality you want BWA aln to run with, default is 15.  This parameter is only applied to bwa aln.  It is not used for BWA_MEM.
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* BWA_MEM_OPTS =
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** Specify any additional bwa mem options using this parameter.
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* SORT_MAX_MEM = 2000000000  
 
** Maximum amount of memory used by samtools sort after running bwa
 
** Maximum amount of memory used by samtools sort after running bwa
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* JAVA_MEM = -Xmx4g
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** Set the maximum size of the java memory allocation pool.  Default is 4g, adjust that as necessary.
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  −
  −
== Running the Mapping Pipeline ==
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=== Command-Line Options ===
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== Running the Alignment Pipeline ==  
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* help - print usage
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* test OUTPUT_DIR - run the test example placing the output in a user specified OUTPUT_DIR.  No other options are required.
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* out_dir OUTPUT_DIR - directory for the output
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** May also be specified via OUT_DIR in the configuration file
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** Required to be set either via command-line or configuration
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* conf CONFIG_FILE - configuration file
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* index_file INDEX_FILE_NAME  - name of the index file
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** May also be specified via INDEX_FILE in the configuration file
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** Required to be set either via command-line or configuration
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* ref_dir REFERENCE_DIR - value to set config key REF_DIR to, overriding other values, REF_DIR can then be used inside config files.
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** May also be specified via REF_DIR in the configuration file
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* fastq FASTQ_PATH - prefix path to the fastq files specified in the INDEX_FILE
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** May also be specified via FASTQ in the configuration file
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* keepTmp - Do not remove the temporary files (removed by default)
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** May also be specified via KEEP_TMP in the configuration file
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* numjobs N - Replace N with the number of jobs that should be run in parallel
      +
=== Command-Line Options ===
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* help - print usage
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* test OUTPUT_DIR - run the test example placing the output in a user specified OUTPUT_DIR.  No other options are required.
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* outdir OUTPUT_DIR - directory for the output
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** May also be specified via OUT_DIR in the configuration file
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** Required to be set either via command-line or configuration
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* conf CONFIG_FILE - configuration file
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* list FASTQ_LIST_FILE_NAME  - name of the fastq list file
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** May also be specified via FASTQ_LIST in the configuration file
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** Required to be set either via command-line or configuration
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* ref_dir REFERENCE_DIR - value to set config key REF_DIR to, overriding other values, REF_DIR can then be used inside config files.
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** May also be specified via REF_DIR in the configuration file
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* ref_prefix REFERENCE_DIR - path to prepend to non-absolute REF paths.
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** May also be specified via REF_PREFIX in the configuration file
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* fastq_prefix FASTQ_PATH - prefix path to the fastq files specified in the FASTQ_LIST
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** May also be specified via FASTQ_PREFIX in the configuration file
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* base_prefix BASE_PATH - prefix path to the prepend to fastq/ref files without absolute paths
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** May also be specified via BASE_PREFIX in the configuration file
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* keepTmp - Do not remove the temporary files (removed by default)
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** May also be specified via KEEP_TMP in the configuration file
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* keepLog - Do not remove the intermediate log files (removed by default)
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** May also be specified via KEEP_LOG in the configuration file
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* numjobs N - Replace N with the number of samples that should be processed in parallel
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* threads N - Replace N with the number of targets in each makefile that should be run in parallel
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* dryrun - Create the Makefile, but do not run it
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* maxlocaljobs N - Replace N with the maximum number of jobs that can be run locally (no batchtype specified).  Default is 10.
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* batchtype TYPE - Tells GotCloud the specified batch type to send jobs to the client nodes
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** May also be specified via BATCH_TYPE in the configuration file
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** Can be: mosix, slurm, slurmi, pbs, sge, sgei
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* batchopts OPTS - Tells GotCloud the options to pass onto the batch system
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** May also be specified via BATCH_OPTS in the configuration file
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* noPhoneHome - disable the phone home logic
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* gotcloudroot DIR - Specifies an alternate path to other gotcloud files rather than using the path to the gotcloud/align.pl.
 
Note: Command-line options take priority over configuration file settings
 
Note: Command-line options take priority over configuration file settings
   −
The mapping pipeline is currently a 2 step process.
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===Running the Alignment Pipeline===  
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Run <code>gotcloud align</code> with the appropriate command-line parameters.  
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===Step 1: Generate the Makefiles, 1 per sample===
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Run bin/gen_biopipeline.pl with the appropriate command-line parameters.
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Example:
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bin/gen_biopipeline.pl --conf config.txt --out_dir output
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This step generates 1 Makefile per sample in the output/Makefiles/ directory, but does not run them.  These Makefiles contain all of the information to run each sample.
     −
Instructions are printed for running the Makefiles.
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Example:
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gotcloud align --conf config.txt --outdir output
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===Step 2: Run the Samples===
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This step generates 1 Makefile per sample in the output/Makefiles/ directory and then automatically runs them.  The Makefiles contain all of the information to run each sample.  
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Run make -f on each file in the Makefiles directory.  Each Makefile is independent and can be run in parallel and across a cloud.
     −
It is recommended that you redirect stdout and stderr to files to save the results.
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If you only want to generate the makefiles and not run them, use the <code>--dryrun</code> option.  It will generate the Makefiles and print instructions for running the Makefiles.  
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On failure, the Makefile should report a message like:
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Each Makefile is independent and can be run in parallel and across a cloud.  
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make: *** [...] Error 1
     −
Where ... is filled in with other text indicating what step failed.
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On success, you will see:
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Processing finished in nn secs with no errors reported
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On success, you should see the following subdirectories under the user specified output directory:
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If processing fails part way through, you can pick up where you left off by rerunning gotcloud or the make command.
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* alignment_recal/
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* Makefiles/
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* QCFiles/ (if all quality control is not disabled)
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* tmp/
     −
You should see a <code>.OK</code> for each Sample in the index file.
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=== Alignment Pipeline Output ===
 +
Upon successful completion of the alignment pipeline, you should see the following files/ subdirectories under the user specified output directory:
 +
* '''bam.list''' - file containing sample->BAM mapping that can be used in other GotCloud pipelines
 +
* '''bams/''' - contains the final BAM and bai (BAM index) files
 +
** '''*.recal.bam'''
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** '''*.recal.bam.bai'''
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** ''*.recal.bam.bai.done'' - temp file indicating this step completed successfully
 +
** ''*.recal.bam.done'' - temp file indicating this step completed successfully
 +
** *.recal.bam.metrics - dedup & recalibration log
 +
** *.recal.bam.qemp - recalibration tables
 +
* Makefiles/ - contains the Makefiles and logs used by GotCloud to run the alignment pipeline
 +
* '''QCFiles/''' - contains quality control results if quality control is not disabled
 +
** VerifyBamID Output - see [[VerifyBamID#A_guideline_to_interpret_output_files|VerifyBamID: A guideline to interpret output files]] for more information
 +
*** *.genoCheck.depthRG - depth distribution of the sequence reads per read group
 +
*** *.genoCheck.depthSM - depth distribution of the sequence reads per sample
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*** ''*.genoCheck.done'' - temp file indicating this step completed successfully
 +
*** *.genoCheck.selfRG - per-readGroup statistics describing how well each lane matches to the annotated sample
 +
*** '''*.genoCheck.selfSM''' - main output file containing the contamination estimate; per-sample statistics describing how well the sample matches to the annotated sample
 +
**** Check the 'FREEMIX' column for genotype-free estimate of contamination 0-1 scale, the lower, the better
 +
**** If [FREEMIX] >= 0.03 and [FREELK1]-[FREELK0] is large, possible contamination
 +
** Qplot Output - see: [[QPLOT#Diagnose_sequencing_quality|QPLOT: Diagnose sequencing quality]] for more info on how to use QPLOT results
 +
*** ''*.qplot.done'' - temp file indicating this step completed successfully
 +
*** '''*.qplot.R''' - Rscript that can be used to generate the pdf graphs
 +
*** '''*.qplot.stats''' - sample statistics
 +
* tmp/ - contains intermediate files (most are deleted unless --keepTmp is specified)
 +
* *.OK - one OK file per sample; indicates the Sample successfully completed alignment
   −
If you do not see these <code>.OK</code> files, then your Mapping Pipeline failed.
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You should also see a <code>.OK</code> for each Sample in the index file.  
   −
On success, the alignment.recal/ directory contains the final BAMs, bais, and md5s.
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If you do not see these <code>.OK</code> files, then your Alignment Pipeline failed.  
   −
If processing fails part way through, you can pick up where you left off by rerunning the make command.
+
'''On success, the bams/ directory contains the final BAMs and bais.'''
Retrieved from "http://genome.sph.umich.edu/wiki/Special:MobileDiff/6480...14062"

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