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Revision as of 18:39, 28 January 2016
, 18:39, 28 January 2016→Optional Configurable Settings
=== Reference Files ===
=== Reference Files ===
See [[GotCloud: Genetic Reference and Resource Files]] for detailed information about the multiple required reference files for the alignment pipeline, including:
* How to obtain default references
* Configuration keys & default values
* How to generate your own references
* How to point GotCloud to your reference files
Required Reference File Types:
* Reference File fasta files
* [[GotCloud: Genetic Reference and Resource Files#Reference fasta Files|Reference fasta Files]]
* [[GotCloud: Genetic Reference and Resource Files#DBSNP VCF Files|DBSNP VCF Files]]
* [[GotCloud: Genetic Reference and Resource Files#HapMap3 VCF Files|HapMap3 VCF Files]]
*** .GCContent can be generated using qplot, see: [[QPLOT#Input_files| QPLOT: Input Files: --gccontent]] and name the resulting file as <code>.fa.GCcontent</code>
*** Use <code>bin/bwa index ref.fa</code> if you need to generate the bwa reference files (.amb, .ann, .bwt, .pac, .sa)
=== Configuration File ===
=== Configuration File ===
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.
{{:GotCloud: Configuration}}
==== Recommended Settings ====
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>.
You can override the defaults by setting in your configuration file:
* to use <code>bwa mem</code> (you do not need to set this in version 1.16 and later since it is the default)
MAP_TYPE = BWA_MEM
* to use <code>bwa aln</code> (you do not need to set this prior to version 1.16 since it is the default)
MAP_TYPE = BWA
==== Additional Required Settings ====
See [[#FASTQ List File|FASTQ List File]] for how to set the index file either via command line options or via configuration.
See [[#Reference Files|Reference Files]] for the required reference file settings.
==== Turning Off Optional Steps====
==== Turning Off Optional Steps====
Quality Control steps can be disabled.
Quality Control steps can be disabled.
To Disable QPLOT, set:
To Disable QPLOT, remove qplot from the PER_MERGE_STEPS configuration by setting:
PER_MERGE_STEPS = verifyBamID index recab
To Disable VerifyBamID, set:
To Disable VerifyBamID, remove qplot from the PER_MERGE_STEPS configuration by setting:
PER_MERGE_STEPS = qplot index recab
==== Optional Configurable Settings ====
==== Optional Configurable Settings ====
* BWA_THREADS = -t N
* 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
* BWA_QUAL = -q N
** 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.
* BWA_MEM_OPTS =
** Specify any additional bwa mem options using this parameter.
* SORT_MAX_MEM = 2000000000
* 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
== Running the Alignment Pipeline ==
== Running the Alignment Pipeline ==
On success, you will see:
On success, you will see:
Processing finished in nn secs with no errors reported
Processing finished in nn secs with no errors reported
* bams/
If processing fails part way through, you can pick up where you left off by rerunning gotcloud or the make command.
* Makefiles/
* QCFiles/ (if all quality control is not disabled)
=== Alignment Pipeline Output ===
* tmp/
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'''
** '''*.recal.bam.bai'''
** ''*.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
*** ''*.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
You should see a <code>.OK</code> for each Sample in the index file.
You should also see a <code>.OK</code> for each Sample in the index file.
If you do not see these <code>.OK</code> files, then your Alignment Pipeline failed.
If you do not see these <code>.OK</code> files, then your Alignment Pipeline failed.
On success, the bams/ directory contains the final BAMs and bais.
'''On success, the bams/ directory contains the final BAMs and bais.'''
