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GotCloud: Alignment Sub-Pipelines (view source)

Revision as of 21:37, 18 March 2015

4,019 bytes added ,  21:37, 18 March 2015
→‎Example Command Line
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====Inputs====
 
====Inputs====
−
* Bam files (stored in a [[#BAM_LIST|BAM_LIST]] file)
+
* Bam files (stored in a [[#BAM_LIST File for recab|BAM_LIST File]])
 
* Reference files
 
* Reference files
 
* (Optional) configuration file to override default options
 
* (Optional) configuration file to override default options
   −
=====BAM_LIST File=====
+
=====BAM_LIST File for recab=====
 
* Each line of the BAM list file represents a single individual
 
* Each line of the BAM list file represents a single individual
   Line 78: Line 78:  
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
 
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
 
|-
 
|-
−
| --list/--bam_list/--bamlist ''file'' || BAM_LIST || path to the [[#BAM_LIST File|BAM_LIST File]] || $(OUT_DIR)/bam.list
+
| --list/--bam_list/--bamlist ''file'' || BAM_LIST || path to the [[#BAM_LIST File for recab|BAM_LIST File]] || $(OUT_DIR)/bam.list
 
|-
 
|-
 
| --numjobs ''#'' || || number of jobs to run in parallel || 0 (generate Makefile of steps, but do not run)
 
| --numjobs ''#'' || || number of jobs to run in parallel || 0 (generate Makefile of steps, but do not run)
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{| class="wikitable" style="margin: 1em 1em 1em 0; background-color: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" border="1"
 
{| class="wikitable" style="margin: 1em 1em 1em 0; background-color: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" border="1"
−
! colspan="4" | Common Options
  −
|-
   
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
 
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
 
|-
 
|-
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  gotcloud pipe –-name recab --numjobs <N>
 
  gotcloud pipe –-name recab --numjobs <N>
   −
== recabQC ==  
+
== recabQC ==
−
== bamQC ==
+
*What it does:
−
== bamQC_createIndex ==
+
# merge BAMs for samples that have multiple BAMs
 +
# dedup and recalibrate
 +
# index the recalibrated BAM
 +
# qplot
 +
# verifyBamID
    +
====Inputs====
 +
* Bam files (stored in a [[#BAM_LIST File for recabQC|BAM_LIST]] file)
 +
* Reference files
 +
* (Optional) configuration file to override default options
    +
=====BAM_LIST File for recabQC=====
 +
* Each line of the BAM list file represents a single individual
    +
Columns:
 +
# sample id
 +
# comma separated population labels (optional column)
 +
# BAM File 1 (preferable to have full paths to BAM files)
 +
# BAM File 2 (if more than 1 BAM per sample)
 +
:...
    +
: # BAM File N (if more than 1 BAM per sample)
 +
[SAMPLE_ID]    [COMMA SEPARATED POPULATION LABELS] [BAM_FILE1] [BAM_FILE2] ...
 +
or
 +
[SAMPLE_ID] [BAM_FILE1] [BAM_FILE2] ...
    +
* Notes:
 +
** tab delimited
 +
** multiple BAMs per individual may be provided, but should all be on the same line of the list file
 +
** population label is optional - it will default to <code>ALL</code>
 +
*** only used by Thunder (part of ldrefine pipeline)
 +
*** if all samples are from the same population, population label can be skipped or you can just specify <code>ALL</code> for the population label for each sample.
    +
====Outputs====
 +
Upon successful completion of the *recabQC* sub-pipeline, you should see the following files/subdirectories under the user specified output directory:
 +
*'''recab/mergedBams/''' - contains merge results
 +
** '''''*/SAMPLE.merged.bam'' - a merged BAM file'''
 +
** ''*/SAMPLE.merged.bam.log'' - merge log
 +
** ''*/SAMPLE.merged.bam.OK'' - temp file indicating the merge step completed successfully
    +
* '''recab/''' - contains recalibration results
 +
** '''''*/SAMPLE.recal.bam'' - a merged, recalibrated, and deduped BAM file'''
 +
** '''''*/SAMPLE.recal.bam.bai'' - an indexed version of the  merged, recalibrated, and deduped BAM file'''
 +
** ''*/SAMPLE.recal.bam.metrics'' - dedup & recalibration log
 +
** ''*/SAMPLE.recal.bam.qemp'' - recalibration tables
 +
** ''*/SAMPLE.recal.bam.done'' - temp file indicating the recalibration step completed successfully
 +
** ''*/SAMPLE.recal.bam.bai.done'' - temp file indicating the indexing step completed successfully
    +
* '''QCFiles/''' - contains quality control results
 +
** VerifyBamID Output - see [[VerifyBamID#A_guideline_to_interpret_output_files|VerifyBamID: A guideline to interpret output files]] for more information
 +
*** ''*/SAMPLE.genoCheck.depthRG'' - depth distribution of the sequence reads per read group
 +
*** ''*/SAMPLE.genoCheck.depthSM'' - depth distribution of the sequence reads per sample
 +
*** ''*/SAMPLE.genoCheck.err'' - log file
 +
*** ''*/SAMPLE.genoCheck.log'' - log file
 +
*** ''*/SAMPLE.genoCheck.OK'' - temp file indicating the VerifyBAMID step completed successfully
 +
*** ''*/SAMPLE.genoCheck.selfRG'' - per-readGroup statistics describing how well each lane matches to the annotated sample
 +
*** '''''*/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
 +
*** ''*/SAMPLE.qplot.OK'' - temp file indicating the qplot step completed successfully
 +
*** '''''*/SAMPLE.qplot.R'' - Rscript that can be used to generate the pdf graphs'''
 +
*** '''''*/SAMPLE.qplot.stats'' - sample statistics'''
    +
You should see .done and .OK files for each SAMPLE in the index file. If you do not see the .done and .OK files, then your *recabQC* sub-pipeline failed.
   −
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.
+
'''On success, the recab/ folder contains the final BAMs and bais, while the QCFiles/ folder contains the quality control output'''
   −
*Run alignment pipeline test:
+
===Command-Line and Configuration Options===
−
gotcloud align --test OUTPUT_DIR
  −
where OUTPUT_DIR is the directory where you want to store the test results
     −
If you see "Successfully ran the test case, congratulations!", then you are ready to align samples.
+
*Required Options
 +
{| class="wikitable" style="margin: 1em 1em 1em 0; background-color: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" border="1"
 +
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
 +
|-
 +
| --list/--bam_list/--bamlist ''file'' || BAM_LIST || path to the [[#BAM_LIST File for recabQC|BAM_LIST File]] || $(OUT_DIR)/bam.list
 +
|-
 +
| --numjobs ''#'' || || number of jobs to run in parallel || 0 (generate Makefile of steps, but do not run)
 +
|}
   −
== Input Data:==
+
*Common Options
−
*Raw Sequence (FASTQ) files
  −
*FASTQ List file mapping fastq pairs to sample (optional: Read Group information)
  −
*Reference files
  −
*(Optional) Configuration file to override default options
     −
=== Raw Sequence (FASTQ) files ===
+
{| class="wikitable" style="margin: 1em 1em 1em 0; background-color: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" border="1"
 +
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
 +
|-
 +
| --outdir ''path'' || OUT_DIR || output directory ||
 +
|-
 +
| --conf ''file'' || || configuration file to use ||
 +
|-
 +
|  || REF_DIR || where the reference/resource files are stored || gotcloud.ref subdirectory within the base GotCloud directory
 +
|-
 +
| || REF || [[GotCloud: Genetic Reference and Resource Files#Reference fasta Files|Reference fasta Files]] || $(REF_DIR)/human.g1k.v37.fa
 +
|-
 +
| || DBSNP_VCF || [[GotCloud: Genetic Reference and Resource Files#DBSNP VCF File|DBSNP VCF Files]] || $(REF_DIR)/dbsnp_135.b37.vcf.gz
 +
|-
 +
| || HM3_VCF || [[GotCloud: Genetic Reference and Resource Files#HapMap3 VCF File|HapMap3 VCF Files]] || $(REF_DIR)/hapmap_3.3.b37.sites.vcf.gz
 +
|}
   −
These are the FASTQ files that need to be mapped to BAM files.  
+
==== Example Configuration File ====
 +
Example configuration file where reference files happen to be stored in /path/reference, and bam list file is stored in in path/freeze5
 +
BAM_LIST = /path/freeze5.bam.list
 +
OUT_DIR = /path/freeze5/output
 +
REF_DIR = /path/reference/
 +
REF = $(REF_DIR)/hs37d5.fa
 +
DBSNP_VCF = $(REF_DIR)/dbsnp_135.b37.sites.vcf.gz
 +
HM3_VCF = $(REF_DIR)/hapmap3_r3_b37.sites.vcf.gz
   −
These files are specified in the [[#FASTQ List File|FASTQ List File]].
+
==== Example Command Line ====
 +
gotcloud pipe –-name recabQC --numjobs <N>
   −
=== FASTQ List File ===  
+
== bamQC ==
−
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.
+
*What it does:
 +
# qplot
 +
# verifyBamID
   −
This file is specified either via the command line parameter <code>--list</code> or via the configuration file setting <code>FASTQ_LIST</code>. 
+
====Inputs====
 +
* Single merged, recalibrated, and deduped BAM file for each subject (stored in a [[#BAM_LIST File for bamQC|BAM_LIST File]])
 +
* BAI file for each subject
 +
* Reference files
 +
* (Optional) configuration file to override default options
   −
The command-line setting takes precedence over the configuration file setting.
+
=====BAM_LIST File for bamQC=====
 +
* Each line of the BAM list file represents a single individual
   −
The FASTQ list is a tab delimited file that starts with a header line.  The columns may be in any order.
+
Columns:
 +
# sample id
 +
# comma separated population labels (optional column)
 +
# BAM File (preferable to have full path to BAM file)
 +
# BAI File (preferable to have full path to BAI file)
   −
Following the header line, there is one line per single-end read and one line per paired-end read (only 1 line per pair).
+
[SAMPLE_ID] [COMMA SEPARATED POPULATION LABELS] [BAM_FILE] [BAI_FILE]
 +
or
 +
[SAMPLE_ID] [BAM_FILE] [BAI_FILE]
   −
Required Column Names:  
+
* Notes:
−
* MERGE_NAME - base name for the resulting BAM file for the sample (used to group multiple fastqs or fastq pairs into a single BAM)
+
** tab delimited
−
** The SAMPLE column can be specified instead of MERGE_NAME.  SAMPLE will be used for both the sample and the base name.
+
** population label is optional - it will default to <code>ALL</code>
−
* FASTQ1 - name of the fastq or the first in the pair if paired-end.  (Only 1 line per pair)
+
*** only used by Thunder (part of ldrefine pipeline)
 +
*** if all samples are from the same population, population label can be skipped or you can just specify <code>ALL</code> for the population label for each sample.
   −
Optional Column Names:
+
====Outputs====
−
* FASTQ2 - name of the 2nd fastq in paired-end reads.  Specify '.' if the column exists, but this line is single-ended.
+
Upon successful completion of the *bamQC* sub-pipeline, you should see the following files/subdirectories under the user specified output directory:
−
* RGID - Read Group ID for this entry
  −
** If this field is not specified, the first line of the fastq will be used to determine the RG.
  −
*** If the first line does not match the expected format for determining RG, incrementing numbers per fastq file will be used.
  −
* SAMPLE - Sample Name for this entry
  −
** If SAMPLE is not specified, MERGE_NAME will be used for the sample name
  −
* LIBRARY - Library for this entry
  −
** If LIBRARY is not specified, the sample name will be used
  −
* CENTER - Center Name for this entry
  −
** If CENTER is not specified, it will default to "unknown"
  −
* PLATFORM - Platform for this entry
  −
** If PLATFORM is not specified, it will default to ILLUMINA
     −
The RGID, SAMPLE, LIBRARY, CENTER, and PLATFORM are used to populate the Read Group information for this entry.
+
* '''QCFiles/''' - contains quality control results
 +
** VerifyBamID Output - see [[VerifyBamID#A_guideline_to_interpret_output_files|VerifyBamID: A guideline to interpret output files]] for more information
 +
*** ''*/SAMPLE.genoCheck.depthRG'' - depth distribution of the sequence reads per read group
 +
*** ''*/SAMPLE.genoCheck.depthSM'' - depth distribution of the sequence reads per sample
 +
*** ''*/SAMPLE.genoCheck.err'' - log file
 +
*** ''*/SAMPLE.genoCheck.log'' - log file
 +
*** ''*/SAMPLE.genoCheck.OK'' - temp file indicating the VerifyBAMID step completed successfully
 +
*** ''*/SAMPLE.genoCheck.selfRG'' - per-readGroup statistics describing how well each lane matches to the annotated sample
 +
*** '''''*/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
 +
*** ''*/SAMPLE.qplot.OK'' - temp file indicating the qplot step completed successfully
 +
*** '''''*/SAMPLE.qplot.R'' - Rscript that can be used to generate the pdf graphs'''
 +
*** '''''*/SAMPLE.qplot.stats'' - sample statistics'''
   −
MERGE_NAME FASTQ1 FASTQ2 RGID SAMPLE LIBRARY CENTER PLATFORM
+
You should see .done and .OK files for each SAMPLE in the index file. If you do not see the .done and .OK files, then your *bamQC* sub-pipeline failed.
−
Sample1 fastq/S1/F1_R1.fastq.gz fastq/S1/F1_R2.fastq.gz RGID1 SampleID1 Lib1 UM ILLUMINA
  −
Sample1 fastq/S1/F2_R1.fastq.gz fastq/S1/F2_R2.fastq.gz RGID1a SampleID1 Lib1 UM ILLUMINA
  −
Sample2 fastq/S2/F1_R1.fastq.gz fastq/S2/F1_R2.fastq.gz RGID2 SampleID2 Lib2 UM ILLUMINA
  −
Sample2 fastq/S2/F2.fastq.gz . RGID2 SampleID2 Lib2 UM ILLUMINA
     −
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.
+
'''On success, the QCFiles/ folder contains the quality control output'''
   −
=== Reference Files ===  
+
===Command-Line and Configuration Options===
−
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:
+
*Required Options
−
* [[GotCloud: Genetic Reference and Resource Files#Reference fasta Files|Reference fasta Files]]
+
{| class="wikitable" style="margin: 1em 1em 1em 0; background-color: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" border="1"
−
* [[GotCloud: Genetic Reference and Resource Files#DBSNP VCF Files|DBSNP VCF Files]]
+
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
−
* [[GotCloud: Genetic Reference and Resource Files#HapMap3 VCF Files|HapMap3 VCF Files]]
+
|-
 +
| --list/--bam_list/--bamlist ''file'' || BAM_LIST || path to the [[#BAM_LIST File for bamQC|BAM_LIST File]] || $(OUT_DIR)/bam.list
 +
|-
 +
| --numjobs ''#'' || || number of jobs to run in parallel || 0 (generate Makefile of steps, but do not run)
 +
|}
   −
=== Configuration File ===
+
*Common Options
−
{{:GotCloud: Configuration}}
     −
==== Recommended Settings ====
+
{| class="wikitable" style="margin: 1em 1em 1em 0; background-color: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" border="1"
 +
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
 +
|-
 +
| --outdir ''path'' || OUT_DIR || output directory ||
 +
|-
 +
| --conf ''file'' || || configuration file to use ||
 +
|-
 +
|  || REF_DIR || where the reference/resource files are stored || gotcloud.ref subdirectory within the base GotCloud directory
 +
|-
 +
| || REF || [[GotCloud: Genetic Reference and Resource Files#Reference fasta Files|Reference fasta Files]] || $(REF_DIR)/human.g1k.v37.fa
 +
|-
 +
| || DBSNP_VCF || [[GotCloud: Genetic Reference and Resource Files#DBSNP VCF File|DBSNP VCF Files]] || $(REF_DIR)/dbsnp_135.b37.vcf.gz
 +
|-
 +
| || HM3_VCF || [[GotCloud: Genetic Reference and Resource Files#HapMap3 VCF File|HapMap3 VCF Files]] || $(REF_DIR)/hapmap_3.3.b37.sites.vcf.gz
 +
|}
   −
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>.   
+
==== Example Configuration File ====
 +
Example configuration file where reference files happen to be stored in /path/reference, and bam list file is stored in in path/freeze5
 +
BAM_LIST = /path/freeze5.bam.list
 +
  OUT_DIR = /path/freeze5/output
 +
REF_DIR = /path/reference/
 +
REF = $(REF_DIR)/hs37d5.fa
 +
DBSNP_VCF = $(REF_DIR)/dbsnp_135.b37.sites.vcf.gz
 +
  HM3_VCF = $(REF_DIR)/hapmap3_r3_b37.sites.vcf.gz
   −
You can override the defaults by setting in your configuration file:
+
==== Example Command Line ====
−
* to use <code>bwa mem</code> (you do not need to set this in version 1.16 and later since it is the default)
+
  gotcloud pipe –-name bamQC --numjobs <N>
−
  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 ====
+
== bamQC_createIndex ==
 +
*What it does:
 +
# creates a BAI file for any BAM that is missing it
 +
# qplot
 +
# verifyBamID
   −
See [[#FASTQ List File|FASTQ List File]] for how to set the index file either via command line options or via configuration.
+
====Inputs====
 +
* Single merged, recalibrated, and deduped BAM file for each subject (stored in a [[#BAM_LIST File for bamQC_createIndex|BAM_LIST File]])
 +
* Reference files
 +
* (Optional) configuration file to override default options
   −
==== Turning Off Optional Steps====  
+
=====BAM_LIST File for bamQC_createIndex=====
−
Quality Control steps can be disabled.
+
* Each line of the BAM list file represents a single individual
   −
To Disable QPLOT, remove qplot from the PER_MERGE_STEPS configuration by setting:  
+
Columns:
−
PER_MERGE_STEPS = verifyBamID index recab
+
# sample id
 +
# comma separated population labels (optional column)
 +
# BAM File (preferable to have full paths to BAM files)
    +
[SAMPLE_ID] [COMMA SEPARATED POPULATION LABELS] [BAM_FILE]
 +
or
 +
[SAMPLE_ID] [BAM_FILE]
   −
To Disable VerifyBamID, remove qplot from the PER_MERGE_STEPS configuration by setting:
+
* Notes:
−
PER_MERGE_STEPS = qplot index recab
+
** tab delimited
 +
** population label is optional - it will default to <code>ALL</code>
 +
*** only used by Thunder (part of ldrefine pipeline)
 +
*** if all samples are from the same population, population label can be skipped or you can just specify <code>ALL</code> for the population label for each sample.
   −
==== Optional Configurable Settings ====  
+
====Outputs====
−
You may want to adjust the amount of memory/threads that are used:
+
Upon successful completion of the *bamQC_createIndex* sub-pipeline, you should see the following files/subdirectories under the user specified output directory:
 +
* A BAI file with the exact same path and name as the BAM file that was input, with *.bai on the end
 +
* '''QCFiles/''' - contains quality control results
 +
** VerifyBamID Output - see [[VerifyBamID#A_guideline_to_interpret_output_files|VerifyBamID: A guideline to interpret output files]] for more information
 +
*** ''*/SAMPLE.genoCheck.depthRG'' - depth distribution of the sequence reads per read group
 +
*** ''*/SAMPLE.genoCheck.depthSM'' - depth distribution of the sequence reads per sample
 +
*** ''*/SAMPLE.genoCheck.err'' - log file
 +
*** ''*/SAMPLE.genoCheck.log'' - log file
 +
*** ''*/SAMPLE.genoCheck.OK'' - temp file indicating the VerifyBAMID step completed successfully
 +
*** ''*/SAMPLE.genoCheck.selfRG'' - per-readGroup statistics describing how well each lane matches to the annotated sample
 +
*** '''''*/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
 +
*** ''*/SAMPLE.qplot.OK'' - temp file indicating the qplot step completed successfully
 +
*** '''''*/SAMPLE.qplot.R'' - Rscript that can be used to generate the pdf graphs'''
 +
*** '''''*/SAMPLE.qplot.stats'' - sample statistics'''
   −
There are additional configurable settings, but these are the ones most likely to be adjusted.  
+
You should see .done and .OK files for each SAMPLE in the index file. If you do not see the .done and .OK files, then your *bamQC_createIndex* sub-pipeline failed.
   −
* BWA_THREADS = -t N
+
'''On success, the QCFiles/ folder contains the quality control output'''
−
** Fill in the N with the number of threads you want BWA to run with, default is 1
  −
* SORT_MAX_MEM = 2000000000
  −
** Maximum amount of memory used by samtools sort after running bwa
     −
== Running the Alignment Pipeline ==  
+
===Command-Line and Configuration Options===
   −
=== Command-Line Options ===
+
*Required Options
−
* help - print usage
+
{| class="wikitable" style="margin: 1em 1em 1em 0; background-color: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" border="1"
−
* test OUTPUT_DIR - run the test example placing the output in a user specified OUTPUT_DIR.  No other options are required.
+
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
−
* outdir OUTPUT_DIR - directory for the output
+
|-
−
** May also be specified via OUT_DIR in the configuration file
+
| --list/--bam_list/--bamlist ''file'' || BAM_LIST || path to the [[#BAM_LIST File for bamQC_createIndex|BAM_LIST File]] || $(OUT_DIR)/bam.list
−
** Required to be set either via command-line or configuration
+
|-
−
* conf CONFIG_FILE - configuration file
+
| --numjobs ''#'' || || number of jobs to run in parallel || 0 (generate Makefile of steps, but do not run)
−
* list FASTQ_LIST_FILE_NAME  - name of the fastq list file
+
|}
−
** May also be specified via FASTQ_LIST in the configuration file
  −
** Required to be set either via command-line or configuration
  −
* ref_dir REFERENCE_DIR - value to set config key REF_DIR to, overriding other values, REF_DIR can then be used inside config files.
  −
** May also be specified via REF_DIR in the configuration file
  −
* ref_prefix REFERENCE_DIR - path to prepend to non-absolute REF paths.
  −
** May also be specified via REF_PREFIX in the configuration file  
  −
* fastq_prefix FASTQ_PATH - prefix path to the fastq files specified in the FASTQ_LIST
  −
** May also be specified via FASTQ_PREFIX in the configuration file
  −
* base_prefix BASE_PATH - prefix path to the prepend to fastq/ref files without absolute paths
  −
** May also be specified via BASE_PREFIX in the configuration file
  −
* keepTmp - Do not remove the temporary files (removed by default)  
  −
** May also be specified via KEEP_TMP in the configuration file
  −
* keepLog - Do not remove the intermediate log files (removed by default)
  −
** May also be specified via KEEP_LOG in the configuration file
  −
* numjobs N - Replace N with the number of samples that should be processed in parallel
  −
* threads N - Replace N with the number of targets in each makefile that should be run in parallel  
  −
* dryrun - Create the Makefile, but do not run it
  −
* maxlocaljobs N - Replace N with the maximum number of jobs that can be run locally (no batchtype specified).  Default is 10.
  −
* batchtype TYPE - Tells GotCloud the specified batch type to send jobs to the client nodes
  −
** May also be specified via BATCH_TYPE in the configuration file
  −
** Can be: mosix, slurm, slurmi, pbs, sge, sgei
  −
* batchopts OPTS - Tells GotCloud the options to pass onto the batch system
  −
** May also be specified via BATCH_OPTS in the configuration file
  −
* noPhoneHome - disable the phone home logic
  −
* 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
     −
===Running the Alignment Pipeline===
+
*Common Options
−
Run <code>gotcloud align</code> with the appropriate command-line parameters.
     −
Example:  
+
{| class="wikitable" style="margin: 1em 1em 1em 0; background-color: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" border="1"
−
  gotcloud align --conf config.txt --outdir output
+
! Command-line Flag !! Configuration Key !! Value Description !! Default Value
 +
|-
 +
| --outdir ''path'' || OUT_DIR || output directory ||
 +
|-
 +
| --conf ''file'' || || configuration file to use ||
 +
|-
 +
| || REF_DIR || where the reference/resource files are stored || gotcloud.ref subdirectory within the base GotCloud directory
 +
|-
 +
| || REF || [[GotCloud: Genetic Reference and Resource Files#Reference fasta Files|Reference fasta Files]] || $(REF_DIR)/human.g1k.v37.fa
 +
|-
 +
| || DBSNP_VCF || [[GotCloud: Genetic Reference and Resource Files#DBSNP VCF File|DBSNP VCF Files]] || $(REF_DIR)/dbsnp_135.b37.vcf.gz
 +
|-
 +
| || HM3_VCF || [[GotCloud: Genetic Reference and Resource Files#HapMap3 VCF File|HapMap3 VCF Files]] || $(REF_DIR)/hapmap_3.3.b37.sites.vcf.gz
 +
|}
   −
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.  
+
==== Example Configuration File ====
 +
Example configuration file where reference files happen to be stored in /path/reference, and bam list file is stored in in path/freeze5
 +
BAM_LIST = /path/freeze5.bam.list
 +
OUT_DIR = /path/freeze5/output
 +
REF_DIR = /path/reference/
 +
REF = $(REF_DIR)/hs37d5.fa
 +
DBSNP_VCF = $(REF_DIR)/dbsnp_135.b37.sites.vcf.gz
 +
  HM3_VCF = $(REF_DIR)/hapmap3_r3_b37.sites.vcf.gz
   −
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.
+
==== Example Command Line ====
−
 
+
gotcloud pipe –-name bamQC_createIndex --numjobs <N>
−
Each Makefile is independent and can be run in parallel and across a cloud.
  −
 
  −
On success, you will see:
  −
Processing finished in nn secs with no errors reported
  −
 
  −
If processing fails part way through, you can pick up where you left off by rerunning gotcloud or the make command.
  −
 
  −
=== 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'''
  −
** '''*.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 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.
  −
 
  −
'''On success, the bams/ directory contains the final BAMs and bais.'''
 
Kleckner
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Retrieved from "http://genome.sph.umich.edu/wiki/Special:MobileDiff/13006...13041"

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