GotCloud: Variant Calling Pipeline

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The Variant Calling Pipeline (previously called 'UMAKE') makes genotype calls from recalibrated BAM files. These genotype calls are output into VCF (Variant Call Format) files.

Running the GotCloud Variant Calling Pipeline

The variant calling pipeline (umake) is run using gotcloud snpcall and gotcloud ldrefine.

Running the Automatic Test

The automatic test runs the variant calling pipeline on a small test set and checks the results against expected results validating that GotCloud is installed correctly.

• Run snpcall pipeline test:

gotcloud snpcall --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 run snpcall on your own samples.
• Run ldrefine pipeline test:

gotcloud snpcall --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 run ldrefine on your own samples.

Overview of Variant Calling Pipeline Steps

Here is an overview of the Variant Calling Pipeline:

For more information on the filters applied during the Variant Calling Pipeline, see, GotCloud: Filters.

Input Data

• Aligned/Processed/Recalibrated BAM files
• BAM list file containing Sample IDs & BAM file names
• Reference files
• (Optional) Configuration file to override default options

BAM Files

The BAM files need to be duplicate-marked and base-quality recalibrated in order to obtain high quality SNP calls. Generating these BAM files from original FASTQs is automatically done as part of the Alignment Pipeline of GotCloud.

BAM List File

• Automatically created when running the GotCloud Alignment Pipeline
• Each line of the BAM list file represents a single individual

Columns:

1. sample id
2. comma separated population labels (optional column)
3. BAM File 1 (preferable to have full paths to BAM files)
4. 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 ALL
    • population is 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 ALL for the population label for each sample.

Reference Files

See GotCloud: Genetic Reference and Resource Files for detailed information about the multiple required reference files for the variant calling 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 fasta Files
• DBSNP VCF Files
• HapMap3 VCF Files
• OMNI VCF Files
• INDEL VCF File(s)

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 must specify their own configuration file specifying just the values different than the defaults.

Comments begin with a #

Format: KEY = value

Where KEY is the item being set and value is its new value

Required User Config Files Settings

The following Config File Settings must be specified by the user:

• CHRS = space separated list of chromosomes you want
• BAM_INDEX = path to the Index File of BAMs

Required on Command-Line or in Config File

The following Command-Line or Config File Settings must be specified by the user:

• --outdir/OUT_DIR= path to desired output directory

Targeted/Exome Sequencing Settings

If you are running Targeted/Exome Sequencing, the user should specify:

• Write loci file when performing pileup
  • WRITE_TARGET_LOCI = TRUE
• Specify the output sub-directory to store target information, for example: targetDir
  • Should not be a full path as this will co under the OUT_DIR directory.
  • TARGET_DIR = targetDir

If all individuals have the same target:

• Specify the single bed file, for example: target.bed
  • UNIFORM_TARGET_BED = target.bed

If not all individuals have the same target:

• Specify the file containing the sample id -> bed map, for example: targetMap.txt
  • MULTIPLE_TARGET_MAP = targetMap.txt
    • Each line of the file contains [SM_ID] [TARGET_BED]

Optional Settings:

• Extend the target region by a given number of bases, for example: 50
  • OFFSET_OFF_TARGET = 50

Configure Reference Files

See Reference Files for information on how to specify the reference files.

Chromosome X Calling

Making calls on the X chromosome requires the user to specifty a PED file with sex information.

• PED_INDEX = pedfile.ped

Example Configuration File

Example configuration file where reference files happen to be stored in /path/reference, and bam index file in path/freeze5

CHRS = 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
BAM_INDEX = /path/freeze5/freeze5.bam.index  ### The BAM index file described above
OUT_DIR = /path/freeze5/output               ### Directory in which to put all gotcloud output
REF = /path/reference/hs37d5.fa              ### Reference sequence
INDEL_PREFIX = /path/reference/1kg.pilot_release.merged.indels.sites.hg19   ### Known indel sites
HM3_VCF = /path/reference/hapmap3_r3_b37.sites.vcf.gz    ### HapMap variants (requires tabix index file in same directory)
DBSNP_VCF = /path/reference/dbsnp_135.b37.sites.vcf.gz   ### dbSNP variants (requires tabix index file in same directory)

Running

Running variant calling is straightforward:

gotcloud snpcall --conf vc.conf --numjobs 2
gotcloud ldrefine --conf vc.conf --numjobs 2

• Replace vc.conf with the path/name of the user's configuration file
  • If you are not overriding any defaults, you can alternatively specify --list path/bam.list replacing path/bam.list with the path/name of your BAM list file.
• Replace 2 following --numjobs with the number of jobs to be run in parallel
• If OUT_DIR is not defined in the configuration file, add --outdir followed by the path to the user's desired output directory.

Running on a Cluster

To run on the Cluster, the following settings need to be added to the configuration file:

BATCH_TYPE = batch_type
BATCH_OPTS = options to your batch system, as you would normally specify them

Alternatively, --batchtype and --batchopts can be specified on the command line.

Valid values for BATCH_TYPE are: mosix, sge, sgei, slurm, slurmi, pbs, local

• If you are at UM and are using flux, you can specify either flux or pbs.
• sgei and slurmi run in interactive mode.
• For any BATCH_TYPEs that run in batch mode, GotCloud generates a script that will wait until the step is complete before returning.
  • In a sense, it "fakes" interactive mode for all batch types since it will not proceed until a command is finished.

Here's the same configuration file we used above but now made to run on a cluster computer with MOSIX.

== Example Configuration File ==
CHRS = 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
BAM_INDEX = /path/freeze5/freeze5.bam.index 
OUT_DIR = /path/freeze5/output              
REF = /path/reference/hs37d5.fa             
INDEL_PREFIX = /path/reference/1kg.pilot_release.merged.indels.sites.hg19
HM3_VCF = /path/reference/hapmap3_r3_b37.sites.vcf.gz
DBSNP_VCF = /path/reference/dbsnp_135.b37.sites.vcf.gz
BATCH_TYPE = mosix             ### Specify MOSIX as the batch system
BATCH_OPTS = -j10,11,12,13     ### Specify available MOSIX compute nodes

Results

If there is a failure, you should see a message like:

make: *** [...] Error 1

Where ... is filled in with other text indicating what step failed.

On SNP Call success, you should see the following output sub-directories under your output directory:

• glfs with a bams & samples subdirectory
• pvcfs with a subdirectory per chromosome and then per region
• split with a subdirectory per chromosome
• vcfs with a subdirectory per chromosome
• (optionally your target directory)

Under the vcf/chrXX directory, there should be:

• chrXX.filtered.sites.vcf
• chrXX.filtered.sites.vcf.norm.log
• chrXX.filtered.sites.vcf.summary
• chrXX.filtered.vcf.gz
• chrXX.filtered.vcf.gz.OK
• chrXX.filtered.vcf.gz.tbi
• chrXX.hardfiltered.sites.vcf
• chrXX.hardfiltered.sites.vcf.log
• chrXX.hardfiltered.sites.vcf.summary
• chrXX.hardfiltered.vcf.gz
• chrXX.hardfiltered.vcf.gz.OK
• chrXX.hardfiltered.vcf.gz.tbi
• chrXX.merged.sites.vcf
• chrXX.merged.stats.vcf
• chrXX.merged.vcf
• chrXX.merged.vcf.OK

The .merged.vcf is the merged together versions of the separate regions in the same chromosome.

The filtered is the merged.vcf after it has been run through filters and is marked with PASS/FAIL.

Under the split/chrXX directory, there should be:

• chrXX.filtered.PASS.split.[N].vcf.gz
• chrXX.filtered.PASS.split.err
• chrXX.filtered.PASS.split.vcflist
• chrXX.filtered.PASS.gz
• subset.OK