Tutorial: GotCloud
Installation
First, make sure GotCloud is installed on your system. Installation instructions here.
Running the Automatic Test
This verifies that GotCloud was installed correctly.
To run the test case automatically, change your current directory to GotCloud's root directory, and type in the following command:
bin/gen_biopipeline.pl --test OUTPUT_DIR
where OUTPUT_DIR is the directory where you want to store the results.
If you see "Test Passed", then you are ready to run a sample.
Aligning a Sample
As an example, we can align the sample fastq files used in the automatic test. They belong to two different samples, which we will call "Sample1" and "Sample2". They are found in {ROOT_DIR}/test/align/fastq. (We will call the directory in which GotCloud is installed "{ROOT_DIR}".)
To make this easier, change to the {ROOT_DIR}/test/align directory. It contains an index file and a configuration file that can be used directly.
Index file
There are four fastq files in {ROOT_DIR}/test/align/fastq/Sample_1 and four fastq files in {ROOT_DIR}/test/align/fastq/Sample_2, both in paired-end format. Normally, we would need to build an index file for these files. Conveniently, an index file (indexFile.txt) already exists for the automatic test samples. It can be found in {ROOT_DIR}/test/align/, and contains the following information in tab-delimited format:
MERGE_NAME FASTQ1 FASTQ2 RGID SAMPLE LIBRARY CENTER PLATFORM Sample1 fastq/Sample_1/File1_R1.fastq.gz fastq/Sample_1/File1_R2.fastq.gz RGID1 SampleID1 Lib1 UM ILLUMINA Sample1 fastq/Sample_1/File2_R1.fastq.gz fastq/Sample_1/File2_R2.fastq.gz RGID1a SampleID1 Lib1 UM ILLUMINA Sample2 fastq/Sample_2/File1_R1.fastq.gz fastq/Sample_2/File1_R2.fastq.gz RGID2 SampleID2 Lib2 UM ILLUMINA Sample2 fastq/Sample_2/File2_R1.fastq.gz fastq/Sample_2/File2_R2.fastq.gz RGID2 SampleID2 Lib2 UM ILLUMINA
If you are in the {ROOT_DIR}/test/align directory, you can use this file as-is. If you prefer, you can create a new index file and change the MERGE_NAME, RGID, SAMPLE, LIBRARY, CENTER, or PLATFORM values. It is recommended that you do not modify existing files in {ROOT_DIR}/test/align.
If you want to run this example from a different directory, make sure the FASTQ1 and FASTQ2 paths are correct. That is, each of the FASTQ1 and FASTQ2 entry in the index file should look like the following:
{ROOT_DIR}/test/align/fastq/Sample_1/File1_R1.fastq.gz
Alternately, if you want to run this example from a different directory, but do not want to edit the index file, you can copy all the fastq files to a new directory with the relative path listed in the index file:
ln -s {ROOT_DIR}/test/align/fastq fastq
This will create a symbolic link to the test fastq directory from your current directory.
(More information about: the index file.)
Configuration file
Similar to the index file, a configuration file (test.conf) already exists for the automatic test samples. It contains the following information:
INDEX_FILE = indexFile.txt ############ # References REF_DIR = $(PIPELINE_DIR)/test/align/chr20Ref AS = NCBI37 FA_REF = $(REF_DIR)/human_g1k_v37_chr20.fa DBSNP_VCF = $(REF_DIR)/dbsnp.b130.ncbi37.chr20.vcf.gz PLINK = $(REF_DIR)/hapmap_3.3.b37.chr20
If you are in the {ROOT_DIR}/test/align directory, you can use this file as-is. If you are using a different index file, make sure your index file is named correctly in the first line. If you are not running this from {ROOT_DIR}/test/align, make sure your configuration and index files are in the same directory.
(More information about: reference files, optional configurable settings, or command-line options.)
Running the alignment pipeline
You are now ready to run the alignment pipeline. This requires two steps: first, generating the Makefiles; and second, running those Makefiles.
Generating the Makefiles
Enter the following command:
{ROOT_DIR}/bin/gen_biopipeline.pl --conf test.conf --out_dir {OUT_DIR}
where {OUT_DIR} is the directory in which you wish to store the resulting BAM files (for example, ~/out).
If everything went well, you will see the following messages:
Finished creating makefile {OUT_DIR}/Makefiles/biopipe_Sample2.Makefile
Finished creating makefile {OUT_DIR}/Makefiles/biopipe_Sample1.Makefile
--------------------------------------------------------------------
Run the following commands:
make -f {OUT_DIR}/Makefiles/biopipe_Sample2.Makefile > {OUT_DIR}/Makefiles/biopipe_Sample2.Makefile.log
make -f {OUT_DIR}/Makefiles/biopipe_Sample1.Makefile > {OUT_DIR}/Makefiles/biopipe_Sample1.Makefile.log
where {OUT_DIR} will be replaced with the directory you entered above.
Running the Makefiles
To run a Makefile, simply enter one-by-one the commands generated in the previous step. If you wish to run the alignment in the background, add "&" after the make command, as follows:
make -f {OUT_DIR}/Makefiles/biopipe_Sample2.Makefile > {OUT_DIR}/Makefiles/biopipe_Sample2.Makefile.log &
The log files for the runs will be found in the Makefiles directory, while the BAM files will be found in the {OUT_DIR}/alignment.recal directory. If you see two BAM files, one for each sample, then you have successfully aligned the fastq files.
Analyzing a Sample
Using UMAKE, you can analyze BAM files by calling SNPs, and generate a VCF file containing the results. Once again, we can analyze BAM files used in the automatic test. For this example, we have 60 BAM files, found in {ROOT_DIR}/test/umake/bams. In addition to the BAM files, you will need three files to run UMAKE: an index file, a configuration file, and a bed file.
Index file
First, you need a list of all the BAM files to be analyzed. Conveniently, the a test index file (umake_test.index) already exists in {ROOT_DIR}/test/umake/. It contains the following information:
NA12272 ALL bams/NA12272.mapped.ILLUMINA.bwa.CEU.low_coverage.20101123.chrom20.20000001.20300000.bam NA12004 ALL bams/NA12004.mapped.ILLUMINA.bwa.CEU.low_coverage.20101123.chrom20.20000001.20300000.bam ... NA12874 ALL bams/NA12874.mapped.LS454.ssaha2.CEU.low_coverage.20101123.chrom20.20000001.20300000.bam
You can use this file directly if you change your current directory to {ROOT_DIR}/test/umake/.
Alternately, if you want to copy and use this index file to a different directory, you can create a symbolic link to the bams folder as follows:
ln -s {ROOT_DIR}/test/umake/bams bams
(More information about: the index file.)
BED file
This file contains a single line:
chr20 20000050 20300000
You can copy this to the current directory and use it as-is.
(More information about: targeted/exome sequencing settings.)
Configuration file
A configuration file (umake_test.conf) already exists in {ROOT_DIR}/test/umake/. It contains the following information:
CHRS = 20 TEST_ROOT = $(UMAKE_ROOT)/test/umake BAM_INDEX = $(TEST_ROOT)/umake_test.index OUT_PREFIX = umake_test REF_ROOT = $(TEST_ROOT)/ref # REF = $(REF_ROOT)/karma.ref/human.g1k.v37.chr20.fa INDEL_PREFIX = $(REF_ROOT)/indels/1kg.pilot_release.merged.indels.sites.hg19 DBSNP_PREFIX = $(REF_ROOT)/dbSNP/dbsnp_135_b37.rod HM3_PREFIX = $(REF_ROOT)/HapMap3/hapmap3_r3_b37_fwd.consensus.qc.poly # RUN_INDEX = TRUE # create BAM index file RUN_PILEUP = TRUE # create GLF file from BAM RUN_GLFMULTIPLES = TRUE # create unfiltered SNP calls RUN_VCFPILEUP = TRUE # create PVCF files using vcfPileup and run infoCollector RUN_FILTER = TRUE # filter SNPs using vcfCooker RUN_SPLIT = TRUE # split SNPs into chunks for genotype refinement RUN_BEAGLE = FALSE # BEAGLE - MUST SET AFTER FINISHING PREVIOUS STEPS RUN_SUBSET = FALSE # SUBSET FOR THUNDER - MAY BE SET WITH BEAGLE STEP TOGETHER RUN_THUNDER = FALSE # THUNDER - MUST SET AFTER FINISHING PREVIOUS STEPS ############################################################################### WRITE_TARGET_LOCI = TRUE # FOR TARGETED SEQUENCING ONLY -- Write loci file when performing pileup UNIFORM_TARGET_BED = $(TEST_ROOT)/umake_test.bed # Targeted sequencing : When all individuals has the same target. Otherwise, comment it out OFFSET_OFF_TARGET = 50 # Extend target by given # of bases MULTIPLE_TARGET_MAP = # Target per individual : Each line contains [SM_ID] [TARGET_BED] TARGET_DIR = target # Directory to store target information SAMTOOLS_VIEW_TARGET_ONLY = TRUE # When performing samtools view, exclude off-target regions (may make command line too long)
If you are running this from a different directory, you will want to change some of the lines as follows:
BAM_INDEX = {CURRENT_DIR}/umake_test.index
UNIFORM_TARGET_BED = {CURRENT_DIR}/umake_test.bed
where {CURRENT_DIR} is the absolute path to the directory that contains the index and bed files.
An additional option can be added in the configuration file:
OUT_DIR = {OUT_DIR}
where {OUT_DIR} is the name of directory in which you want the output to be stored. If you do not specify this in the configuration file, you will need to add an extra parameter when you run UMAKE in the next step.
(More information about: the configuration file, reference files.)
Running UMAKE
If you added an OUT_DIR line to the configuration file, you can run UMAKE with the following command:
{ROOT_DIR}/bin/umake.pl --conf umake_test.conf --snpcall --numjobs 2
If you have not added an OUT_DIR line to the configuration file, you can specify the output directory directly with the following command:
{ROOT_DIR}/bin/umake.pl --conf umake_test.conf --outdir {OUT_DIR} --snpcall --numjobs 2
where {OUT_DIR} is the directory in which you want the output to be stored.
Either command will perform SNP calling on the test samples. If you find the resulting VCF files located in {OUT_DIR}/vcfs/chr20, then you have successfully called the SNPs from the test BAM files.
