Tutorial: GotCloud

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GotCloud Tutorial

In this tutorial, we illustrate some of the essential steps in the analysis of next generation sequence data.

We will start with a set of sequence reads and associated base quality scores stored in fastq file.

The mapping pipeline will find the most likely genomic location for each read producing a BAM file.

The variant calling pipeline generates an initial list of polymorphic sites and genotypes stored in a VCF file and then uses haplotype information to refine these genotypes in an updated VCF file.

Example Dataset

Our dataset consists of 60 individuals from GBR sequenced by the 1000 Genomes Project. These individuals have been sequenced to an average depth of about 4x.

To conserve time and disk-space, our analysis will focus on a small region on chromosome 20, 42900000 - 43200000. We will first map the reads for a single individual (labeled TBD). We will then combine the results with mapped reads from the other 59 individuals to generate a list of polymorphic sites and estimate accurate genotypes at each of these sites.

The example dataset we'll be using is included in this tar-ball TBD.

Required Software

In order to run this tutorial, you need to make sure you have GotCloud is installed on your system.

Installation instructions here.

Mapping Reads

The first step in processing next generation sequence data is mapping the reads to the reference genome, generating per sample BAM files.

The mapping pipeline has multiple built-in steps to generate BAMs:

  1. Align the fastqs to the reference genome
    • handles both single & paired end
  2. Merge the results from multiple fastqs into 1 file per sample
  3. Mark Duplicate Reads are marked
  4. Recalibrate Base Qualities

This processing results in 1 BAM file per sample.

The mapping pipeline also includes Quality Control (QC) steps:

  1. Visualization of various quality measures (QPLOT)
  2. Screen for sample contamination & swap (VerifyBamID)

Run the mapping pipeline:

gen_biopipeline.pl --conf GBR60map.conf --out_dir mappingResults

TBD - add link explaining the contents of the .conf & .index files.

Upon successful completion of the mapping pipeline, you will see the following message:

Commands finished in nn secs with no errors reported

The final BAM files produced by the mapping pipeline can be found in the files:

ls mappingResults/alignment.recal/*.recal.bam

Index files (.bai) for these BAMs are also in that directory.

The QC files for verifyBamID are:

ls mappingResults/QCFiles/*.genoCheck.selfRG mappingResults/QCFiles/*.genoCheck.selfSM 

Understanding VerifyBamID output

The QC files for qplot are:

ls mappingResults/QCFiles/*.qplot.R mappingResults/QCFiles/*.qplot.stats 

Understanding QPLOT output

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:


Alternately, if you want to run this example from a different directory, but do not want to edit the index file, you can create a relative path to the test fastq files so their path agrees with that 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
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.

To run the alignment pipeline, 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:

Created {OUT_DIR}/Makefiles/biopipe_Sample2.Makefile
Created {OUT_DIR}/Makefiles/biopipe_Sample1.Makefile
Submitted 2  commands
Waiting for commands to complete... . .  Commands finished in 33 secs with no errors reported

The aligned BAM files are found in {OUT_DIR}/alignment.recal/

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, which can be found in {ROOT_DIR}/test/umake/bams. These contain sequence information for a targeted region in chromosome 20.

In addition to the BAM files, you will need three files to run UMAKE: an index file, a configuration file, and a bed file (needed to analyze BAM files from targeted/exome sequencing).

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 = $(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
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

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:


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.

Further Information

Mapping (Alignment) Pipeline

Variant Calling Pipeline (UMAKE)