Variant Calling Pipeline using GATK4

This is an updated version of the variant calling pipeline post published in 2016 (link). This updated version employs GATK4 and is available as a containerized Nextflow script on GitHub.

Identifying genomic variants, including single nucleotide polymorphisms (SNPs) and DNA insertions and deletions (indels), from next generation sequencing data is an important part of scientific discovery. At the NYU Center for Genomics and Systems Biology (CGSB) this task is central to many research programs. For example, the Carlton Lab analyzes SNP data to study population genetics of the malaria parasites Plasmodium falciparum and Plasmodium vivax. The Gresham Lab uses SNP and indel data to study adaptive evolution in yeast, and the Lupoli Lab in the Department of Chemistry uses variant analysis to study antibiotic resistance in E. coli. 

To facilitate this research, a bioinformatics pipeline has been developed to enable researchers to accurately and rapidly identify, and annotate, sequence variants. The pipeline employs the Genome Analysis Toolkit 4 (GATK4) to perform variant calling and is based on the best practices for variant discovery analysis outlined by the Broad Institute. Once SNPs have been identified, SnpEff is used to annotate, and predict, variant effects. 

This pipeline is intended for calling variants in samples that are clonal – i.e. a single individual.  The frequencies of variants in these samples are expected to be 1 (for haploids or homozygous diploids) or 0.5 (for heterozygous diploids).  To call variants in samples that are heterogeneous, such as human tumors and mixed microbial populations, in which allele frequencies vary continuously between 0 and 1 researcher should use GATK4 Mutect2 which is designed to identify subclonal events (workflow coming soon). 

Base Quality Score Recalibration (BQSR) is an important step for accurate variant detection that aims to minimize the effect of technical variation on base quality scores (measured as Phred scores). As with the original pipeline (link), this pipeline assumes that a ‘gold standard’ set of SNPS and indels are not available for BQSR.  In the absence of a gold standard the pipeline performs an initial step detecting variants without performing BQSR, and then uses the identified SNPs as input for BQSR before calling variants again. This process is referred to as bootstrapping and is the procedure recommended by the Broad Institute’s best practices for variant discovery analysis when a gold standard is not available.

This pipeline uses nextflow, a framework that enables reproducible and portable workflows (https://www.nextflow.io/).  The full pipeline and instructions on how to use the Nextflow script are described below.

Script Location

greene

/scratch/work/cgsb/scripts/variant_calling/gatk4/main.nf

1. You don’t need to copy the script, but you should copy the file nextflow.config from the above directory into a new project directory.
   (/scratch/work/cgsb/scripts/variant_calling/gatk4/nextflow.config)
2. If you want to copy and run the main.nf script locally, you must copy the /bin directory as well. This needs to be in the same directory as main.nf.
3. You will set parameters for your analysis in the nextflow.config file
4. See How to Use and Examples below to learn how to configure and run the script. 
5. This Nextflow script uses pre-installed software modules on the NYU HPC, and is tailored to the unique file naming scheme used by the Genomics Core at the NYU CGSB.

github

https://github.com/gencorefacility/variant-calling-pipeline-gatk4

1. This version of the script is configured for standard Illumina filenames
2. This version of the script does not use software modules local to the NYU HPC, it is instead packaged with a Docker container which has all tools used in the pipeline. Integration with Docker allows for completely self-contained and truly reproducible analysis. This example shows how to run the workflow using the container. The container is hosted on Docker at: https://hub.docker.com/r/gencorefacility/variant-calling-pipeline-gatk4

Full List of Tools Used in this Pipeline

• GATK4
• BWA
• Picard Tools
• Samtools
• SnpEff
• R (dependency for some GATK steps)

How to Use This Script

Setting Up Nextflow

This pipeline is written in Nextflow, a computational pipeline framework. The easiest way to get setup with Nextflow is with conda. On the NYU HPC, conda is already installed and available as a module.

module load anaconda3/2019.10

Note: Use module avail anaconda to check for the latest conda version. 

Update: NYU HPC users can load Nextflow using the module load command. There is no need to install via conda. Simply load the module and skip to “Setting Up The Script” below.

For users outside NYU, see the conda installation instructions, then come back and follow the rest of the instructions below.

Once you have conda loaded, make sure you add the bioconda channel (required for Nextflow) as well as the other channels bioconda depends on. It is important to add them in this order so that the priority is set correctly (if you get warnings that a channel exists and is being moved to the top, that’s OK keep going):

conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge

Create a conda environment for Nextflow. I call it ‘nf-env’ but you can call it anything you like.

conda create --name nf-env

(If you get a message asking you to update conda, press enter to proceed and allow your conda environment to be created.)

We’ll install Nextflow inside this newly created environment. Anytime we want to use Nextflow, we’ll need to load the environment first using the following command:

conda activate nf-env

When a conda environment has been loaded, you’ll see its name in parenthesis at the command prompt, for example:

(nf-env) [user@log-1 ~]$

Once you’ve activated your conda environment, install nextflow using:

conda install nextflow

(When you’re finished with nextflow, deactivate the environment using conda deactivate)

Setting Up The Script

Once you have Nextflow setup, we can run the pipeline. The pipeline requires six mandatory input parameters to run. The values can be hard coded into a config file, or provided as command line arguments (examples below).

1) params.reads The directory with your FastQ libraries to be processed, including a glob path matcher. Must include a paired end group pattern matcher (i.e. {1,2}).
Example:
params.reads = "/path/to/data/*_n0{1,2}_*.fastq.gz"
or
params.reads = "/path/to/data/*_R{1,2}_*.fastq.gz"

2) params.ref Reference genome to use, in fasta format. If you’re at the NYU CGSB, there’s a good chance your genome of interest is located in the Shared Genome Resource (/scratch/work/cgsb/genomes/Public/). Selecting a reference genome from within the Shared Genome Resource ensures that all index files and reference dictionaries required by BWA, Picard, GATK, etc. are available. If you’re using your own reference data, you will need to build index files for BWA (using BWA), a fasta index (using samtools), and a reference dictionary (using Picard Tools). These files need to be located in the same directory as the reference fasta file.

3) params.snpeff_dbThe name of the SnpEff database to use. To determine the name of the SnpEff DB to use, ensure you have snpeff installed/loaded and then issue the databases command.

# On the NYU HPC you can load SnpEff using module load
module load snpeff/4.3i

# $SNPEFF_JAR is the path to the snpEff.jar file.
# On the NYU HPC this is automatically set to the
# snpEff.jar path when the module is loaded
java -jar $SNPEFF_JAR databases | grep -i SPECIES_NAME

From the list of returned results, select the database you wish to use. The value of the first column is the value to input for params.snpeff_db.

For example if you wanted to use the Arabidopsis Thaliana SnpEff database:

java -jar $SNPEFF_JAR databases | grep -i thaliana

Output:

athalianaTair10   Arabidopsis_Thaliana http://downloads.sourceforge.net/...

Then:

params.snpeff_db='athalianaTair10'

Note: If your organism is not available in SnpEff, it is possible to create a custom SnpEff Database if a reference fasta and gff file are available.

4) params.outdir By default, output files will go into params.outdir/out, temporary GATK files will go into params.outdir/gatk_temp, SnpEff database files will go into params.outdir/snpeff_db, and Nextflow will run all analysis in params.outdir/nextflow_work_dir.These individual paths can be overridden by specifying the params.out, params.tmpdir, params.snpeff_data, and the workDir parameters respectively. Note: There is no  params. prefix for workDir.

5) params.pl Platform. E.g. Illumina (required for read groups)

6) params.pm Machine. E.g. nextseq (required for read groups)

Examples

Note: If you are working on the NYU HPC, you should run all nextflow jobs in an interactive slurm session. This is because Nextflow uses some resources in managing your workflow. 

Once you have set up your nextflow environment you can perform your analysis in multiple ways.  Below I describe four different ways this can be done.

Scenario 1: Hard code all parameters in config file

Copy the nextflow.config file into a new directory (see Script Location). Edit the first six parameters in this file, e.g.:

params.reads = "/path/to/reads/*_n0{1,2}_*.fastq.gz"
params.ref = "/path/to/ref.fa"
params.outdir = "/scratch/user/gatk4/"
params.snpeff_db = "athalianaTair10"
params.pl = "illumina"
params.pm = "nextseq"

After activating your conda environment with Nextflow installed, run:

nextflow run main.nf

By default, Nextflow will look for a config file called nextflow.config inside the directory from which Nextflow was launched. You can specify a different config file on the command line using -c:

nextflow run main.nf -c your.config

Note: Parameters hard coded in the config can be overridden by arguments passed through the command line. See following scenario.

Scenario 2: Call the script providing all required parameters through the command line (after activating your Nextflow conda environment)

nextflow run main.nf \
    --reads "/path/to/reads/*_n0{1,2}_*.fastq.gz" \
    --ref "/path/to/ref.fa" \
    --outdir "/scratch/user/gatk4/" \
    --snpeff_db "athalianaTair10" \
    --pl "illumina" \
    --pm "illumina"

Note: You are able to set/override any and all parameters in this way.

Scenario 3: Call directly from GitHub
Nextflow allows you to run scripts hosted on GitHub directly. There is no need to download the script (main.nf) to your local computer. Rather, you can call the variant calling pipeline script directly from github by entering the following command:

nextflow run gencorefacility/variant-calling-pipeline-gatk4

In this scenario you still need the config file (which you can download from the git repo) to set your parameters (see Scenario 1), or pass parameters through the command line (see Scenario 2). 

Scenario 4: Use with a Docker container

All software tools used in the pipeline have been packaged in a Docker container. You can instruct Nextflow to use a containerized Docker image for the analysis by passing the -with-docker or -with-singularity flags and the container. Calling the script directly from GitHub and telling it to use this docker image looks like this: 

nextflow run gencorefacility/variant-calling-pipeline-gatk4 -with-docker gencorefacility/variant-calling-pipeline-gatk4

Launching Nextflow

Once Nextflow begins running, it will actively manage and monitor your jobs. If you’re working on the HPC at NYU, the corresponding nextflow.config file contains the line process.executor = 'slurm' which instructs Nextflow to submit jobs to SLURM. You can monitor your jobs in real time using watch and squeue:

watch squeue -u <netID>

Note: Since Nextflow actively manages your jobs, the session in which Nextflow is running must remain open. For example, if Nextflow is running and you lose power or network, Nextflow will be terminated. To avoid this, I recommend running Nextflow inside a tmux session, using nohup, or even submitting the nextflow script as a SLURM job. My preference is to use tmux (terminal multiplexer). 

Enter the command tmux to enter a tmux session. Launch an interactive slurm session if you are on the NYU HPC. Activate your conda environment. Now, you can launch your Nextflow script.

“Detach” from the tmux session at any time by pressing “ctrl” + “B” together, then pressing “D” (for detach). If you ever lose power, connection, or detach, you can reattach your tmux session by typing tmux a. You will find your session running just as when you last left it. 

If your Nextflow script does get interrupted for some reason, you can always resume the pipeline from the last successfully completed step using the -resume flag:

nextflow run main.nf -resume

Workflow Overview

Step 1	Alignment – Map to Reference
Tool	BWA MEM
Input	.fastq files reference genome
Output	aligned_reads.sam
Notes	-Y tells BWA to use soft clipping for supplementary alignments -K tells BWA to process INT input bases in each batch regardless of nThreads (for reproducibility) Readgroup info is provided with the -R flag. This information is key for downstream GATK functionality. GATK will not work without a read group tag.
Command	bwa mem \ -K 100000000 \ -Y \ -R '@RG\tID:sample_1\tLB:sample_1\tPL:ILLUMINA\tPM:HISEQ\tSM:sample_1' \ ref.fa \ sample_1_reads_1.fq \ sample_1_reads_2.fq \ > aligned_reads.sam

Step 2	Mark Duplicates + Sort
Tool	GATK4 MarkDuplicatesSpark
Input	aligned_reads.sam
Output	sorted_dedup_reads.bam sorted_dedup_reads.bam.bai dedup_metrics.txt
Notes	In GATK4, the Mark Duplicates and Sort Sam steps have been combined into one step using the MarkDuplicatesSpark tool. In addition, the BAM index file (.bai) is created as well by default. The tool is optimized to run on queryname-grouped alignments (that is, all reads with the same queryname are together in the input file). The output of BWA is query-grouped, however if provided coordinate-sorted alignments, the tool will spend additional time first queryname sorting the reads internally. Due to MarkDuplicatesSpark queryname-sorting coordinate-sorted inputs internally at the start, the tool produces identical results regardless of the input sort-order. That is, it will flag duplicates sets that include secondary, and supplementary and unmapped mate records no matter the sort-order of the input. This differs from how Picard MarkDuplicates behaves given the differently sorted inputs. (i.e. coordinate sorted vs queryname sorted).  You don’t need a Spark cluster to run Spark-enabled GATK tools. If you’re working on a “normal” machine (even just a laptop) with multiple CPU cores, the GATK engine can still use Spark to create a virtual standalone cluster in place, and set it to take advantage of however many cores are available on the machine — or however many you choose to allocate. (https://gatk.broadinstitute.org/hc/en-us/articles/360035890591?id=11245)
Command	gatk \         MarkDuplicatesSpark \         -I aligned_reads.sam \         -M dedup_metrics.txt \         -O sorted_dedup_reads.bam

Step 3	Collect Alignment & Insert Size Metrics
Tool	Picard Tools, R, Samtools
Input	sorted_dedup_reads.bam reference genome
Output	alignment_metrics.txt, insert_metrics.txt, insert_size_histogram.pdf, depth_out.txt
Command	java -jar picard.jar \         CollectAlignmentSummaryMetrics \         R=ref.fa \         I=sorted_dedup_reads.bam \         O=alignment_metrics.txt java -jar picard.jar \ CollectInsertSizeMetrics \         INPUT=sorted_dedup_reads.bam \         OUTPUT=insert_metrics.txt \         HISTOGRAM_FILE=insert_size_histogram.pdf samtools depth -a sorted_dedup_reads.bam > depth_out.txt

Step 4	Call Variants
Tool	GATK4
Input	sorted_dedup_reads.bam reference genome
Output	raw_variants.vcf
Notes	First round of variant calling. The variants identified in this step will be filtered and provided as input for Base Quality Score Recalibration (BQSR)
Command	gatk HaplotypeCaller \         -R ref.fa \         -I sorted_dedup_reads.bam \         -o raw_variants.vcf

Step 5	Extract SNPs & Indels
Tool	GATK4
Input	raw_variants.vcf reference genome
Output	raw_indels.vcf raw_snps.vcf
Notes	This step separates SNPs and Indels so they can be processed and used independently
Command	gatk SelectVariants \         -R ref.fa \         -V raw_variants.vcf \         -select-type SNP \         -o raw_snps.vcf gatk SelectVariants \         -R ref.fa \         -V raw_variants.vcf \         -select-type INDEL \         -o raw_indels.vcf

Step 6	Filter SNPs
Tool	GATK4
Input	raw_snps.vcf reference genome
Output	filtered_snps.vcf filtered_snps.vcf.idx
Notes	The filters below are a good starting point provided by the Broad. You will need to experiment a little to find the values that are appropriate for your data, and to produce the tradeoff between sensitivity and specificity that is right for your analysis. QD < 2.0: This is the variant confidence (from the QUAL field) divided by the unfiltered depth of non-hom-ref samples. This annotation is intended to normalize the variant quality in order to avoid inflation caused when there is deep coverage. For filtering purposes it is better to use QD than either QUAL or DP directly. FS > 60.0: This is the Phred-scaled probability that there is strand bias at the site. Strand Bias tells us whether the alternate allele was seen more or less often on the forward or reverse strand than the reference allele. When there is little to no strand bias at the site, the FS value will be close to 0. MQ < 40.0: This is the root mean square mapping quality over all the reads at the site. Instead of the average mapping quality of the site, this annotation gives the square root of the average of the squares of the mapping qualities at the site. It is meant to include the standard deviation of the mapping qualities. Including the standard deviation allows us to include the variation in the dataset. A low standard deviation means the values are all close to the mean, whereas a high standard deviation means the values are all far from the mean.When the mapping qualities are good at a site, the MQ will be around 60. SOR > 4.0: This is another way to estimate strand bias using a test similar to the symmetric odds ratio test. SOR was created because FS tends to penalize variants that occur at the ends of exons. Reads at the ends of exons tend to only be covered by reads in one direction and FS gives those variants a bad score. SOR will take into account the ratios of reads that cover both alleles. MQRankSum < -8.0: Compares the mapping qualities of the reads supporting the reference allele and the alternate allele. ReadPosRankSum < -8.0: Compares whether the positions of the reference and alternate alleles are different within the reads. Seeing an allele only near the ends of reads is indicative of error, because that is where sequencers tend to make the most errors. Learn more about hard filtering: https://gatk.broadinstitute.org/hc/en-us/articles/360035890471-Hard-filtering-germline-short-variants Note: SNPs which are ‘filtered out’ at this step will remain in the filtered_snps.vcf file, however they will be marked as ‘_filter’, while SNPs which passed the filter will be marked as ‘PASS’. We need to extract and provide only the passing SNPs to the BQSR tool, we do this in the next step (step 9).
Command	gatk VariantFiltration \         -R ref.fa \         -V raw_snps.vcf \         -O filtered_snps.vcf \         -filter-name "QD_filter" -filter "QD < 2.0" \         -filter-name "FS_filter" -filter "FS > 60.0" \         -filter-name "MQ_filter" -filter "MQ < 40.0" \         -filter-name "SOR_filter" -filter "SOR > 4.0" \         -filter-name "MQRankSum_filter" -filter "MQRankSum < -12.5" \         -filter-name "ReadPosRankSum_filter" -filter "ReadPosRankSum < -8.0"

Step 7	Filter Indels
Tool	GATK4
Input	raw_indels.vcf reference genome
Output	filtered_indels.vcf filtered_indels.vcf.idx
Notes	The filters below are a good starting point provided by the Broad. You will need to experiment a little to find the values that are appropriate for your data, and to produce the tradeoff between sensitivity and specificity that is right for your analysis. QD < 2.0: This is the variant confidence (from the QUAL field) divided by the unfiltered depth of non-hom-ref samples. This annotation is intended to normalize the variant quality in order to avoid inflation caused when there is deep coverage. For filtering purposes it is better to use QD than either QUAL or DP directly. FS > 200.0: This is the Phred-scaled probability that there is strand bias at the site. Strand Bias tells us whether the alternate allele was seen more or less often on the forward or reverse strand than the reference allele. When there is little to no strand bias at the site, the FS value will be close to 0. SOR > 10.0: This is another way to estimate strand bias using a test similar to the symmetric odds ratio test. SOR was created because FS tends to penalize variants that occur at the ends of exons. Reads at the ends of exons tend to only be covered by reads in one direction and FS gives those variants a bad score. SOR will take into account the ratios of reads that cover both alleles. Learn more about hard filtering: https://gatk.broadinstitute.org/hc/en-us/articles/360035890471-Hard-filtering-germline-short-variants Note: Indels which are ‘filtered out’ at this step will remain in the filtered_snps.vcf file, however they will be marked as ‘_filter’, while SNPs which passed the filter will be marked as ‘PASS’. We need to extract and provide only the passing indels to the BQSR tool, we do this next.
Command	gatk VariantFiltration \         -R ref.fa \         -V raw_indels.vcf \         -O filtered_indels.vcf \         -filter-name "QD_filter" -filter "QD < 2.0" \         -filter-name "FS_filter" -filter "FS > 200.0" \         -filter-name "SOR_filter" -filter "SOR > 10.0"

Step 8	Exclude Filtered Variants
Tool	GATK4
Input	filtered_snps.vcf filtered_indels.vcf
Output	bqsr_snps.vcf bqsr_indels.vcf
Notes	We need to extract only the passing variants and provide this as input to BQSR (next step).
Command	gatk SelectVariants \         --exclude-filtered \         -V filtered_snps.vcf \         -O bqsr_snps.vcf gatk SelectVariants \         --exclude-filtered \         -V filtered_indels.vcf \         -O bqsr_indels.vcf

Step 9	Base Quality Score Recalibration (BQSR) #1
Tool	GATK4
Input	sorted_dedup_reads.bam (from step 2) bqsr_snps.vcf bqsr_indels.vcf reference genome
Output	recal_data.table
Notes	BQSR is performed twice. The second pass is optional, only required to produce a recalibration report.
Command	gatk BaseRecalibrator \         -R ref.fa \         -I sorted_dedup_reads.bam \         --known-sites bqsr_snps.vcf \         --known-sites bqsr_indels.vcf \         -O recal_data.table

Step 10	Apply BQSR
Tool	GATK4
Input	recal_data.table sorted_dedup_reads.bam reference genome
Output	recal_reads.bam
Notes	This step applies the recalibration computed in the first BQSR step to the bam file. This recalibrated bam file is now analysis-ready.
Command	gatk ApplyBQSR \         -R ref.fa \         -I sorted_dedup_reads.bam \         -bqsr recal_data.table \         -O recal_reads.bam \

Step 11	Base Quality Score Recalibration (BQSR) #2
Tool	GATK4
Input	recal_reads.bam bqsr_snps.vcf bqsr_indels.vcf reference genome
Output	post_recal_data.table
Notes	This round of BQSR is optional. It is required if you want to produce a recalibration report with the Analyze Covariates step (next). For this round of BQSR, you provide the recalibrated reads obtained from the Apply BQSR step above as input.
Command	gatk BaseRecalibrator \         -R ref.fa \         -I recal_reads.bam \         --known-sites bqsr_snps.vcf \         --known-sites bqsr_indels.vcf \         -O post_recal_data.table

Step 12	Analyze Covariates
Tool	GATK4
Input	recal_data.table post_recal_data.table
Output	recalibration_plots.pdf
Notes	This step produces a recalibration report based on the output from the two BQSR runs
Command	gatk AnalyzeCovariates \                -before recal_data.table \         -after post_recal_data.table \         -plots recalibration_plots.pdf

Step 13	Call Variants
Tool	GATK4
Input	recal_reads.bam reference genome
Output	raw_variants_recal.vcf
Notes	Second round of variant calling performed using recalibrated (analysis-ready) bam
Command	gatk HaplotypeCaller \         -R ref.fa \         -I recal_reads.bam \         -o raw_variants_recal.vcf

Step 14	Extract SNPs & Indels
Tool	GATK4
Input	raw_variants_recal.vcf reference genome
Output	raw_indels_recal.vcfraw_snps_recal.vcf
Notes	This step separates SNPs and Indels so they can be processed and analyzed independently
Command	gatk SelectVariants \         -R ref.fa \         -V raw_variants_recal.vcf \         -select-type SNP \         -o raw_snps_recal.vcf gatk SelectVariants \         -R ref.fa \         -V raw_variants.vcf \         -select-type INDEL \         -o raw_indels_recal.vcf

Step 15	Filter SNPs
Tool	GATK4
Input	raw_snps_recal.vcf reference genome
Output	filtered_snps_final.vcf filtered_snps_final.vcf.idx
Notes	The filters below are a good starting point provided by the Broad. You will need to experiment a little to find the values that are appropriate for your data, and to produce the tradeoff between sensitivity and specificity that is right for your analysis. QD < 2.0: This is the variant confidence (from the QUAL field) divided by the unfiltered depth of non-hom-ref samples. This annotation is intended to normalize the variant quality in order to avoid inflation caused when there is deep coverage. For filtering purposes it is better to use QD than either QUAL or DP directly. FS > 60.0: This is the Phred-scaled probability that there is strand bias at the site. Strand Bias tells us whether the alternate allele was seen more or less often on the forward or reverse strand than the reference allele. When there is little to no strand bias at the site, the FS value will be close to 0. MQ < 40.0: This is the root mean square mapping quality over all the reads at the site. Instead of the average mapping quality of the site, this annotation gives the square root of the average of the squares of the mapping qualities at the site. It is meant to include the standard deviation of the mapping qualities. Including the standard deviation allows us to include the variation in the dataset. A low standard deviation means the values are all close to the mean, whereas a high standard deviation means the values are all far from the mean.When the mapping qualities are good at a site, the MQ will be around 60. SOR > 4.0: This is another way to estimate strand bias using a test similar to the symmetric odds ratio test. SOR was created because FS tends to penalize variants that occur at the ends of exons. Reads at the ends of exons tend to only be covered by reads in one direction and FS gives those variants a bad score. SOR will take into account the ratios of reads that cover both alleles. MQRankSum < -8.0: Compares the mapping qualities of the reads supporting the reference allele and the alternate allele. ReadPosRankSum < -8.0: Compares whether the positions of the reference and alternate alleles are different within the reads. Seeing an allele only near the ends of reads is indicative of error, because that is where sequencers tend to make the most errors. Learn more about hard filtering: https://gatk.broadinstitute.org/hc/en-us/articles/360035890471-Hard-filtering-germline-short-variants Note: SNPs which are ‘filtered out’ at this step will remain in the filtered_snps.vcf file, however they will be marked as ‘_filter’, while SNPs which passed the filter will be marked as ‘PASS’.
Command	gatk VariantFiltration \        -R ref.fa \         -V raw_snps_recal.vcf \         -O filtered_snps_final.vcf \         -filter-name "QD_filter" -filter "QD < 2.0" \         -filter-name "FS_filter" -filter "FS > 60.0" \         -filter-name "MQ_filter" -filter "MQ < 40.0" \         -filter-name "SOR_filter" -filter "SOR > 4.0" \         -filter-name "MQRankSum_filter" -filter "MQRankSum < -12.5" \         -filter-name "ReadPosRankSum_filter" -filter "ReadPosRankSum < -8.0"

Step 16	Filter Indels
Tool	GATK4
Input	raw_indels_recal.vcf reference genome
Output	filtered_indels_final.vcf filtered_indels_final.vcf.idx
Notes	The filters below are a good starting point provided by the Broad. You will need to experiment a little to find the values that are appropriate for your data, and to produce the tradeoff between sensitivity and specificity that is right for your analysis. QD < 2.0: This is the variant confidence (from the QUAL field) divided by the unfiltered depth of non-hom-ref samples. This annotation is intended to normalize the variant quality in order to avoid inflation caused when there is deep coverage. For filtering purposes it is better to use QD than either QUAL or DP directly. FS > 200.0: This is the Phred-scaled probability that there is strand bias at the site. Strand Bias tells us whether the alternate allele was seen more or less often on the forward or reverse strand than the reference allele. When there is little to no strand bias at the site, the FS value will be close to 0. SOR > 10.0: This is another way to estimate strand bias using a test similar to the symmetric odds ratio test. SOR was created because FS tends to penalize variants that occur at the ends of exons. Reads at the ends of exons tend to only be covered by reads in one direction and FS gives those variants a bad score. SOR will take into account the ratios of reads that cover both alleles. Learn more about hard filtering: https://gatk.broadinstitute.org/hc/en-us/articles/360035890471-Hard-filtering-germline-short-variants Note: Indels which are ‘filtered out’ at this step will remain in the filtered_snps.vcf file, however they will be marked as ‘_filter’, while SNPs which passed the filter will be marked as ‘PASS’.
Command	gatk VariantFiltration \         -R ref.fa \         -V raw_indels_recal.fa \         -O filtered_indels_final.vcf \         -filter-name "QD_filter" -filter "QD < 2.0" \         -filter-name "FS_filter" -filter "FS > 200.0" \         -filter-name "SOR_filter" -filter "SOR > 10.0"

Step 17	Annotate SNPs and Predict Effects
Tool	SnpEff
Input	filtered_snps_final.vcf
Output	filtered_snps_final.ann.vcf snpeff_summary.html snpEff_genes.txt
Command	java -jar snpEff.jar -v \         <snpeff_db> \         filtered_snps_final.vcf > $filtered_snps_final.ann.vcf

Step 18	Compile Statistics
Tool	parse_metrics.sh (in /bin)
Input	sample_id_alignment_metrics.txt sample_id_insert_metrics.txt sample_id_dedup_metrics.txt sample_id_raw_snps.vcf sample_id_filtered_snps.vcf sample_id_raw_snps_recal.vcf sample_id_filtered_snps_final.vcf sample_id_depth_out.txt
Output	report.csv
Command	parse_metrics.sh sample_id > sample_id_report.csv
Notes	A single report file is generated with summary statistics for each library processed containing the following pieces of information: # of Reads # of Aligned Reads % Aligned # Aligned Bases Read Length % Paired % Duplicate Mean Insert Size # SNPs, # Filtered SNPs # SNPs after BQSR, # Filtered SNPs after BQSR Average Coverage Give the script a sample id and it will look for the corresponding sample_id_* files (listed in the input section above) and print the statistics and header to stdout.