# Tagged with #callset 7 documentation articles | 0 announcements | 2 forum discussions

Created 2013-09-11 21:54:51 | Updated 2015-05-22 22:46:00 | Tags: vqsr callset filtering hard-filtering

### The problem:

Our preferred method for filtering variants after the calling step is to use VQSR, a.k.a. recalibration. However, it requires well-curated training/truth resources, which are typically not available for organisms other than humans, and it also requires a large amount of variant sites to operate properly, so it is not suitable for some small-scale experiments such as targeted gene panels or exome studies with fewer than 30 exomes. For the latter, it is sometimes possible to pad your cohort with exomes from another study (especially for humans -- use 1000 Genomes or ExAC!) but again for non-human organisms it is often not possible to do this.

### The solution: hard-filtering

So, if this is your case and you are sure that you cannot use VQSR, then you will need to use the VariantFiltration tool to manually filter your variants. To do this, you will need to compose filter expressions as explained here, here and here based on the recommendations detailed further below.

### But first, some caveats

Let's be painfully clear about this: there is no magic formula that will give you perfect results. Filtering variants manually, using thresholds on annotation values, is subject to all sorts of caveats. The appropriateness of both the annotations and the threshold values is very highly dependent on the specific callset, how it was called, what the data was like, what organism it belongs to, etc.

HOWEVER, because we want to help and people always say that something is better than nothing (not necessarily true, but let's go with that for now), we have formulated some generic recommendations that should at least provide a starting point for people to experiment with their data.

In case you didn't catch that bit in bold there, we're saying that you absolutely SHOULD NOT expect to run these commands and be done with your analysis. You absolutely SHOULD expect to have to evaluate your results critically and TRY AGAIN with some parameter adjustments until you find the settings that are right for your data.

In addition, please note that these recommendations are mainly designed for dealing with very small data sets (in terms of both number of samples or size of targeted regions). If you are not using VQSR because you do not have training/truth resources available for your organism, then you should expect to have to do even more tweaking on the filtering parameters.

### Filtering recommendations

Here are some recommended arguments to use with VariantFiltration when ALL other options are unavailable to you. Note that these JEXL expressions will tag as filtered any sites where the annotation value matches the expression. So if you use the expression QD < 2.0, any site with a QD lower than 2 will be tagged as failing that filter.

#### For SNPs:

• QD < 2.0
• MQ < 40.0
• FS > 60.0
• SOR > 4.0
• HaplotypeScore > 13.0 only for variants output byUnifiedGenotyper; for HaplotypeCaller's output it is not informative
• MQRankSum < -12.5
• ReadPosRankSum < -8.0

#### For indels:

• QD < 2.0
• ReadPosRankSum < -20.0
• InbreedingCoeff < -0.8
• FS > 200.0
• SOR > 10.0

### And now some more IMPORTANT caveats (don't skip this!)

• The InbreedingCoeff statistic is a population-level calculation that is only available with 10 or more samples. If you have fewer samples you will need to omit that particular filter statement.

• For shallow-coverage (<10x), it is virtually impossible to use manual filtering to reliably separate true positives from false positives. You really, really, really should use the protocol involving variant quality score recalibration. If you can't do that, maybe you need to take a long hard look at your experimental design. In any case you're probably in for a world of pain.

• The maximum DP (depth) filter only applies to whole genome data, where the probability of a site having exactly N reads given an average coverage of M is a well-behaved function. First principles suggest this should be a binomial sampling but in practice it is more a Gaussian distribution. Regardless, the DP threshold should be set a 5 or 6 sigma from the mean coverage across all samples, so that the DP > X threshold eliminates sites with excessive coverage caused by alignment artifacts. Note that for exomes, a straight DP filter shouldn't be used because the relationship between misalignments and depth isn't clear for capture data.

### Finally, a note of hope

Some bits of this article may seem harsh, or depressing. Sorry. We believe in giving you the cold hard truth.

HOWEVER, we do understand that this is one of the major points of pain that GATK users encounter -- along with understanding how VQSR works, so really, whichever option you go with, you're going to suffer.

And we do genuinely want to help. So although we can't look at every single person's callset and give an opinion on how it looks (no, seriously, don't ask us to do that), we do want to hear from you about how we can best help you help yourself. What information do you feel would help you make informed decisions about how to set parameters? Are the meanings of the annotations not clear? Would knowing more about how they are computed help you understand how you can use them? Do you want more math? Less math, more concrete examples?

Tell us what you'd like to see here, and we'll do our best to make it happen. (no unicorns though, we're out of stock)

We also welcome testimonials from you. We are one small team; you are a legion of analysts all trying different things. Please feel free to come forward and share your findings on what works particularly well in your hands.

Created 2012-07-23 18:15:52 | Updated 2015-05-15 08:07:03 | Tags: fastareference vcf utilities callset

This is a classic problem: you get some VCF files from collaborators, you try to use them with your own data, and GATK fails with a big fat error saying that the references don't match.

### Solution

So what do you do? If you can, you find a version of the VCF file that is derived from the right reference. If you're working with human data and the VCF in question is just a common resource like dbsnp, you're in luck -- we provide versions of dbsnp and similar resources derived from the major human reference builds in our resource bundle (see FAQs for access details).

location: ftp.broadinstitute.org


If that's not an option, then you'll have to "liftover" -- specifically, liftover the mismatching VCF to the reference you need to work with. The procedure for doing so is described below.

### Liftover procedure

This procedure involves three steps:

1. Run GATK LiftoverVariants on your VCF file
2. Run a script to sort the lifted-over file
3. Filter out records whose REF field does not match the new reference

We provide a script that performs those three steps for you, called liftOverVCF.pl, which is available in our public source repository under the 'perl' directory. Instructions for pulling down our source are available here.

The example below shows how you would run the script:

./liftOverVCF.pl \
-vcf calls.b36.vcf \                    # input vcf
-chain b36ToHg19.broad.over.chain \     # chain file
-out calls.hg19.vcf \                   # output vcf
-gatk gatk_source \                     # path to source code
-newRef Homo_sapiens_assembly19 \       # path to new reference base name (without extension)
-oldRef human_b36_both \                # path to old reference prefix (without extension)
-tmp /broad/shptmp [defaults to /tmp]   # temp file location (defaults to /tmp)


We provide several chain files to liftover between the major human reference builds, also in our resource bundle (mentioned above) in the Liftover_Chain_Files directory. If you are working with non-human organisms, we can't help you -- but others may have chain files, so ask around in your field.

Note that if you're at the Broad, you can access chain files to liftover from b36/hg18 to hg19 on the humgen server.

/humgen/gsa-hpprojects/GATK/data/Liftover_Chain_Files/


Created 2012-07-23 17:50:07 | Updated 2015-05-15 23:55:20 | Tags: selectvariants jexl vcf callset

### This document describes why you might want to extract a subset of variants from a callset and how you would achieve this.

Often, a VCF containing many samples and/or variants will need to be subset in order to facilitate certain analyses (e.g. comparing and contrasting cases vs. controls; extracting variant or non-variant loci that meet certain requirements, displaying just a few samples in a browser like IGV, etc.). The GATK tool that we use the most for subsetting calls in various ways is SelectVariants; it enables easy and convenient subsetting of VCF files according to many criteria.

Select Variants operates on VCF files (also sometimes referred to as ROD in our documentation, for Reference Ordered Data) provided at the command line using the GATK's built in --variant option. You can provide multiple VCF files for Select Variants, but at least one must be named 'variant' and this will be the file (or set of files) from which variants will be selected. Other files can be used to modify the selection based on concordance or discordance between the callsets (see --discordance / --concordance arguments in the tool documentation).

There are many options for setting the selection criteria, depending on what you want to achieve. For example, given a single VCF file, one or more samples can be extracted from the file, based either on a complete sample name, or on a pattern match. Variants can also be selected based on annotated properties, such as depth of coverage or allele frequency. This is done using JEXL expressions; make sure to read the linked document for details, especially the section on working with complex expressions.

Note that in the output VCF, some annotations such as AN (number of alleles), AC (allele count), AF (allele frequency), and DP (depth of coverage) are recalculated as appropriate to accurately reflect the composition of the subset callset. See further below for an explanation of how that works.

### Command-line arguments

For a complete, detailed argument reference, refer to the GATK document page here.

### Subsetting by sample and ALT alleles

SelectVariants now keeps (r5832) the alt allele, even if a record is AC=0 after subsetting the site down to selected samples. For example, when selecting down to just sample NA12878 from the OMNI VCF in 1000G (1525 samples), the resulting VCF will look like:

1       82154   rs4477212       A       G       .       PASS    AC=0;AF=0.00;AN=2;CR=100.0;DP=0;GentrainScore=0.7826;HW=1.0     GT:GC   0/0:0.7205
1       534247  SNP1-524110     C       T       .       PASS    AC=0;AF=0.00;AN=2;CR=99.93414;DP=0;GentrainScore=0.7423;HW=1.0  GT:GC   0/0:0.6491
1       565286  SNP1-555149     C       T       .       PASS    AC=2;AF=1.00;AN=2;CR=98.8266;DP=0;GentrainScore=0.7029;HW=1.0   GT:GC   1/1:0.3471
1       569624  SNP1-559487     T       C       .       PASS    AC=2;AF=1.00;AN=2;CR=97.8022;DP=0;GentrainScore=0.8070;HW=1.0   GT:GC   1/1:0.3942


Although NA12878 is 0/0 at the first sites, ALT allele is preserved in the VCF record. This is the correct behavior, as reducing samples down shouldn't change the character of the site, only the AC in the subpopulation. This is related to the tricky issue of isPolymorphic() vs. isVariant().

• isVariant => is there an ALT allele?

• isPolymorphic => is some sample non-ref in the samples?

For clarity, in previous versions of SelectVariants, the first two monomorphic sites lose the ALT allele, because NA12878 is hom-ref at this site, resulting in VCF that looks like:

1       82154   rs4477212       A       .       .       PASS    AC=0;AF=0.00;AN=2;CR=100.0;DP=0;GentrainScore=0.7826;HW=1.0     GT:GC   0/0:0.7205
1       534247  SNP1-524110     C       .       .       PASS    AC=0;AF=0.00;AN=2;CR=99.93414;DP=0;GentrainScore=0.7423;HW=1.0  GT:GC   0/0:0.6491
1       565286  SNP1-555149     C       T       .       PASS    AC=2;AF=1.00;AN=2;CR=98.8266;DP=0;GentrainScore=0.7029;HW=1.0   GT:GC   1/1:0.3471
1       569624  SNP1-559487     T       C       .       PASS    AC=2;AF=1.00;AN=2;CR=97.8022;DP=0;GentrainScore=0.8070;HW=1.0   GT:GC   1/1:0.3942


If you really want a VCF without monomorphic sites, use the option to drop monomorphic sites after subsetting.

### How do the AC, AF, AN, and DP fields change?

Let's say you have a file with three samples. The numbers before the ":" will be the genotype (0/0 is hom-ref, 0/1 is het, and 1/1 is hom-var), and the number after will be the depth of coverage.

BOB        MARY        LINDA
1/0:20     0/0:30      1/1:50


In this case, the INFO field will say AN=6, AC=3, AF=0.5, and DP=100 (in practice, I think these numbers won't necessarily add up perfectly because of some read filters we apply when calling, but it's approximately right).

Now imagine I only want a file with the samples "BOB" and "MARY". The new file would look like:

BOB        MARY
1/0:20     0/0:30


The INFO field will now have to change to reflect the state of the new data. It will be AN=4, AC=1, AF=0.25, DP=50.

Let's pretend that MARY's genotype wasn't 0/0, but was instead "./." (no genotype could be ascertained). This would look like

BOB        MARY
1/0:20     ./.:.


with AN=2, AC=1, AF=0.5, and DP=20.

For information on how to construct regular expressions for use with this tool, see the method article on variant filtering with JEXL, or "Summary of regular-expression constructs" section here for more hardcore reading.

Created 2012-07-23 17:45:57 | Updated 2015-05-16 02:26:36 | Tags: combinevariants vcf callset

### Solutions for combining variant callsets depending on purpose

There are three main reasons why you might want to combine variants from different files into one, and the tool to use depends on what you are trying to achieve.

1. The most common case is when you have been parallelizing your variant calling analyses, e.g. running HaplotypeCaller per-chromosome, producing separate VCF files (or gVCF files) per-chromosome. For that case, you can use a tool called CatVariants to concatenate the files. There are a few important requirements (e.g. the files should contain all the same samples, and distinct intervals) which you can read about on the tool's documentation page.

2. The second case is when you have been using HaplotypeCaller in -ERC GVCF or -ERC BP_RESOLUTION to call variants on a large cohort, producing many gVCF files. We recommend combining the output gVCF in batches of e.g. 200 before putting them through joint genotyping with GenotypeGVCFs (for performance reasons), which you can do using CombineGVCFs, which is specific for handling gVCF files.

3. The third case is when you want to combine variant calls that were produced from the same samples but using different methods, for comparison. For example, if you're evaluating variant calls produced by different variant callers, different workflows, or the same but using different parameters. This produces separate callsets for the same samples, which are then easier to compare if you combine them into a single file. For that purpose, you can use CombineVariants, which is capable of merging VCF records intelligently, treating the same samples as separate or not as desired, combining annotations as appropriate. This is the case that requires the most preparation and forethought because there are many options that may be used to adapt the behavior of the tool.

There is also one reason you might want to combine variants from different files into one, that we do not recommend following. That is, if you have produced variant calls from various samples separately, and want to combine them for analysis. This is how people used to do variant analysis on large numbers of samples, but we don't recommend proceeding this way because that workflow suffers from serious methodological flaws. Instead, you should follow our recommendations as laid out in the Best Practices documentation.

### Merging records across VCFs with CombineVariants

Here we provide some more information and a worked out example to illustrate the third case because it is less straightforward than the other two.

A key point to understand is that CombineVariants will include a record at every site in all of your input VCF files, and annotate in which input callsets the record is present, pass, or filtered in in the set attribute in the INFO field (see below). In effect, CombineVariants always produces a union of the input VCFs. Any part of the Venn of the N merged VCFs can then be extracted specifically using JEXL expressions on the set attribute using SelectVariants. If you want to extract just the records in common between two VCFs, you would first CombineVariants the two files into a single VCF, and then run SelectVariants to extract the common records with -select 'set == "Intersection"', as worked out in the detailed example below.

#### Handling PASS/FAIL records at the same site in multiple input files

The -filteredRecordsMergeType argument determines how CombineVariants handles sites where a record is present in multiple VCFs, but it is filtered in some and unfiltered in others, as described in the tool documentation page linked above.

#### Understanding the set attribute

The set property of the INFO field indicates which call set the variant was found in. It can take on a variety of values indicating the exact nature of the overlap between the call sets. Note that the values are generalized for multi-way combinations, but here we describe only the values for 2 call sets being combined.

• set=Intersection : occurred in both call sets, not filtered out

• set=NAME : occurred in the call set NAME only

• set=NAME1-filteredInNAME : occurred in both call sets, but was not filtered in NAME1 but was filtered in NAME2

• set=filteredInAll : occurred in both call sets, but was filtered out of both

For three or more call sets combinations, you can see records like NAME1-NAME2 indicating a variant occurred in both NAME1 and NAME2 but not all sets.

You specify the NAME of a callset is by using the following syntax in your command line: -V:omni 1000G_omni2.5.b37.sites.vcf.

#### Emitting minimal VCF output

You can add the -minimalVCF argument to CombineVariants if you want to eliminate unnecessary information from the INFO field and genotypes. In that case, the only fields emitted will be GT:GQ for genotypes and the keySet for INFO.

An even more extreme output format is -sites_only (a general engine capability listed in the CommandLineGATK documentation) where the genotypes for all samples are completely stripped away from the output format. Enabling this option results in a significant performance speedup as well.

#### Requiring sites to be present in a minimum number of callsets

Sometimes you may want to combine several data sets but you only keep sites that are present in at least 2 of them. To do so, simply add the -minN (or --minimumN) command, followed by an integer if you want to only output records present in at least N input files. In our example, you would add -minN 2 to the command line.

#### Example: intersecting two VCFs

In the following example, we use CombineVariants and SelectVariants to obtain only the sites in common between the OMNI 2.5M and HapMap3 sites in the GSA bundle.

# combine the data
java -Xmx2g -jar dist/GenomeAnalysisTK.jar -T CombineVariants -R bundle/b37/human_g1k_v37.fasta -L 1:1-1,000,000 -V:omni bundle/b37/1000G_omni2.5.b37.sites.vcf -V:hm3 bundle/b37/hapmap_3.3.b37.sites.vcf -o union.vcf

# select the intersection
java -Xmx2g -jar dist/GenomeAnalysisTK.jar -T SelectVariants -R ~/Desktop/broadLocal/localData/human_g1k_v37.fasta -L 1:1-1,000,000 -V:variant union.vcf -select 'set == "Intersection";' -o intersect.vcf


This results in two vcf files, which look like:

# contents of union.vcf
1       990839  SNP1-980702     C       T       .       PASS    AC=150;AF=0.05384;AN=2786;CR=100.0;GentrainScore=0.7267;HW=0.0027632264;set=Intersection
1       990882  SNP1-980745     C       T       .       PASS    CR=99.79873;GentrainScore=0.7403;HW=0.005225421;set=omni
1       990984  SNP1-980847     G       A       .       PASS    CR=99.76005;GentrainScore=0.8406;HW=0.26163524;set=omni
1       992265  SNP1-982128     C       T       .       PASS    CR=100.0;GentrainScore=0.7412;HW=0.0025895447;set=omni
1       992819  SNP1-982682     G       A       .       id50    CR=99.72961;GentrainScore=0.8505;HW=4.811053E-17;set=FilteredInAll
1       993987  SNP1-983850     T       C       .       PASS    CR=99.85935;GentrainScore=0.8336;HW=9.959717E-28;set=omni
1       994391  rs2488991       G       T       .       PASS    AC=1936;AF=0.69341;AN=2792;CR=99.89378;GentrainScore=0.7330;HW=1.1741E-41;set=filterInomni-hm3
1       996184  SNP1-986047     G       A       .       PASS    CR=99.932205;GentrainScore=0.8216;HW=3.8830226E-6;set=omni
1       998395  rs7526076       A       G       .       PASS    AC=2234;AF=0.80187;AN=2786;CR=100.0;GentrainScore=0.8758;HW=0.67373306;set=Intersection
1       999649  SNP1-989512     G       A       .       PASS    CR=99.93262;GentrainScore=0.7965;HW=4.9767335E-4;set=omni

# contents of intersect.vcf
1       950243  SNP1-940106     A       C       .       PASS    AC=826;AF=0.29993;AN=2754;CR=97.341675;GentrainScore=0.7311;HW=0.15148845;set=Intersection
1       957640  rs6657048       C       T       .       PASS    AC=127;AF=0.04552;AN=2790;CR=99.86667;GentrainScore=0.6806;HW=2.286109E-4;set=Intersection
1       959842  rs2710888       C       T       .       PASS    AC=654;AF=0.23559;AN=2776;CR=99.849;GentrainScore=0.8072;HW=0.17526293;set=Intersection
1       977780  rs2710875       C       T       .       PASS    AC=1989;AF=0.71341;AN=2788;CR=99.89077;GentrainScore=0.7875;HW=2.9912625E-32;set=Intersection
1       985900  SNP1-975763     C       T       .       PASS    AC=182;AF=0.06528;AN=2788;CR=99.79926;GentrainScore=0.8374;HW=0.017794203;set=Intersection
1       987200  SNP1-977063     C       T       .       PASS    AC=1956;AF=0.70007;AN=2794;CR=99.45917;GentrainScore=0.7914;HW=1.413E-42;set=Intersection
1       987670  SNP1-977533     T       G       .       PASS    AC=2485;AF=0.89196;AN=2786;CR=99.51427;GentrainScore=0.7005;HW=0.24214932;set=Intersection
1       990417  rs2465136       T       C       .       PASS    AC=1113;AF=0.40007;AN=2782;CR=99.7599;GentrainScore=0.8750;HW=8.595538E-5;set=Intersection
1       990839  SNP1-980702     C       T       .       PASS    AC=150;AF=0.05384;AN=2786;CR=100.0;GentrainScore=0.7267;HW=0.0027632264;set=Intersection
1       998395  rs7526076       A       G       .       PASS    AC=2234;AF=0.80187;AN=2786;CR=100.0;GentrainScore=0.8758;HW=0.67373306;set=Intersection


Created 2012-07-23 17:36:42 | Updated 2013-06-10 18:24:01 | Tags: official snpeff variantannotator vcf tooltips callset annotation

### IMPORTANT NOTE: This document is out of date and will be replaced soon. In the meantime, you can find accurate information on how to run SnpEff in a compatible way with GATK in the SnpEff documentation, and instructions on what steps are necessary in the presentation on Functional Annotation linked in the comments below.

Our testing has shown that not all combinations of snpEff/database versions produce high-quality results. Be sure to read this document completely to familiarize yourself with our recommended best practices BEFORE running snpEff.

### Introduction

Until recently we were using an in-house annotation tool for genomic annotation, but the burden of keeping the database current and our lack of ability to annotate indels has led us to employ the use of a third-party tool instead. After reviewing many external tools (including annoVar, VAT, and Oncotator), we decided that SnpEff best meets our needs as it accepts VCF files as input, can annotate a full exome callset (including indels) in seconds, and provides continually-updated transcript databases. We have implemented support in the GATK for parsing the output from the SnpEff tool and annotating VCFs with the information provided in it.

### SnpEff Setup and Usage

Download the SnpEff core program. If you want to be able to run VariantAnnotator on the SnpEff output, you'll need to download a version of SnpEff that VariantAnnotator supports from this page (currently supported versions are listed below). If you just want the most recent version of SnpEff and don't plan to run VariantAnnotator on its output, you can get it from here.

After unzipping the core program, open the file snpEff.config in a text editor, and change the "database_repository" line to the following:

database_repository = http://sourceforge.net/projects/snpeff/files/databases/


java -jar snpEff.jar download GRCh37.64


You can find a list of available databases here. The human genome databases have GRCh or hg in their names. You can also download the databases directly from the SnpEff website, if you prefer.

The download command by default puts the databases into a subdirectory called data within the directory containing the SnpEff jar file. If you want the databases in a different directory, you'll need to edit the data_dir entry in the file snpEff.config to point to the correct directory.

Run SnpEff on the file containing your variants, and redirect its output to a file. SnpEff supports many input file formats including VCF 4.1, BED, and SAM pileup. Full details and command-line options can be found on the SnpEff home page.

### Supported SnpEff Versions

If you want to take advantage of SnpEff integration in the GATK, you'll need to run SnpEff version **2.0.5*. Note: newer versions are currently unsupported by the GATK, as we haven't yet had the reources to test it.

### Current Recommended Best Practices When Running SnpEff

These best practices are based on our analysis of various snpEff/database versions as described in detail in the Analysis of SnpEff Annotations Across Versions section below.

• We recommend using only the GRCh37.64 database with SnpEff 2.0.5. The more recent GRCh37.65 database produces many false-positive Missense annotations due to a regression in the ENSEMBL Release 65 GTF file used to build the database. This regression has been acknowledged by ENSEMBL and is supposedly fixed as of 1-30-2012; however as we have not yet tested the fixed version of the database we continue to recommend using only GRCh37.64 for now.

• We recommend always running with -onlyCoding true with human databases (eg., the GRCh37.* databases). Setting -onlyCoding false causes snpEff to report all transcripts as if they were coding (even if they're not), which can lead to nonsensical results. The -onlyCoding false option should only be used with databases that lack protein coding information.

• Do not trust annotations from versions of snpEff prior to 2.0.4. Older versions of snpEff (such as 2.0.2) produced many incorrect annotations due to the presence of a certain number of nonsensical transcripts in the underlying ENSEMBL databases. Newer versions of snpEff filter out such transcripts.

### Analyses of SnpEff Annotations Across Versions

See our analysis of the SNP annotations produced by snpEff across various snpEff/database versions here.

• Both snpEff 2.0.2 + GRCh37.63 and snpEff 2.0.5 + GRCh37.65 produce an abnormally high Missense:Silent ratio, with elevated levels of Missense mutations across the entire spectrum of allele counts. They also have a relatively low (~70%) level of concordance with the 1000G Gencode annotations when it comes to Silent mutations. This suggests that these combinations of snpEff/database versions incorrectly annotate many Silent mutations as Missense.

• snpEff 2.0.4 RC3 + GRCh37.64 and snpEff 2.0.5 + GRCh37.64 produce a Missense:Silent ratio in line with expectations, and have a very high (~97%-99%) level of concordance with the 1000G Gencode annotations across all categories.

See our comparison of SNP annotations produced using the GRCh37.64 and GRCh37.65 databases with snpEff 2.0.5 here

• The GRCh37.64 database gives good results on the condition that you run snpEff with the -onlyCoding true option. The -onlyCoding false option causes snpEff to mark all transcripts as coding, and so produces many false-positive Missense annotations.

• The GRCh37.65 database gives results that are as poor as those you get with the -onlyCoding false option on the GRCh37.64 database. This is due to a regression in the ENSEMBL release 65 GTF file used to build snpEff's GRCh37.65 database. The regression has been acknowledged by ENSEMBL and is due to be fixed shortly.

See our analysis of the INDEL annotations produced by snpEff across snpEff/database versions here

• snpEff's indel annotations are highly concordant with those of a high-quality set of genomic annotations from the 1000 Genomes project. This is true across all snpEff/database versions tested.

### Example SnpEff Usage with a VCF Input File

Below is an example of how to run SnpEff version 2.0.5 with a VCF input file and have it write its output in VCF format as well. Notice that you need to explicitly specify the database you want to use (in this case, GRCh37.64). This database must be present in a directory of the same name within the data_dir as defined in snpEff.config.

java -Xmx4G -jar snpEff.jar eff -v -onlyCoding true -i vcf -o vcf GRCh37.64 1000G.exomes.vcf > snpEff_output.vcf


In this mode, SnpEff aggregates all effects associated with each variant record together into a single INFO field annotation with the key EFF. The general format is:

EFF=Effect1(Information about Effect1),Effect2(Information about Effect2),etc.


And here is the precise layout with all the subfields:

EFF=Effect1(Effect_Impact|Effect_Functional_Class|Codon_Change|Amino_Acid_Change|Gene_Name|Gene_BioType|Coding|Transcript_ID|Exon_ID),Effect2(etc...


It's also possible to get SnpEff to output in a (non-VCF) text format with one Effect per line. See the SnpEff home page for full details.

### Adding SnpEff Annotations using VariantAnnotator

Once you have a SnpEff output VCF file, you can use the VariantAnnotator walker to add SnpEff annotations based on that output to the input file you ran SnpEff on.

There are two different options for doing this:

#### Option 1: Annotate with only the highest-impact effect for each variant

NOTE: This option works only with supported SnpEff versions as explained above. VariantAnnotator run as described below will refuse to parse SnpEff output files produced by other versions of the tool, or which lack a SnpEff version number in their header.

The default behavior when you run VariantAnnotator on a SnpEff output file is to parse the complete set of effects resulting from the current variant, select the most biologically-significant effect, and add annotations for just that effect to the INFO field of the VCF record for the current variant. This is the mode we plan to use in our Production Data-Processing Pipeline.

When selecting the most biologically-significant effect associated with the current variant, VariantAnnotator does the following:

• Prioritizes the effects according to the categories (in order of decreasing precedence) "High-Impact", "Moderate-Impact", "Low-Impact", and "Modifier", and always selects one of the effects from the highest-priority category. For example, if there are three moderate-impact effects and two high-impact effects resulting from the current variant, the annotator will choose one of the high-impact effects and add annotations based on it. See below for a full list of the effects arranged by category.

• Within each category, ties are broken using the functional class of each effect (in order of precedence: NONSENSE, MISSENSE, SILENT, or NONE). For example, if there is both a NON_SYNONYMOUS_CODING (MODERATE-impact, MISSENSE) and a CODON_CHANGE (MODERATE-impact, NONE) effect associated with the current variant, the annotator will select the NON_SYNONYMOUS_CODING effect. This is to allow for more accurate counts of the total number of sites with NONSENSE/MISSENSE/SILENT mutations. See below for a description of the functional classes SnpEff associates with the various effects.

• Effects that are within a non-coding region are always considered lower-impact than effects that are within a coding region.

Example Usage:

java -jar dist/GenomeAnalysisTK.jar \
-T VariantAnnotator \
-R /humgen/1kg/reference/human_g1k_v37.fasta \
-A SnpEff \
--variant 1000G.exomes.vcf \        (file to annotate)
--snpEffFile snpEff_output.vcf \    (SnpEff VCF output file generated by running SnpEff on the file to annotate)
-L 1000G.exomes.vcf \
-o out.vcf


VariantAnnotator adds some or all of the following INFO field annotations to each variant record:

• SNPEFF_EFFECT - The highest-impact effect resulting from the current variant (or one of the highest-impact effects, if there is a tie)
• SNPEFF_IMPACT - Impact of the highest-impact effect resulting from the current variant (HIGH, MODERATE, LOW, or MODIFIER)
• SNPEFF_FUNCTIONAL_CLASS - Functional class of the highest-impact effect resulting from the current variant (NONE, SILENT, MISSENSE, or NONSENSE)
• SNPEFF_CODON_CHANGE - Old/New codon for the highest-impact effect resulting from the current variant
• SNPEFF_AMINO_ACID_CHANGE - Old/New amino acid for the highest-impact effect resulting from the current variant
• SNPEFF_GENE_NAME - Gene name for the highest-impact effect resulting from the current variant
• SNPEFF_GENE_BIOTYPE - Gene biotype for the highest-impact effect resulting from the current variant
• SNPEFF_TRANSCRIPT_ID - Transcript ID for the highest-impact effect resulting from the current variant
• SNPEFF_EXON_ID - Exon ID for the highest-impact effect resulting from the current variant

Example VCF records annotated using SnpEff and VariantAnnotator:

1   874779  .   C   T   279.94  . AC=1;AF=0.0032;AN=310;BaseQRankSum=-1.800;DP=3371;Dels=0.00;FS=0.000;HRun=0;HaplotypeScore=1.4493;InbreedingCoeff=-0.0045;
SNPEFF_EFFECT=SYNONYMOUS_CODING;SNPEFF_EXON_ID=exon_1_874655_874840;SNPEFF_FUNCTIONAL_CLASS=SILENT;SNPEFF_GENE_BIOTYPE=protein_coding;SNPEFF_GENE_NAME=SAMD11;
SNPEFF_IMPACT=LOW;SNPEFF_TRANSCRIPT_ID=ENST00000342066

1   874816  .   C   CT  2527.52 .   AC=15;AF=0.0484;AN=310;BaseQRankSum=-11.876;DP=4718;FS=48.575;HRun=1;HaplotypeScore=91.9147;InbreedingCoeff=-0.0520;
SNPEFF_FUNCTIONAL_CLASS=NONE;SNPEFF_GENE_BIOTYPE=protein_coding;SNPEFF_GENE_NAME=SAMD11;SNPEFF_IMPACT=HIGH;SNPEFF_TRANSCRIPT_ID=ENST00000342066


#### Option 2: Annotate with all effects for each variant

VariantAnnotator also has the ability to take the EFF field from the SnpEff VCF output file containing all the effects aggregated together and copy it verbatim into the VCF to annotate.

Here's an example of how to do this:

java -jar dist/GenomeAnalysisTK.jar \
-T VariantAnnotator \
-R /humgen/1kg/reference/human_g1k_v37.fasta \
-E resource.EFF \
--variant 1000G.exomes.vcf \      (file to annotate)
--resource snpEff_output.vcf \    (SnpEff VCF output file generated by running SnpEff on the file to annotate)
-L 1000G.exomes.vcf \
-o out.vcf


Of course, in this case you can also use the VCF output by SnpEff directly, but if you are using VariantAnnotator for other purposes anyway the above might be useful.

### List of Genomic Effects

Below are the possible genomic effects recognized by SnpEff, grouped by biological impact. Full descriptions of each effect are available on this page.

#### High-Impact Effects

• SPLICE_SITE_ACCEPTOR
• SPLICE_SITE_DONOR
• START_LOST
• EXON_DELETED
• FRAME_SHIFT
• STOP_GAINED
• STOP_LOST

#### Moderate-Impact Effects

• NON_SYNONYMOUS_CODING
• CODON_CHANGE (note: this effect is used by SnpEff only for MNPs, not SNPs)
• CODON_INSERTION
• CODON_CHANGE_PLUS_CODON_INSERTION
• CODON_DELETION
• CODON_CHANGE_PLUS_CODON_DELETION
• UTR_5_DELETED
• UTR_3_DELETED

#### Low-Impact Effects

• SYNONYMOUS_START
• NON_SYNONYMOUS_START
• START_GAINED
• SYNONYMOUS_CODING
• SYNONYMOUS_STOP
• NON_SYNONYMOUS_STOP

#### Modifiers

• NONE
• CHROMOSOME
• CUSTOM
• CDS
• GENE
• TRANSCRIPT
• EXON
• INTRON_CONSERVED
• UTR_5_PRIME
• UTR_3_PRIME
• DOWNSTREAM
• INTRAGENIC
• INTERGENIC
• INTERGENIC_CONSERVED
• UPSTREAM
• REGULATION
• INTRON

### Functional Classes

SnpEff assigns a functional class to certain effects, in addition to an impact:

• NONSENSE: assigned to point mutations that result in the creation of a new stop codon
• MISSENSE: assigned to point mutations that result in an amino acid change, but not a new stop codon
• SILENT: assigned to point mutations that result in a codon change, but not an amino acid change or new stop codon
• NONE: assigned to all effects that don't fall into any of the above categories (including all events larger than a point mutation)

The GATK prioritizes effects with functional classes over effects of equal impact that lack a functional class when selecting the most significant effect in VariantAnnotator. This is to enable accurate counts of NONSENSE/MISSENSE/SILENT sites.

Created 2012-07-23 17:28:06 | Updated 2013-03-19 17:57:23 | Tags: official varianteval vcf tooltips callset

For a complete, detailed argument reference, refer to the technical documentation page.

## Modules

You can find detailed information about the various modules here.

### Stratification modules

• AlleleFrequency
• AlleleCount
• CompRod
• Contig
• CpG
• Degeneracy
• EvalRod
• Filter
• FunctionalClass
• JexlExpression
• Novelty
• Sample

### Evaluation modules

• CompOverlap
• CountVariants

Note that the GenotypeConcordance module has been rewritten as a separate walker tool (see its Technical Documentation page).

## A useful analysis using VariantEval

We in GSA often find ourselves performing an analysis of 2 different call sets. For SNPs, we often show the overlap of the sets (their "venn") and the relative dbSNP rates and/or transition-transversion ratios. The picture provided is an example of such a slide and is easy to create using VariantEval. Assuming you have 2 filtered VCF callsets named 'foo.vcf' and 'bar.vcf', there are 2 quick steps.

#### Combine the VCFs

java -jar GenomeAnalysisTK.jar \
-R ref.fasta \
-T CombineVariants \
-V:FOO foo.vcf \
-V:BAR bar.vcf \
-priority FOO,BAR \
-o merged.vcf


#### Run VariantEval

java -jar GenomeAnalysisTK.jar \
-T VariantEval \
-R ref.fasta \
-D dbsnp.vcf \
-select 'set=="Intersection"' -selectName Intersection \
-select 'set=="FOO"' -selectName FOO \
-select 'set=="FOO-filterInBAR"' -selectName InFOO-FilteredInBAR \
-select 'set=="BAR"' -selectName BAR \
-select 'set=="filterInFOO-BAR"' -selectName InBAR-FilteredInFOO \
-select 'set=="FilteredInAll"' -selectName FilteredInAll \
-o merged.eval.gatkreport \
-eval merged.vcf \
-l INFO


### Checking the possible values of 'set'

It is wise to check the actual values for the set names present in your file before writing complex VariantEval commands. An easy way to do this is to extract the value of the set fields and then reduce that to the unique entries, like so:

java -jar GenomeAnalysisTK.jar -T VariantsToTable -R ref.fasta -V merged.vcf -F set -o fields.txt
grep -v 'set' fields.txt | sort | uniq -c


This will provide you with a list of all of the possible values for 'set' in your VCF so that you can be sure to supply the correct select statements to VariantEval.

## Reading the VariantEval output file

The VariantEval output is formatted as a GATKReport.

### Understanding Genotype Concordance values from Variant Eval

The VariantEval genotype concordance module emits information the relationship between the eval calls and genotypes and the comp calls and genotypes. The following three slides provide some insight into three key metrics to assess call sensitivity and concordance between genotypes.

##:GATKReport.v0.1 GenotypeConcordance.sampleSummaryStats&#160;: the concordance statistics summary for each sample
GenotypeConcordance.sampleSummaryStats  CompRod   CpG      EvalRod  JexlExpression  Novelty  percent_comp_ref_called_var  percent_comp_het_called_het  percent_comp_het_called_var  percent_comp_hom_called_hom  percent_comp_hom_called_var  percent_non-reference_sensitivity  percent_overall_genotype_concordance  percent_non-reference_discrepancy_rate
GenotypeConcordance.sampleSummaryStats  compOMNI  all      eval     none            all      0.78                         97.65                        98.39                        99.13                        99.44                        98.80                              99.09                                 3.60


The key outputs:

• percent_overall_genotype_concordance
• percent_non_ref_sensitivity_rate
• percent_non_ref_discrepancy_rate

All defined below.

Created 2012-07-23 16:49:34 | Updated 2015-05-15 08:33:12 | Tags: variantrecalibrator vqsr applyrecalibration vcf callset variantrecalibration

This document describes what Variant Quality Score Recalibration (VQSR) is designed to do, and outlines how it works under the hood. For command-line examples and recommendations on what specific resource datasets and arguments to use for VQSR, please see this FAQ article.

As a complement to this document, we encourage you to watch the workshop videos available on our Events webpage.

Slides that explain the VQSR methodology in more detail as well as the individual component variant annotations can be found here in the GSA Public Drop Box.

Detailed information about command line options for VariantRecalibrator can be found here.

Detailed information about command line options for ApplyRecalibration can be found here.

### Introduction

The purpose of variant recalibration is to assign a well-calibrated probability to each variant call in a call set. This enables you to generate highly accurate call sets by filtering based on this single estimate for the accuracy of each call.

The approach taken by variant quality score recalibration is to develop a continuous, covarying estimate of the relationship between SNP call annotations (QD, SB, HaplotypeScore, HRun, for example) and the the probability that a SNP is a true genetic variant versus a sequencing or data processing artifact. This model is determined adaptively based on "true sites" provided as input (typically HapMap 3 sites and those sites found to be polymorphic on the Omni 2.5M SNP chip array, for humans). This adaptive error model can then be applied to both known and novel variation discovered in the call set of interest to evaluate the probability that each call is real. The score that gets added to the INFO field of each variant is called the VQSLOD. It is the log odds ratio of being a true variant versus being false under the trained Gaussian mixture model.

The variant recalibrator contrastively evaluates variants in a two step process, each performed by a distinct tool:

• VariantRecalibrator
Create a Gaussian mixture model by looking at the annotations values over a high quality subset of the input call set and then evaluate all input variants. This step produces a recalibration file.

• ApplyRecalibration
Apply the model parameters to each variant in input VCF files producing a recalibrated VCF file in which each variant is annotated with its VQSLOD value. In addition, this step will filter the calls based on this new lod score by adding lines to the FILTER column for variants that don't meet the specified lod threshold.

Please see the VQSR tutorial for step-by-step instructions on running these tools.

#### How VariantRecalibrator works in a nutshell

The tool takes the overlap of the training/truth resource sets and of your callset. It models the distribution of these variants relative to the annotations you specified, and attempts to group them into clusters. Then it uses the clustering to assign VQSLOD scores to all variants. Variants that are closer to the heart of a cluster will get a higher score than variants that are outliers.

#### How ApplyRecalibration works in a nutshell

During the first part of the recalibration process, variants in your callset were given a score called VQSLOD. At the same time, variants in your training sets were also ranked by VQSLOD. When you specify a tranche sensitivity threshold with ApplyRecalibration, expressed as a percentage (e.g. 99.9%), what happens is that the program looks at what is the VQSLOD value above which 99.9% of the variants in the training callset are included. It then takes that value of VQSLOD and uses it as a threshold to filter your variants. Variants that are above the threshold pass the filter, so the FILTER field will contain PASS. Variants that are below the threshold will be filtered out; they will be written to the output file, but in the FILTER field they will have the name of the tranche they belonged to. So VQSRTrancheSNP99.90to100.00 means that the variant was in the range of VQSLODs corresponding to the remaining 0.1% of the training set, which are basically considered false positives.

### Interpretation of the Gaussian mixture model plots

The variant recalibration step fits a Gaussian mixture model to the contextual annotations given to each variant. By fitting this probability model to the training variants (variants considered to be true-positives), a probability can be assigned to the putative novel variants (some of which will be true-positives, some of which will be false-positives). It is useful for users to see how the probability model was fit to their data. Therefore a modeling report is automatically generated each time VariantRecalibrator is run (in the above command line the report will appear as path/to/output.plots.R.pdf). For every pair-wise combination of annotations used in modeling, a 2D projection of the Gaussian mixture model is shown.

The figure shows one page of an example Gaussian mixture model report that is automatically generated by the VQSR from the example HiSeq call set. This page shows the 2D projection of mapping quality rank sum test versus Haplotype score by marginalizing over the other annotation dimensions in the model.

In each page there are four panels which show different ways of looking at the 2D projection of the model. The upper left panel shows the probability density function that was fit to the data. The 2D projection was created by marginalizing over the other annotation dimensions in the model via random sampling. Green areas show locations in the space that are indicative of being high quality while red areas show the lowest probability areas. In general putative SNPs that fall in the red regions will be filtered out of the recalibrated call set.

The remaining three panels give scatter plots in which each SNP is plotted in the two annotation dimensions as points in a point cloud. The scale for each dimension is in normalized units. The data for the three panels is the same but the points are colored in different ways to highlight different aspects of the data. In the upper right panel SNPs are colored black and red to show which SNPs are retained and filtered, respectively, by applying the VQSR procedure. The red SNPs didn't meet the given truth sensitivity threshold and so are filtered out of the call set. The lower left panel colors SNPs green, grey, and purple to give a sense of the distribution of the variants used to train the model. The green SNPs are those which were found in the training sets passed into the VariantRecalibrator step, while the purple SNPs are those which were found to be furthest away from the learned Gaussians and thus given the lowest probability of being true. Finally, the lower right panel colors each SNP by their known/novel status with blue being the known SNPs and red being the novel SNPs. Here the idea is to see if the annotation dimensions provide a clear separation between the known SNPs (most of which are true) and the novel SNPs (most of which are false).

An example of good clustering for SNP calls from the tutorial dataset is shown to the right. The plot shows that the training data forms a distinct cluster at low values for each of the two statistics shown (haplotype score and mapping quality bias). As the SNPs fall off the distribution in either one or both of the dimensions they are assigned a lower probability (that is, move into the red region of the model's PDF) and are filtered out. This makes sense as not only do higher values of HaplotypeScore indicate a lower chance of the data being explained by only two haplotypes but also higher values for mapping quality bias indicate more evidence of bias between the reference bases and the alternative bases. The model has captured our intuition that this area of the distribution is highly enriched for machine artifacts and putative variants here should be filtered out!

### Tranches and the tranche plot

The recalibrated variant quality score provides a continuous estimate of the probability that each variant is true, allowing one to partition the call sets into quality tranches. The main purpose of the tranches is to establish thresholds within your data that correspond to certain levels of sensitivity relative to the truth sets. The idea is that with well calibrated variant quality scores, you can generate call sets in which each variant doesn't have to have a hard answer as to whether it is in or out of the set. If a very high accuracy call set is desired then one can use the highest tranche, but if a larger, more complete call set is a higher priority than one can dip down into lower and lower tranches. These tranches are applied to the output VCF file using the FILTER field. In this way you can choose to use some of the filtered records or only use the PASSing records.

The first tranche (from the bottom, with the highest value of truth sensitivity but usually the lowest values of novel Ti/Tv) is exceedingly specific but less sensitive, and each subsequent tranche in turn introduces additional true positive calls along with a growing number of false positive calls. Downstream applications can select in a principled way more specific or more sensitive call sets or incorporate directly the recalibrated quality scores to avoid entirely the need to analyze only a fixed subset of calls but rather weight individual variant calls by their probability of being real. An example tranche plot, automatically generated by the VariantRecalibrator walker, is shown below.

This is an example of a tranches plot generated for a HiSeq call set. The x-axis gives the number of novel variants called while the y-axis shows two quality metrics -- novel transition to transversion ratio and the overall truth sensitivity.

Note that the tranches plot is not applicable for indels and will not be generated when the tool is run in INDEL mode.

### Ti/Tv-free recalibration

We use a Ti/Tv-free approach to variant quality score recalibration. This approach requires an additional truth data set, and cuts the VQSLOD at given sensitivities to the truth set. It has several advantages over the Ti/Tv-targeted approach:

• The truth sensitivity (TS) approach gives you back the novel Ti/Tv as a QC metric
• The truth sensitivity (TS) approach is conceptual cleaner than deciding on a novel Ti/Tv target for your dataset
• The TS approach is easier to explain and defend, as saying "I took called variants until I found 99% of my known variable sites" is easier than "I took variants until I dropped my novel Ti/Tv ratio to 2.07"

We have used hapmap 3.3 sites as the truth set (genotypes_r27_nr.b37_fwd.vcf), but other sets of high-quality (~99% truly variable in the population) sets of sites should work just as well. In our experience, with HapMap, 99% is a good threshold, as the remaining 1% of sites often exhibit unusual features like being close to indels or are actually MNPs, and so receive a low VQSLOD score.
Note that the expected Ti/Tv is still an available argument but it is only used for display purposes.

### Finally, a couple of Frequently Asked Questions

#### - Can I use the variant quality score recalibrator with my small sequencing experiment?

This tool is expecting thousands of variant sites in order to achieve decent modeling with the Gaussian mixture model. Whole exome call sets work well, but anything smaller than that scale might run into difficulties.

One piece of advice is to turn down the number of Gaussians used during training. This can be accomplished by adding --maxGaussians 4 to your command line.

maxGaussians is the maximum number of different "clusters" (=Gaussians) of variants the program is "allowed" to try to identify. Lowering this number forces the program to group variants into a smaller number of clusters, which means there will be more variants in each cluster -- hopefully enough to satisfy the statistical requirements. Of course, this decreases the level of discrimination that you can achieve between variant profiles/error modes. It's all about trade-offs; and unfortunately if you don't have a lot of variants you can't afford to be very demanding in terms of resolution.

#### - Why don't all the plots get generated for me?

The most common problem related to this is not having Rscript accessible in your environment path. Rscript is the command line version of R that gets installed right alongside. We also make use of the ggplot2 library so please be sure to install that package as well. See the Common Problems section of the Guide for more details.

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Created 2013-03-04 12:53:20 | Updated 2013-03-04 13:58:54 | Tags: merge callset

Hi all,

I would appreciate your thoughts on the following pipeline:
I'm currently working on a number of WGS of non-human vertebrates. My approach for calling variants is to maximize the sensitivity of the calls by using two callers (GATK's UnifiedGenotyper + samtools' mpileup) per chromosome regardless of / ingnoring all filters. Next, I would like to merge (not intersect) the two vcf files (GATK+samtools) per each chromosome, then merge (not intersect) all the vcf files pertaining to all chromosomes in order to retrieve a final vcf dataset per individual:

For merging the GATK and samtools:

$java -Xmx10g -jar GenomeAnalysisTK.jar -T CombineVariants -R ref.fasta --variant:GATK chr#.GATK.vcf --variant:samtools chr#.samtools.vcf -o chr#.GATK_samtools.union.vcf -genotypeMergeOptions PRIORITIZE -priority GATK,samtools --filteredrecordsmergetype KEEP_UNCONDITIONAL  For merging all chromosomes per individual: $ java -Xmx10g -jar GenomeAnalysisTK.jar -T CombineVariants -R ref.fasta
--variant:chr1 chr1.GATK_samtools.union.vcf --variant:chr2 chr2.GATK_samtools.union.vcf --variant:chr3 chr3.GATK_samtools.union.vcf
-o Individual1.union.vcf
-genotypeMergeOptions PRIORITIZE -priority chr1,chr2,chr3 --filteredrecordsmergetype KEEP_UNCONDITIONAL


Finally I would like to intersect between two individuals and keep only the variants that are common to both individuals:

Uniting / merging two individuals:

$java -Xmx10g -jar GenomeAnalysisTK.jar -T CombineVariants -R ref.fasta --variant:individual1 Individual1.union.vcf --variant:Individual2 Individual2.union.vcf -o Individual1_2.union.vcf -genotypeMergeOptions PRIORITIZE -priority Indiviual1,Individual2 --filteredrecordsmergetype KEEP_UNCONDITIONAL  Intersecting the two indiviuals in order to keep only common variants: $  java -Xmx10g -jar GenomeAnalysisTK.jar -T SelectVariants -R ref.fasta
--variant Individual1_2.union.vcf -select 'set == "Intersection";'
-o Intersected.vcf


Am I doing this right? I'm afraid I may be losing variants or something else along this pipeline. Remember that I want to keep only the common variants while ignoring the filters in order to increase sensitivity as much as possible.

Thanks!

Sagi

Created 2013-02-06 05:56:12 | Updated 2013-02-06 16:21:59 | Tags: best-practices callset

Hi, I am estimating SNP number for a genome of a speceis.

1. After mapping the illumina reads to a reference seqence (of a related species), I used picard to remove duplicates and used samtools (mpileup) to convert the bam file to a fastq file.
2. I also use GATK to do local reglimant and recalication from the mappnig bam file. Then I used samtools (mpileup) to convert the bam files output by GATK to a fastq file.

I found that the number of SNP in the fastq going through GATK is 10 times more than the first fastq. Interestingly, if I use picard to do duplicats-removomg again to the GATK bam and used samtools to convert the bam to fastq file. The SNP jumps back to 10 time fewer.

What can be the reason that the SNP number can be 10 time different between the two methods? Actually, I expect the GATK output file has fewer SNP given the effect of recaliraiton or relingement. But the result is opposite.

Chih-Ming