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All analyses done with the GATK typically involve several (though not necessarily all) of the following inputs:
Reference genome sequence
This article describes the corresponding file formats that are acceptable for use with the GATK.
The GATK requires the reference sequence in a single reference sequence in FASTA format, with all contigs in the same file. The GATK requires strict adherence to the FASTA standard. All the standard IUPAC bases are accepted, but keep in mind that non-standard bases (i.e. other than ACGT, such as W for example) will be ignored (i.e. those positions in the genome will be skipped).
Some users have reported having issues with reference files that have been stored or modified on Windows filesystems. The issues manifest as "10" characters (corresponding to encoded newlines) inserted in the sequence, which cause the GATK to quit with an error. If you encounter this issue, you will need to re-download a valid master copy of the reference file, or clean it up yourself.
Gzipped fasta files will not work with the GATK, so please make sure to unzip them first. Please see this article for more information on preparing FASTA reference sequences for use with the GATK.
If you are using human data, your reads must be aligned to one of the official b3x (e.g. b36, b37) or hg1x (e.g. hg18, hg19) references. The names and order of the contigs in the reference you used must exactly match that of one of the official references canonical orderings. These are defined by historical karotyping of largest to smallest chromosomes, followed by the X, Y, and MT for the b3x references; the order is thus 1, 2, 3, ..., 10, 11, 12, ... 20, 21, 22, X, Y, MT. The hg1x references differ in that the chromosome names are prefixed with "chr" and chrM appears first instead of last. The GATK will detect misordered contigs (for example, lexicographically sorted) and throw an error. This draconian approach, though unnecessary technically, ensures that all supplementary data provided with the GATK works correctly. You can use ReorderSam to fix a BAM file aligned to a missorted reference sequence.
Our Best Practice recommendation is that you use a standard GATK reference from the GATK resource bundle.
The only input format for sequence reads that the GATK itself supports is the [Sequence Alignment/Map (SAM)] format. See [SAM/BAM] for more details on the SAM/BAM format as well as Samtools and Picard, two complementary sets of utilities for working with SAM/BAM files.
If you don't find the information you need in this section, please see our FAQs on BAM files.
If you are starting out your pipeline with raw reads (typically in FASTQ format) you'll need to make sure that when you map those reads to the reference and produce a BAM file, the resulting BAM file is fully compliant with the GATK requirements. See the Best Practices documentation for detailed instructions on how to do this.
In addition to being in SAM format, we require the following additional constraints in order to use your file with the GATK:
The file must be binary (with
.bam file extension).
The file must be indexed.
Below is an example well-formed SAM field header and fields (with @SQ dictionary truncated to show only the first two chromosomes for brevity):
@HD VN:1.0 GO:none SO:coordinate @SQ SN:1 LN:249250621 AS:NCBI37 UR:file:/lustre/scratch102/projects/g1k/ref/main_project/human_g1k_v37.fasta M5:1b22b98cdeb4a9304cb5d48026a85128 @SQ SN:2 LN:243199373 AS:NCBI37 UR:file:/lustre/scratch102/projects/g1k/ref/main_project/human_g1k_v37.fasta M5:a0d9851da00400dec1098a9255ac712e @RG ID:ERR000162 PL:ILLUMINA LB:g1k-sc-NA12776-CEU-1 PI:200 DS:SRP000031 SM:NA12776 CN:SC @RG ID:ERR000252 PL:ILLUMINA LB:g1k-sc-NA12776-CEU-1 PI:200 DS:SRP000031 SM:NA12776 CN:SC @RG ID:ERR001684 PL:ILLUMINA LB:g1k-sc-NA12776-CEU-1 PI:200 DS:SRP000031 SM:NA12776 CN:SC @RG ID:ERR001685 PL:ILLUMINA LB:g1k-sc-NA12776-CEU-1 PI:200 DS:SRP000031 SM:NA12776 CN:SC @PG ID:GATK TableRecalibration VN:v2.2.16 CL:Covariates=[ReadGroupCovariate, QualityScoreCovariate, DinucCovariate, CycleCovariate], use_original_quals=true, defau t_read_group=DefaultReadGroup, default_platform=Illumina, force_read_group=null, force_platform=null, solid_recal_mode=SET_Q_ZERO, window_size_nqs=5, homopolymer_nback=7, except on_if_no_tile=false, pQ=5, maxQ=40, smoothing=137 UR:file:/lustre/scratch102/projects/g1k/ref/main_project/human_g1k_v37.fasta M5:b4eb71ee878d3706246b7c1dbef69299 @PG ID:bwa VN:0.5.5 ERR001685.4315085 16 1 9997 25 35M * 0 0 CCGATCTCCCTAACCCTAACCCTAACCCTAACCCT ?8:C7ACAABBCBAAB?CCAABBEBA@ACEBBB@? XT:A:U XN:i:4 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 RG:Z:ERR001685 NM:i:6 MD:Z:0N0N0N0N1A0A28 OQ:Z:>>:>2>>>>>>>>>>>>>>>>>>?>>>>??>???> ERR001689.1165834 117 1 9997 0 * = 9997 0 CCGATCTAGGGTTAGGGTTAGGGTTAGGGTTAGGG >7AA<@@C?@?B?B??>9?B??>A?B???BAB??@ RG:Z:ERR001689 OQ:Z:>:<<8<<<><<><><<>7<>>>?>>??>??????? ERR001689.1165834 185 1 9997 25 35M = 9997 0 CCGATCTCCCTAACCCTAACCCTAACCCTAACCCT 758A:?>>8?=@@>>?;4<>=??@@==??@?==?8 XT:A:U XN:i:4 SM:i:25 AM:i:0 X0:i:1 X1:i:0 XM:i:2 XO:i:0 XG:i:0 RG:Z:ERR001689 NM:i:6 MD:Z:0N0N0N0N1A0A28 OQ:Z:;74>7><><><>>>>><:<>>>>>>>>>>>>>>>> ERR001688.2681347 117 1 9998 0 * = 9998 0 CGATCTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG 5@BA@A6B???A?B??>B@B??>B@B??>BAB??? RG:Z:ERR001688 OQ:Z:=>>>><4><<?><??????????????????????
The GATK requires that the BAM file be sorted in the same order as the reference. Unfortunately, many BAM files have headers that are sorted in some other order -- lexicographical order is a common alternative. To resort the BAM file please use ReorderSam.
The GATK accept interval files for processing subsets of the genome in several different formats. Please see the FAQs on interval lists for details.
The GATK can associate arbitrary reference ordered data (ROD) files with named tracks for all tools. Some tools require specific ROD data files for processing, and developers are free to write tools that access arbitrary data sets using the ROD interface. The general ROD system has the following syntax:
name is the name in the GATK tool (like "eval" in VariantEval),
type is the type of the file, such as VCF or dbSNP, and
file is the path to the file containing the ROD data.
The GATK supports several common file formats for reading ROD data:
VCF : VCF type, the recommended format for representing variant loci and genotype calls. The GATK will only process valid VCF files; VCFTools provides the official VCF validator. See here for a useful poster detailing the VCF specification.
UCSC formated dbSNP : dbSNP type, UCSC dbSNP database output
Note that we no longer support the PED format. See here for converting .ped files to VCF.
Geraldine Van der Auwera, PhD