From 847f24a348fa2cf348e2fcd37c510eed0c2802c8 Mon Sep 17 00:00:00 2001 From: Jonn Smith Date: Fri, 1 Feb 2019 11:11:14 -0500 Subject: [PATCH] Added in Markdown files for Funcotator forum posts. Added readme about where the forum posts live. --- docs/funcotator/forum_info/README.md | 5 + .../forum_post_annotation_specifications.md | 513 ++++++++++++++ ...orum_post_human_reference_discrepancies.md | 379 +++++++++++ .../forum_info/forum_post_tutorial.md | 635 ++++++++++++++++++ 4 files changed, 1532 insertions(+) create mode 100644 docs/funcotator/forum_info/README.md create mode 100644 docs/funcotator/forum_info/forum_post_annotation_specifications.md create mode 100644 docs/funcotator/forum_info/forum_post_human_reference_discrepancies.md create mode 100644 docs/funcotator/forum_info/forum_post_tutorial.md diff --git a/docs/funcotator/forum_info/README.md b/docs/funcotator/forum_info/README.md new file mode 100644 index 00000000000..9d7ae879d29 --- /dev/null +++ b/docs/funcotator/forum_info/README.md @@ -0,0 +1,5 @@ +The Links to the forum posts are as follows: +* forum\_post\_annotation\_specifications.md - https://gatkforums.broadinstitute.org/dsde/discussion/23389/funcotator-annotation-specifications +* forum\_post\_human\_reference\_discrepancies.md - https://gatkforums.broadinstitute.org/dsde/discussion/23390/grch37-hg19-b37-humang1kv37-human-reference-discrepancies +* forum\_post\_tutorial.md - https://gatkforums.broadinstitute.org/gatk/discussion/11193/funcotator-information-and-tutorial + diff --git a/docs/funcotator/forum_info/forum_post_annotation_specifications.md b/docs/funcotator/forum_info/forum_post_annotation_specifications.md new file mode 100644 index 00000000000..6850996e1b3 --- /dev/null +++ b/docs/funcotator/forum_info/forum_post_annotation_specifications.md @@ -0,0 +1,513 @@ + +## 0 - Introduction +This page details the specification of the annotations that [Funcotator](https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_funcotator_Funcotator.php "Funcotator") can create. + +Detailed information on [Funcotator](https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_funcotator_Funcotator.php "Funcotator") can be found in [this forum post](https://gatkforums.broadinstitute.org/dsde/discussion/11193/funcotator-information-and-tutorial "this forum post"). + + +### 0.1 - Table of Contents +0. [0.0 Introduction](#0) + 1. [0.1 Table of Contents](#0.1) +1. [1.0 Annotation Specification](#1) + 1. [1.1 Annotations for Pre-Packaged Data Sources](#1.1) + 1. [1.1.1 Gencode Annotation Specification](#1.1.1) + 1. [hugoSymbol](#f1) + 2. [ncbiBuild](#f2) + 3. [chromosome](#f3) + 4. [start](#f4) + 5. [end](#f5) + 6. [variantClassification](#f6) + 7. [secondaryVariantClassification](#f7) + 8. [variantType](#f8) + 9. [refAllele](#f9) + 10. [tumorSeqAllele1](#f10) + 11. [tumorSeqAllele2](#f11) + 12. [genomeChange](#f12) + 13. [annotationTranscript](#f13) + 14. [transcriptStrand](#f14) + 15. [transcriptExon](#f15) + 16. [transcriptPos](#f16) + 17. [cDnaChange](#f17) + 18. [codonChange](#f18) + 19. [proteinChange](#f19) + 20. [gcContent](#f20) + 21. [referenceContext](#f21) + 22. [otherTranscripts](#f22) + + +## 1.0 Annotation Specification + + +## 1.1 Annotations for Pre-Packaged Data Sources +The pre-packaged data sources will create a set of baseline, or default annotations for an input data set. +Most of these data sources copy and paste values from their source files into the output of Funcotator to create annotations. In this sense they are trivial data sources. + + +### 1.1.1 - Gencode Annotation Specification +Funcotator performs some processing on the input data to create the Gencode annotations. Gencode is currently required, so Funcotator will create these annotations for all input variants. + +The order and a specification of the Gencode annotations that Funcotator creates is as follows: + + + +1. **hugoSymbol** +Type: _String_ +The name of the gene in which the annotated variant allele occurs. If the variant allele occurs outside of any known gene boundaries, then this field is set to "Unknown". + + + +2. **ncbiBuild** +Type: _String_ +The reference which was used to create this Gencode annotation. Current valid values are: `hg19` or `hg38`. + + + +3. **chromosome** +Type: _String_ +The contig in which the variant occurs. Will always correspond to the contig in the variant position. + + + +4. **start** +Type: _Integer_ +The start position in genomic coordinates of the variant allele being annotated (1-based, inclusive). Will always correspond to the start in the variant position. + + +5. **end** +Type: _Integer_ +The end position in genomic coordinates of the variant allele being annotated (1-based, inclusive). Will always correspond to the position last base in the variant allele. + + + +6. **variantClassification** +Type: _String_ +The classification of the variant being annotated. Will always be one of the following: + +* `COULD_NOT_DETERMINE` + Variant classification could not be determined. +
+ +* `INTRON` + Variant lies between exons within the bounds of the chosen transcript. + Only valid for Introns. +
+ +* `FIVE_PRIME_UTR` + Variant is on the 5'UTR for the chosen transcript. + Only valid for UTRs. +
+ +* `THREE_PRIME_UTR` + Variant is on the 3'UTR for the chosen transcript + Only valid for UTRs. +
+ +* `IGR` + Intergenic region. Does not overlap any transcript. + Only valid for IGRs. +
+ +* `FIVE_PRIME_FLANK` + The variant is upstream of the chosen transcript + Only valid for IGRs. +
+ +* `THREE_PRIME_FLANK` + The variant is downstream of the chosen transcript + Only valid for IGRs. +
+ +* `MISSENSE` + The point mutation alters the protein structure by one amino acid. + Can occur in Coding regions or Introns. +
+ +* `NONSENSE` + A premature stop codon is created by the variant. + Can occur in Coding regions or Introns. +
+ +* `NONSTOP` + Variant removes stop codon. + Can occur in Coding regions or Introns. +
+ +* `SILENT` + Variant is in coding region of the chosen transcript, but protein structure is identical. + Can occur in Coding regions or Introns. +
+ +* `SPLICE_SITE` + The variant is within a configurable number of bases of a splice site. See the secondary classification to determine if it lies on the exon or intron side. + Can occur in Coding regions or Introns. +
+ +* `IN_FRAME_DEL` + Deletion that keeps the sequence in frame. + Can occur in Coding regions or Introns. +
+ +* `IN_FRAME_INS` + Insertion that keeps the sequence in frame. + Can occur in Coding regions or Introns. +
+ +* `FRAME_SHIFT_INS` + Insertion that moves the coding sequence out of frame. + Can occur in Coding regions or Introns. +
+ +* `FRAME_SHIFT_DEL` + Deletion that moves the sequence out of frame. + Can occur in Coding regions or Introns. +
+ +* `START_CODON_SNP` + Point mutation that overlaps the start codon. + Can occur in Coding regions. +
+ +* `START_CODON_INS` + Insertion that overlaps the start codon. + Can occur in Coding regions. +
+ +* `START_CODON_DEL` + Deletion that overlaps the start codon. + Can occur in Coding regions. +
+ +* `DE_NOVO_START_IN_FRAME` + New start codon is created by the given variant using the chosen transcript. + However, it is in frame relative to the coded protein, meaning that if the coding sequence were extended + then the new start codon would be in frame with the + existing start and stop codons. + + This can only occur in a 5' UTR. +
+ +* `DE_NOVO_START_OUT_FRAME` + New start codon is created by the given variant using the chosen transcript. + However, it is out of frame relative to the coded protein, meaning that if the coding sequence were extended + then the new start codon would NOT be in frame with + the existing start and stop codons. + + This can only occur in a 5' UTR. +
+ +* `RNA` + Variant lies on one of the RNA transcripts. + (special catch-all case) +
+ +* `LINCRNA` + Variant lies on one of the lincRNAs. + (special catch-all case) +
+ + + +7. **secondaryVariantClassification** +Type: _String_ +Additional variant classification information for variant alleles that have a `VariantClassification` of `SPLICE_SITE`. +For a variant allele with the `VariantClassification` of `SPLICE_SITE`, this will indicate the specific classification of the variant. +For all variants that do **not** have the `VariantClassification` of `SPLICE_SITE`, this will be the empty string. + + + +8. **variantType** +Type: _String_ +Basic information about the variant allele being annotated. Can be one of: +* `INS` - The variant allele is some kind of insertion. +* `DEL` - The variant allele is some kind of deletion. +* `SNP` - The variant allele is a single nucleotide polymorphism. +* `DNP` - The variant allele is a di-nucleotide polymorphism. +* `TNP` - The variant allele is a tri-nucleotide polymorphism. +* `ONP` - The variant allele is an oligo-nucleotide polymorphism (Synonymous with MNP). +* `MNP` - The variant allele is a multi-nucleotide polymorphism (Synonymous with ONP). +* `NA` - The variant allele type cannot be determined. + + + + +9. **refAllele** +Type: _String_ +The reference allele for the position at which this this variant allele occurs. +For insertions, this will be set to `-`. + + + +10. **tumorSeqAllele1** +Type: _String_ +Always the same as the reference allele. This field is a hold-over required for MAF annotations. +For insertions, this will be set to `-`. + + + +11. **tumorSeqAllele2** +Type: _String_ +The variant allele being annotated. This field only includes the bases that are different from the reference. +For the input VCF records, this field may slightly differ from the alternate allele reported in the base data for the `VariantContext`. +For deletions, this will be set to `-`. + + + +12. **genomeChange** +Type: _String_ +A _String_ summarizing the change resulting from this variant allele within the context of the whole genome sequence. +Generally the format of this field is: +`g.[CONTIG]:[POSITION][BASES CHANGED]` + +The format of this field slightly varies based on `VariantType`: + + +* **_Insertion_** + `g.[CONTIG]:[POSITION OF BASE PRIOR TO INSERTION]_[POSITION OF BASE AFTER INSERTION]ins[BASES INSERTED]` + E.g.: + `g.chr19:2018023_2018024insAATCG` + This indicates that the bases AATCG were inserted between bases 2018023 and 2018024 on chromosome 19. +
+ +* **_Deletion_** + `g.[CONTIG]:[POSITION OF BASE DELETED]del[BASE DELETED]` + E.g.: + `g.chr19:2018023delT` + This indicates that the base T was deleted at position 2018023 on chromosome 19. + _OR_ + `g.[CONTIG]:[POSITION OF FIRST BASE DELETED]_[POSITION OF LAST BASE DELETED]del[BASES DELETED]` + E.g.: + `g.chr19:2018023_2018025delTTG` + This indicates that the bases TTG were deleted starting at position 2018023 and ending at position 2018025 on chromosome 19. +
+ +* **_SNP_** + `g.[CONTIG]:[POSITION OF BASE ALTERED][REFERENCE BASE]>[ALTERNATE BASE]` + E.g.: + `g.chr19:2018023T>G` + This indicates that the base T was changed to G at position 2018023 on chromosome 19. +
+ +* **_MNP (including DNPs, TNPs)_** + `g.[CONTIG]:[POSITION OF FIRST BASE ALTERED]_[POSITION OF LAST BASE ALTERED][REFERENCE BASES]>[ALTERNATE BASES]` + E.g.: + `g.chr19:2018023_2018025TTG>GAT` + This indicates that the bases TTG were changed to GAT from position 2018023 to position 2018025 on chromosome 19. + + + + + +13. **annotationTranscript** +Type: _String_ +The ID of the transcript chosen for the detailed Gencode annotation reporting. +E.g.: `ENST00000435064.1` +If the variant allele does not occur within the bounds of any transcript (e.g. is of type `IGR`), then this field is empty. + + + +14. **transcriptStrand** +Type: _String_ +The strand direction associated with the transcript on which this variant allele occurs. +Either `+` or `-`. + + + +15. **transcriptExon** +Type: _Integer_ or Empty +The exon number on the transcript in which this variant allele occurs (1-based). Corresponds directly to the Gencode exon number. +If the variant does not occur in the expressed transcript of the corresponding gene (e.g. is of type `INTRON` or `IGR`), then this field is empty. + + + +16. **transcriptPos** +Type: _Integer_ or Empty +Position in the chosen transcript of the variant allele. All positions listed are 1-based and inclusive (meaning that the first base in the transcript starts at position 1). +For variant alleles that occur at a single base, the format is simply the position at which that variant occurs in the transcript (e.g. `1294`) +For variant alleles spanning multiple bases, the format is: +`[START]_[END]` +E.g.: `1236_1237` +If the variant does not occur in the expressed transcript of the corresponding gene (e.g. is of type`INTRON` or `IGR`), then this field is empty. + + + +17. **cDnaChange** +Type: _String_ +A _String_ that summarizes the change resulting from this variant allele in the coding sequence for the transcript in which it occurs. +Positions in this field are _**relative to the start of the transcript (1-based, inclusive)**_ unless otherwise noted. +Generally the format of this field is: +`c.[POSITION][BASES CHANGED]` + +The format of this field slightly varies based on `VariantType`, the number of affected bases, and whether the variant allele is a `SPLICE_SITE`: + + +* _**Insertions**_ + `c.[POSITION OF BASE PRIOR TO INSERTION]_[POSITION OF BASE AFTER INSERTION]ins[BASES INSERTED]` + E.g.: + `c.2018_2019insAA` + This indicates that the bases AA were inserted between bases 2018 and 2019 in the transcript associated with this variant allele. + +* _**Deletions of One Base**_ + `c.[POSITION OF BASE DELETED]del[BASE DELETED]` + E.g.: + `c2018delT` + This indicates that the base T was deleted at position 2018 in the transcript associated with this variant allele. + +* _**Deletions of Multiple Bases**_ + `c.[POSITION OF FIRST BASE DELETED]_[POSITION OF LAST BASE DELETED]del[BASES DELETED]` + E.g.: + `c2018_2022delTTCAG` + This indicates that the bases TTCAG were deleted from position 2018 to position 2022 in the transcript associated with this variant allele. + +* _**SNPs**_ + `c.[POSITION OF BASE CHANGED]>[NEW BASE]` + E.g.: + `c.1507T>G` + This indicates that the base T was changed to G at position 1507 in the transcript associated with this variant allele. + +* _**MNPs (including DNPs, TNPs)**_ + `c.[POSITION OF FIRST BASE CHANGED]_[POSITION OF LAST BASE CHANGED]>[NEW BASES]` + E.g.: + `c.12899_12900AG>TA` + This indicates that the bases AG were changed to TA from position 12899 to position 12900 in the transcript associated with this variant allele. + +* _**INTRONIC Variants**_ + For variants occuring in INTRONs, the cDNA string position indicates the offset from the exon boundary for the start of the variant. The whole string takes the form: + `c.e[EXON NUMBER][+|-][BASES FROM EXON][REF ALLELE]>[ALT ALLELE]` + E.g.: + `c.e81-4TAA>A` + This indicates that the bases TAA were changed to A starting four bases before exon 81 in the transcript associated with this variant allele. + + +If the variant does not occur in the expressed transcript of the corresponding gene (e.g. is of type `IGR`), then this field is empty. + + + +18. **codonChange** +Type: _String_ +A _String_ that representing the codon-aligned change resulting from this variant allele in the coding sequence for the transcript in which it occurs. +Positions in this field are relative to the start of the transcript (1-based, inclusive) and aligned to the coding sequenceunless otherwise noted. +Unlike the cDnaChange, the bases reported in the codonChange string will always have a length evenly divisble by 3 (except for frameshifts) and represent what the codons would be if the variant alternate allele were expressed in the reference sequence. +Capitalized bases represent the bases changed by the variant alternate allele. Lower-case bases represent reference bases. +Generally the format of this field is: +`c.[POSITION][BASES CHANGED]` +The format of this field slightly varies based on `VariantType`, the number of affected bases, and whether the variant allele occurs in an Intron: + + +* _**In-Frame Insertions**_ + `c.([POSITION OF FIRST BASE IN FIRST CODON IN THE REFERENCE AFFECTED BY THIS VARIANT]-[POSITION OF LAST BASE IN LAST CODON IN THE REFERENCE AFFECTED BY THIS VARIANT][REFERENCE CODONS]>[EXPRESSED CODONS]` + E.g.: + `c.(19-21)ctt>ctCGTt` + This indicates that the bases CGT were inserted before the 6th codon (starting at base 19, ending at base 21) in the transcript associated with this variant allele, and the resulting expressed codons would be `ctCGTt`. + +* _**In-Frame Deletions of Complete Codons (codon-aligned deletions)**_ + `c.([POSITION OF FIRST BASE IN FIRST CODON DELETED]-[POSITION OF LAST BASE IN LAST CODON DELETED][REFERENCE CODONS]del` + E.g.: + `c.(997-999)gcadel` + This indicates that the 332nd codon (starting at base 997, ending at base 999) was deleted in the transcript associated with this variant allele, and the deleted codon bases are `gca`. + +* _**In-Frame Deletions Spanning Multiple Codons**_ + `c.([POSITION OF FIRST BASE IN FIRST CODON DELETED]-[POSITION OF LAST BASE IN LAST CODON DELETED][REFERENCE CODONS]>[EXPRESSED CODONS]` + E.g.: + `c.(997-1002)gcactc>gtc` + This indicates that bases in the 332nd codon (starting at base 997) and 333rd codon (ending at base 1002) were deleted in the transcript associated with this variant allele, and the resulting expressed codon would be gtc. + +* _**Frame Shift Insertions and Deletions**_ + `c.([POSITION OF FIRST BASE IN LAST CORRECTLY EXPRESSED/REFERENCE CODON]-[POSITION OF LAST BASE IN LAST CORRECTLY EXPRESSED/REFERENCE CODON][REFERENCE CODONS]fs` + E.g.: + `c.(997-999)gcafs` + This indicates that bases just _**AFTER**_ the 332nd codon (starting at base 997, ending at base 999) were inserted or deleted in the transcript associated with this variant allele resulting in a frame shift, and that the last correctly transcribed codon would be codon 332 (starting at base 997, ending at base 999), `gca`. + +* _**SNPs / MNPs**_ + `c.([POSITION OF FIRST BASE IN FIRST CODON IN THE REFERENCE AFFECTED BY THIS VARIANT]-[POSITION OF LAST BASE IN LAST CODON IN THE REFERENCE AFFECTED BY THIS VARIANT][REFERENCE CODONS]>[EXPRESSED CODONS]` + E.g. 1: + `c.(39871-39873)cCC>cTT` + This indicates that the bases `CC`were changed to `TT` in the 13290th codon (starting at base 39871, ending at base 39873) in the transcript associated with this variant allele, and the resulting expressed codon would be `cTT`. + E.g. 2: + `c.(4-9)ctAAgc>ctGCgc` + This indicates that the bases `AA` starting in the 2nd codon (starting at base 4) and ending in the 3rd codon (ending at base 9) were changed to `GC` in the transcript associated with this variant allele, and the resulting expressed codons would be `ctGCgc`. + + + + + +19. **proteinChange** +Type: _String_ +A short string representing the predicted amino acid sequence change in the product of the gene transcript in which this variant alternate allele occurs. +Positions in this field are relative to the start of the amino acid sequence (1-based, inclusive) resulting from decoding the codons in the transcript in which this variant alternate allele occursunless otherwise noted. +Amino acid abbreviations are the standard letters as can be found in this table with the exception of the stop codon, which is represented by `*`. +It is important to note that the positions and amino acids reported in this string may not directly align to the codons in which the variant alternate allele occurs. This is most often due to the variant occurring in a set of tandem repeats which would cause the amino acid change to be "pushed" to the end of the tandem repeats. +For protein change strings in the Mitochondrial contig, the mitochondrial genetic code is used (rather than the standard code). +The format of this field takes two forms: + + +* **_Protein Changes with One Amino Acid Changed_** + `p.[REFERENCE AMINO ACID][POSITION][PREDICTED EXPRESSED AMINO ACID]` + E.g. 1: + `p.V5T` + The amino acid at protein position 5 was `V` (Valine) in the reference and would become `T` (Threonine) with the variant alternate allele expressed. + E.g. 2: + `p.R2R` + The amino acid at protein position 2 was `R` (Argenine) in the reference and would become `R` (Argenine) with the variant alternate allele expressed (no change in amino acid sequence / silent variant classification). + +* _**Protein Changes with Multiple Amino Acids Changed**_ + `p.[FIRST AFFECTED AMINO ACID POSITION]_[LAST AFFECTED AMINO ACID POSITION][REFERENCE AMINO ACIDS]>[PREDICTED EXPRESSED AMINO ACIDS]` + E.g.: + `p.100_101Q*>FL` + The amino acid sequence starting at protein position 100 and ending at protein position 101 was `Q*` (Glutamine,STOP) in the reference and would become `FL` (Phenylalanine,Leucine) with the variant alternate allele expressed. + +If the variant alternate allele does not occur in a coding region, this field will be empty. + + + +20. **gcContent** +Type: _Double_ +Represents the fraction of Guanine and Cytosine bases in a window of a given size around a variant. This window size does not include any bases in the variant alternate allele itself. By default the window size is 200 bases. + + + +21. **referenceContext** +Type: _String_ +The _**strand-correct**_ reference coding sequence in a given window around the reference allele. By default the window size is `10 bases`. +E.g. For the reference context around a variant with the reference allele `C` on the `+` strand: +``` + [REF ALLELE] + | + v + GAACCCACGTCGGTGAGGGCC + |________| |________| + v v + 10 bases 10 bases + (window size) (window size) +``` +_**Strand-correct**_ specifically means that if the strand of this transcript is determined to be `-` then the sequence is reverse complemented. +E.g. For the reference context around a variant with the reference allele `C` on the `-` strand: +``` + [REF ALLELE] + | + v + CACGAAAGTCGTTGCGGATCT + |________| |________| + v v + 10 bases 10 bases + (window size) (window size) +``` + + +22. **otherTranscripts** +Type: _String_ +A summary of the other transcripts in which this variant occurs, which were not chosen for detailed reporting due to the transcript selection scheme. +Each other transcript is represented by a condensed string that indicates how that transcript would be affected by this variant alternate allele. +Each other transcript takes the form: +`[HUGO SYMBOL]_[TRANSCRIPT ID]_[VARIANT CLASSIFICATION]_[PROTEIN CHANGE STRING]` +E.g.: +`SDF4_ENST00000263741.7_MISSENSE_p.R243Q` +If the other transcript does not have a protein change string, then that part is not rendered. +In the event that there are multiple other transcripts, these transcripts are separated by `/` +E.g.: +`SDF4_ENST00000263741.7_MISSENSE_p.R243Q/TNFRSF4_ENST00000379236.3_FIVE_PRIME_FLANK` +If this variant alternate allele occurs in only one transcript, this field will be empty. + + +Other annotations will follow the Gencode annotations and will be based on the data sources included in the data sources directory. +
+ diff --git a/docs/funcotator/forum_info/forum_post_human_reference_discrepancies.md b/docs/funcotator/forum_info/forum_post_human_reference_discrepancies.md new file mode 100644 index 00000000000..cde651c3c61 --- /dev/null +++ b/docs/funcotator/forum_info/forum_post_human_reference_discrepancies.md @@ -0,0 +1,379 @@ + +## 0 - Introduction +This page explains the discrepancies between the different "hg19" references. + +There are 4 common "hg19" references, and they are NOT directly interchangeable: +* **hg19** (`ucsc.hg19.fasta`, MD5sum: `a244d8a32473650b25c6e8e1654387d6`) +* **b37** (`Homo_sapiens_assembly19.fasta`, MD5sum: `886ba1559393f75872c1cf459eb57f2d`) +* **GRCh37** (`GRCh37.p13.genome.fasta`, MD5sum: `c140882eb2ea89bc2edfe934d51b66cc`) +* **humanG1Kv37** (`human_g1k_v37.fasta`, MD5sum: `0ce84c872fc0072a885926823dcd0338`) + + +### 0.1 - Table of Contents +0. [0.0 Introduction](#0) + 1. [0.1 Table of Contents](#0.1) +1. [hg19](#hg19) +2. [GRCh37](#grch37) +3. [b37](#b37) +4. [HumanG1Kv37](#humanG1Kv37) +5. [Reference Comparison Table](#comparison) +6. [Additional Information](#additional) + + +## GRCh37 +The Genome Reference Consortium Human Build 37, *GRCh37*, (`GRCh37.p13.genome.fasta`, MD5sum: `c140882eb2ea89bc2edfe934d51b66cc`) is a _Homo Sapiens_ genome reference file built by the Genome Reference Consortium. This is a baseline human genome reference and serves as the basis for the other three references in this comparison. + +For more information on GRCh37, visit the [official Genome Reference Consortium website](https://www.ncbi.nlm.nih.gov/grc). + + +## hg19 +The University of California at Santa Cruz (UCSC) has created a reference based on [GRCh37](#grch37). This reference is often referred to as *hg19* (`ucsc.hg19.fasta`, MD5sum: `a244d8a32473650b25c6e8e1654387d6`). + +This reference contains some alterations from the baseline reference from the Genome Reference Consortium. These alterations largely consist of contig name changes, however there are *known sequence differences on some contigs as well*. + +For details see the [comparison table](#comparison). + + +## b37 +The Broad Institute created a human genome reference file based on [GRCh37](#grch37). This reference is often referred to as *b37* (`Homo_sapiens_assembly19.fasta`, MD5sum: `886ba1559393f75872c1cf459eb57f2d`). + +**When people at The Broad Institute's Genomics Platform refer to the _hg19_ reference, they are actually referring to `b37`.** + +This reference contains some alterations from the baseline reference from the Genome Reference Consortium. These alterations largely consist of contig name changes, however there are *known sequence differences on some contigs as well*. + +Anecdotally the changes are for bases for which there was low confidence. Those low confidence bases were then masked out in the `b37` reference to be the `IUPAC` symbol for any base. However, _there does not seem to be a detailed comparison readily available_. + +For details see the [comparison table](#comparison). + + +## HumanG1Kv37 +The *humanG1Kv37* (`human_g1k_v37.fasta`, MD5sum: `0ce84c872fc0072a885926823dcd0338`) reference is equivalent to [b37](#b37), with the exception that it does not contain the decoy sequence for human herpesvirus 4 type 1 (named _NC_007605_). + + +## Reference Comparison Table + +The specific differences between these four references are detailed in the following table. + +The contigs with identical MD5sums are specified in each row. In the case that the MD5sum does not match between the references (indicating a sequence difference), the row will have a blank entry for that contig (`----`). + +Primary contigs with differing MD5sums are highlighted in red. +Alternate contigs with differing MD5sums are highlighted in orange. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
MD5HumanG1Kv37 ContigB37 ContigHG19 ContigGRCh37 Contig
06cbf126247d89664a4faebad130fe9cGL000202.1GL000202.1chr11_gl000202_randomGL000202.1
0996b4475f353ca98bacb756ac479140GL000244.1GL000244.1chrUn_gl000244GL000244.1
118a25ca210cfbcdfb6c2ebb249f9680GL000235.1GL000235.1chrUn_gl000235GL000235.1
131b1efc3270cc838686b54e7c34b17bGL000238.1GL000238.1chrUn_gl000238GL000238.1
1c1b2cd1fccbc0a99b6a447fa24d1504GL000226.1GL000226.1chrUn_gl000226GL000226.1
1d708b54644c26c7e01c2dad5426d38cGL000218.1GL000218.1chrUn_gl000218GL000218.1
1d78abec37c15fe29a275eb08d5af236GL000249.1GL000249.1chrUn_gl000249GL000249.1
2f8694fc47576bc81b5fe9e7de0ba49eGL000242.1GL000242.1chrUn_gl000242GL000242.1
3238fb74ea87ae857f9c7508d315babbGL000221.1GL000221.1chrUn_gl000221GL000221.1
325ba9e808f669dfeee210fdd7b470acGL000192.1GL000192.1chr1_gl000192_randomGL000192.1
399dfa03bf32022ab52a846f7ca35b30GL000223.1GL000223.1chrUn_gl000223GL000223.1
3e06b6741061ad93a8587531307057d8GL000232.1GL000232.1chrUn_gl000232GL000232.1
43f69e423533e948bfae5ce1d45bd3f1GL000206.1GL000206.1chr17_gl000206_randomGL000206.1
445a86173da9f237d7bcf41c6cb8cc62GL000240.1GL000240.1chrUn_gl000240GL000240.1
46c2032c37f2ed899eb41c0473319a69GL000214.1GL000214.1chrUn_gl000214GL000214.1
563531689f3dbd691331fd6c5730a88bGL000212.1GL000212.1chrUn_gl000212GL000212.1
569af3b73522fab4b40995ae4944e78eGL000199.1GL000199.1chr9_gl000199_randomGL000199.1
5a8e43bec9be36c7b49c84d585107776GL000248.1GL000248.1chrUn_gl000248GL000248.1
5d9ec007868d517e73543b005ba48535GL000195.1GL000195.1chr7_gl000195_randomGL000195.1
5eb3b418480ae67a997957c909375a73GL000215.1GL000215.1chrUn_gl000215GL000215.1
63945c3e6962f28ffd469719a747e73cGL000225.1GL000225.1chrUn_gl000225GL000225.1
642a232d91c486ac339263820aef7fe0GL000216.1GL000216.1chrUn_gl000216GL000216.1
6ac8f815bf8e845bb3031b73f812c012GL000194.1GL000194.1chr4_gl000194_randomGL000194.1
6d243e18dea1945fb7f2517615b8f52eGL000217.1GL000217.1chrUn_gl000217GL000217.1
6f5efdd36643a9b8c8ccad6f2f1edc7bGL000197.1GL000197.1chr8_gl000197_randomGL000197.1
6fe9abac455169f50470f5a6b01d0f59GL000222.1GL000222.1chrUn_gl000222GL000222.1
75e4c8d17cd4addf3917d1703cacaf25GL000200.1GL000200.1chr9_gl000200_randomGL000200.1
7daaa45c66b288847b9b32b964e623d3GL000211.1GL000211.1chrUn_gl000211GL000211.1
7de00226bb7df1c57276ca6baabafd15GL000247.1GL000247.1chrUn_gl000247GL000247.1
7e0e2e580297b7764e31dbc80c2540ddXXchrXchrX
7fed60298a8d62ff808b74b6ce820001GL000233.1GL000233.1chrUn_gl000233GL000233.1
851106a74238044126131ce2a8e5847cGL000210.1GL000210.1chr21_gl000210_randomGL000210.1
868e7784040da90d900d2d1b667a1383GL000198.1GL000198.1chr9_gl000198_randomGL000198.1
89bc61960f37d94abf0df2d481ada0ecGL000245.1GL000245.1chrUn_gl000245GL000245.1
93f998536b61a56fd0ff47322a911d4bGL000234.1GL000234.1chrUn_gl000234GL000234.1
96358c325fe0e70bee73436e8bb14dbdGL000203.1GL000203.1chr17_gl000203_randomGL000203.1
99795f15702caec4fa1c4e15f8a29c07GL000239.1GL000239.1chrUn_gl000239GL000239.1
9d424fdcc98866650b58f004080a992aGL000213.1GL000213.1chrUn_gl000213GL000213.1
a4aead23f8053f2655e468bcc6ecdcebGL000227.1GL000227.1chrUn_gl000227GL000227.1
aa81be49bf3fe63a79bdc6a6f279abf6GL000208.1GL000208.1chr19_gl000208_randomGL000208.1
b4eb71ee878d3706246b7c1dbef69299GL000230.1GL000230.1chrUn_gl000230GL000230.1
ba8882ce3a1efa2080e5d29b956568a4GL000231.1GL000231.1chrUn_gl000231GL000231.1
c5a17c97e2c1a0b6a9cc5a6b064b714fGL000228.1GL000228.1chrUn_gl000228GL000228.1
cc34279a7e353136741c9fce79bc4396GL000243.1GL000243.1chrUn_gl000243GL000243.1
d0f40ec87de311d8e715b52e4c7062e1GL000229.1GL000229.1chrUn_gl000229GL000229.1
d22441398d99caf673e9afb9a1908ec5GL000205.1GL000205.1chr17_gl000205_randomGL000205.1
d5b2fc04f6b41b212a4198a07f450e20GL000224.1GL000224.1chrUn_gl000224GL000224.1
d75b436f50a8214ee9c2a51d30b2c2ccGL000191.1GL000191.1chr1_gl000191_randomGL000191.1
d92206d1bb4c3b4019c43c0875c06dc0GL000196.1GL000196.1chr8_gl000196_randomGL000196.1
dbb6e8ece0b5de29da56601613007c2aGL000193.1GL000193.1chr4_gl000193_randomGL000193.1
dfb7e7ec60ffdcb85cb359ea28454ee9GL000201.1GL000201.1chr9_gl000201_randomGL000201.1
e0c82e7751df73f4f6d0ed30cdc853c0GL000237.1GL000237.1chrUn_gl000237GL000237.1
e4afcd31912af9d9c2546acf1cb23af2GL000246.1GL000246.1chrUn_gl000246GL000246.1
ef4258cdc5a45c206cea8fc3e1d858cfGL000241.1GL000241.1chrUn_gl000241GL000241.1
efc49c871536fa8d79cb0a06fa739722GL000204.1GL000204.1chr17_gl000204_randomGL000204.1
f3814841f1939d3ca19072d9e89f3fd7GL000207.1GL000207.1chr18_gl000207_randomGL000207.1
f40598e2a5a6b26e84a3775e0d1e2c81GL000209.1GL000209.1chr19_gl000209_randomGL000209.1
f977edd13bac459cb2ed4a5457dba1b3GL000219.1GL000219.1chrUn_gl000219GL000219.1
fc35de963c57bf7648429e6454f1c9dbGL000220.1GL000220.1chrUn_gl000220GL000220.1
fdcd739913efa1fdc64b6c0cd7016779GL000236.1GL000236.1chrUn_gl000236GL000236.1
1b22b98cdeb4a9304cb5d48026a8512811chr1chr1
a0d9851da00400dec1098a9255ac712e22chr2chr2
23dccd106897542ad87d2765d28a19a144chr4chr4
0740173db9ffd264d728f32784845cd755chr5chr5
1d3a93a248d92a729ee764823acbbc6b66chr6chr6
618366e953d6aaad97dbe4777c29375e77chr7chr7
96f514a9929e410c6651697bded59aec88chr8chr8
3e273117f15e0a400f01055d9f39376899chr9chr9
988c28e000e84c26d552359af1ea2e1d1010chr10chr10
98c59049a2df285c76ffb1c6db8f8b961111chr11chr11
51851ac0e1a115847ad36449b00158641212chr12chr12
283f8d7892baa81b510a015719ca7b0b1313chr13chr13
98f3cae32b2a2e9524bc19813927542e1414chr14chr14
e5645a794a8238215b2cd77acb95a0781515chr15chr15
fc9b1a7b42b97a864f56b348b06095e61616chr16chr16
351f64d4f4f9ddd45b35336ad97aa6de1717chr17chr17
b15d4b2d29dde9d3e4f93d1d0f2cbc9c1818chr18chr18
1aacd71f30db8e561810913e0b72636d1919chr19chr19
0dec9660ec1efaaf33281c0d5ea2560f2020chr20chr20
2979a6085bfe28e3ad6f552f361ed74d2121chr21chr21
a718acaa6135fdca8357d5bfe94211dd2222chr22chr22
1fa3474750af0948bdf97d5a0ee52e51YY--------
6743bd63b3ff2b5b8985d8933c53290a----NC_007605--------
c68f52674c9fb33aef52dcf399755519MTMT----chrM
fdfd811849cc2fadebc929bb925902e533--------
094d037050cad692b57ea12c4fef790f--------chr6_qbl_hap6GL000255.1
18c17e1641ef04873b15f40f6c8659a4--------chr6_cox_hap2GL000251.1
1e86411d73e6f00a10590f976be01623--------chrYchrY
2a3c677c426a10e137883ae1ffb8da3f--------chr6_dbb_hap3GL000252.1
3b6d666200e72bcc036bf88a4d7e0749--------chr6_ssto_hap7GL000256.1
641e4338fa8d52a5b781bd2a2c08d3c3--------chr3chr3
9d51d4152174461cd6715c7ddc588dc8--------chr6_mann_hap4GL000253.1
d2ed829b8a1628d16cbeee88e88e39eb--------chrM----
d89517b400226d3b56e753972a7cad67--------chr17_ctg5_hap1GL000258.1
efed415dd8742349cb7aaca054675b9a--------chr6_mcf_hap5GL000254.1
fa24f81b680df26bcfb6d69b784fbe36--------chr4_ctg9_hap1GL000257.1
fe71bc63420d666884f37a3ad79f3317--------chr6_apd_hap1GL000250.1
0386df1d3476e6649f919195cc072fc7------------GL383574.1
03de7a950b56720768373120bbddf693------------GL383558.1
03f5fa89e52d0fe155d2e3968bf2eeb7------------GL339450.1
049056a72b5aee0b3f876ddf554f0208------------JH720447.1
063358c8e7f81361b959efab7b3f15cc------------GL383565.1
07f56906bd56829f146dc0bf4b158603------------GL383538.1
09ce2d45f1f973e347e22ad1e3cf06fb------------GL383568.1
09d4cb1070e1c521d6e86e7038824c1c------------GL383544.1
0aee7c3e4bcc4c942230508c7836069b------------GL582967.1
0bdfd2a40e1ceab32d71d6f1c9a6ca32------------JH636056.1
0c787911df2449cbba8609bebf897ecb------------GL383541.1
0d12851232bcd8250e6dd61e3e7fd6a2------------JH636054.1
0db85f8e0ff66a470b46801c9892e471------------JH806589.1
0de582e28ae8c127d978b43e12a4f499------------KE332500.1
0f0364ed52ebe7757feea96ce623239f------------GL383529.1
10b523cdfd4f3707276ec92f0f9cddfb------------JH159133.1
1135da7b213739cfb3bdf2741c0c8083------------KE332506.1
122be4e189778434d8845fd5fd2c9a6b------------GL949748.1
12406aad3f3da31bda9c21a1aa0e16b6------------GL383539.1
12a3180640a49f33c960eb12ca61a6c4------------GL383542.1
13a8dc0d93c1bf1ae397593eba841721------------JH806573.1
1452be48789c27311d94561610f6d5af------------JH159132.1
157eecba5817aa1781a7bc4a9b60f933------------JH720446.1
15a2182cf9d3a55a7809adadc0775e03------------JH636060.1
16197ace4bfafdc2354857b98fc2a794------------GL582971.1
1668b0eb03be297f66837b46b5b73ac7------------JH806600.2
16a9ef53c176dcd2cf029940cbc29382------------JH720452.1
18c01f6e62136005ce1b2f2f33173f02------------GL582974.1
18fd9605f12ec0982adcf9e908f53331------------GL383523.1
190b4d20cab29ddba5f2495a7117f7cf------------JH636055.1
1919a95f3ea48fde56ba925295086028------------GL383582.2
192dead6bc331a0dcbd1ba9d3d8a6f80------------JH806581.1
1b6fa375fdf382778e6645d822d12254------------GL339449.2
1d217e666c48ef7f2ec81946f4f4bfcc------------GL383521.1
1d2933992f087a832718c9de19d4ceab------------KE332499.1
20d5046bbd2a21729fdd64fa94bdd5a1------------GL949742.1
20da91baf79b2e14b605a8ebe1f3704e------------GL877872.1
2118ff7bca8f75acc4629ab88bae1c2e------------GL582968.1
23aea04f46682e2a2be1a5ff3934a9fe------------GL383540.1
24f6ccbfc261e62451042a9713be6280------------JH636059.1
2536c2286fdeb98404ac410dbf528a3e------------GL949745.1
260aca5e1ff29b6ed3d3cd9e438f4219------------JH636053.3
2948653361f974fbed3e26a4dfbf332c------------GL383528.1
2cfa9ec8f70be88f95411dac6efb24c1------------JH159141.2
2e0bec27cfa9b440c746be52187fab0b------------GL383560.1
2f4f58e3b3a95bed1132833156340778------------GL383555.1
2fc316247e162f76a01012bbd9b665e6------------JH806603.1
321b3324431ae40e90a4117ecc07e93c------------JH591185.1
32ceefe714becfd36f207c5bffca4ba7------------GL877871.1
3342f6c21fa2dc364925712e0d52ed2d------------JH159148.1
348782844074360bdc8b6416e16cc5d0------------GL383549.1
349e96f115f829409bd1087b5fb684ca------------GL383519.1
35889722e6212fc9499e06e630268101------------JH806588.1
369f03e72d44461eab4542c58f3b5dcc------------KE332501.1
384b5b32f0ea2cfd15ac268a2ce07909------------JH159146.1
3893d44dfad7ce35744b2bde1e43bbd3------------GL383576.1
38e72cd57edb75d967ac2613d61d297d------------GL383563.2
3b40b7fdb005a1ce00efaa3310148852------------KE332502.1
3c5f20fb0744b7658d37d4ed79a286d1------------GL383520.1
3dd30a7638c3a3c518fc15571546b1be------------GL877875.1
3e0825dd23c9fce74a88d863e33c42b7------------JH159149.1
40015159c7da8f06875bb558587e3f07------------GL383571.1
404580d8ad56ded0fb33642c8b99c28b------------GL383537.1
4112dee892050e18ad279b8ebdcc5d48------------GL383578.1
41cf432193561894813a30da6e682e5b------------KB021647.1
447fe0ff3103170150280c775095eebf------------JH806593.1
44d5da56e5ec6ae0b9ebd354e9b47cfa------------JH159150.3
474baa8f6c6684c55bbc2a10bfa84baf------------GL949747.1
4791ba11d768da2cc1346d37a558047a------------GL582972.1
485c442c93fe19514153702f0c84d952------------GL582966.2
4a3d54bda53308ca941d6d0e794b05cb------------GL383554.1
4ad67c3a4e85f8b2cd54a8ef2aab4426------------GL383557.1
4bc4f02a4fca2c9d70646455bee8066e------------JH806596.1
50fd52ddb8ad2b024fb8b83a5c90a642------------JH806577.1
5404455aab275489bc8e6c9fb3ead5cb------------GL383573.1
54abe159678a84e88ceb2d5271027628------------KB663609.1
5835d9de56b65cefb9406d104d64531e------------GL383536.1
5950c02594cedbdf0fea5e8335e7cf80------------JH591181.2
5b90c3ac4e5938b400fcc2c29f3017bc------------GL949744.1
5b9d9fb059071e552bba531b81bd3472------------KB663606.1
5c3a364520bf7ed46894abdce8f6e032------------GL877876.1
5eb6da458990f121fae13ff83a4bcbca------------JH806576.1
5f9dc3f86463d08a1383cca5f285b7ad------------GL383570.1
5fae03628eb9a445571bac107823b394------------KE332505.1
620913159e2fbd4e931ac120e3c584c9------------GL383526.1
659b65783878ace88f4c4b165f239363------------JH636052.4
675046e52613269a7c2e803525bb5a33------------JH720445.1
67f26a755ca4c6ca9a8f567d80d15fb9------------JH159131.1
68391fb8f16a37b63f607b76702de3b1------------GL383583.1
69490aa24b00717f2b11c095a5339516------------JH720454.3
6b604cf3e324680b72716e814d805944------------GL383530.1
6b862a953dfe724a1f48eaf12a3b948a------------JH806578.1
6c22616c927261b8e5fc90028c780f00------------JH720444.2
6cba57c0e509ab785d3869134979b668------------GL383548.1
6d728406957c5c7fb158dbdb7efef2b7------------GL383527.1
6d85d704338ba29941aca4d278c7eb4a------------KB663608.1
6fbc7007a5ff8aae8b28ca52dd6d5571------------GL383531.1
71a0d13c09c3e7ee64c7740e1425f20e------------JH591183.1
73b240dd73b8bddcab281e265c9d759a------------GL383559.2
73d39b5d51e6e2e8d9549bb85d7dae04------------JH806579.1
741179e4ee12c60fbcc6eba4a5c7695b------------JH806598.1
74cff045a9cd92b7f571a756f248d16a------------GL383524.1
7b556f03729e304a286c8d7ef0f0c10e------------GL383547.1
7b6d6d01c18e91fc07f727ade2450f46------------JH806580.1
7d007a35ff02e56325881c68bb17b565------------GL949752.1
7e0afbdc97540aa0b101228b7bd331fb------------JH806591.1
8213c58e2c1c22397f0ad9d0d901bbdf------------JH159135.2
856a46516332f58a35eeb4f84d17febc------------JH159142.2
883b29a1e5975e0f3139c183fbe2596d------------GL383581.1
8a92722deabdf885d1aebfa8881d5903------------GL949753.1
8ac2dc8046e4bd0d6d46e827ff05ecd1------------JH806575.1
8ac9fb9d942dba38bfd30f8d767f4bba------------JH159136.1
8b1d46e46d3083625eac92e9363773dd------------KB663607.2
8d13c3e7cbb2b7e1a3225c5a54fe8f44------------GL383569.1
8e1004755b0574b2f855130c943fbd8e------------KB663603.1
8e4f862d5b37504199902c6685b7fee5------------GL383533.1
8ede6eec21d781c22a9801f51433fcd6------------JH806584.1
8fc7aaa775b43df3d77c9782a140a981------------GL383572.1
902d62224f09e59cb9c6c44f71b5fca3------------GL383580.1
90ad438579d919fd20c42bb4f48de64b------------JH591184.1
9133580f75d0ffa745af12953d65a4db------------GL383550.1
93001afcfc8594885490513c4ffe243e------------GL582977.2
93a798f03267e553445c7456c6f7ee49------------JH159143.1
94409f94ca59e67f811cd36ab133a82c------------GL877873.1
955e16dfcdb2d28a334349dfa39f2ed4------------KB021645.1
976c3a7c4051dd9ce879833f4a764289------------JH806590.2
978987018f1a910273ebcc387e038de8------------GL383518.1
9bb3fbcd1fc9c35884e0987755c55667------------GL949741.1
9bed9883963242dd74b883218d5f17bf------------GL383532.1
9d197695e8a47d4c30c891a53a0fd588------------JH720451.1
9dcedb7219aa23057244ca9a446f01ac------------JH806592.1
9e1fc7ed55646756ce109b12b82ff192------------JH159147.1
a009cf3116a844d7b2d467e672931bc5------------GL383564.1
a0584071d5a8e88fda38d4cca38704cb------------KB663605.1
a0bce2b33eb96adcb750622527225e7d------------JH720443.2
a0f25165c6537c9861cc1231f710e99f------------GL383566.1
a1dab5e9bbedd3539ace29af1f9d6139------------JH806595.1
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a2ecd2eb53eb1737423d5a637e4374a9------------JH636058.1
a3bf927c2422ea0a661640669efd1081------------GL383577.1
a4053747fc0cf1e03fa6ae9cd5f821d0------------KB021648.1
aa5b0a15acec3c6177db764bd103d8a0------------JH720453.1
ab73a8d586ef4fc44dd063730b6aef39------------GL582973.1
abb9297c8b9dfc3013d416c803ff486c------------JH806601.1
ac9c384b2fc322b684128f1baf75785e------------JH591182.1
adb23c033121d433739de02cfa00c9fb------------JH806582.2
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ba6a3b1599661e674918200a8d1333d3------------JH806594.1
bc6f64b0c4c934c2cea52bbe98639c79------------JH806583.1
bc79d1abee7076ea672293e12bd7ccb9------------GL383517.1
bd742a610e4bbc28fc00aaf71dfdc15d------------KB021644.1
be51fd8c00d62c3efc077a8e882062a4------------JH806586.1
bed6a2667e8452a176e93e921e0c21f6------------GL383556.1
c27dc6fea378fecf178a44682257c25e------------GL383545.1
c28f12c6ee0dec4cc6995766a710960c------------GL383552.1
c6ff49147dedce02366d6ade10580611------------GL383534.2
c86ffa095c924372aa455e43e61c96e8------------JH159145.1
ca0e3270f27bbee944844e44ec76659d------------JH806602.1
caebc01e3f44f7b2a559179b0261b77e------------GL383535.1
cca1c60136ec678eeef374134cd07a90------------GL877877.2
cdab95f32513753b3c0add3014afad3b------------JH806587.1
d08cc284ad35f0bd1eafb443c23ad8bd------------JH159138.1
d0b63f9cef6c4d382e49636465eab851------------GL877870.2
d0caa7bf982cf1e6ca8c8b833f56a21c------------JH806597.1
d4e2cf05984db16a78c953b898f5a86e------------GL582969.1
d76e635e75bc038782fb3d0c195d33fb------------GL949746.1
d8ef242a7373ff5657c8311b92dabfde------------JH591186.1
d9015dd9a0916a98ed8ab99fd3cdd012------------GL383567.1
d96719c32333013a51c4d6d3261f984f------------GL383551.1
d97cf75e24ed1370388fedf523faa7ab------------KE332496.1
da648c938f1bb43b41d254bd9a015cfb------------JH159139.1
dada6dd12ec844a3a13f547f4946428e------------JH159140.1
dd0bc538e31f35af2073daec1f378147------------JH159144.1
dd784bb8074d6f5b949464ffea8c6901------------JH720449.1
dd8730d9d33765ff135fcfadb8810280------------GL383575.2
df3e809f9a87f792218db18db51f6ad4------------GL383522.1
e0da36f2d1d2c6092f13d5bee52537e0------------GL383516.1
e0e934bd79ff323b31f4c9b80fb80a5c------------JH159134.2
e11adfbb638e60f61d7e8ef6647f30f2------------JH720448.1
e2cd68e2099fbd7cee557d6a7910768f------------KB021646.2
e363729ea23dad7c6802e7b439b4f668------------KE332497.1
e5b96eb9510763261839281c198607dd------------GL582979.2
e5cd94b0e0668debf81b82f405597b28------------GL582970.1
e6c232469067e8cadfa852a2ea5513b7------------GL949749.1
e8c870267b2a5261edb9d51d0efd6469------------GL582975.1
ebf72aeb4d53f0fd56e2e72967751f8a------------GL383562.1
ed6bcd4459b3bc6b366ce00262952f57------------GL949743.1
ed6fb45e0a25c31903cbb0f78d9d487e------------GL383546.1
edf086bce359065367b105cae0abfeee------------GL383579.1
edf41bfaf2584364bb4c5a645d73d53c------------GL383553.2
f2bfb99f84f2dd2ea538fe69ee786a0d------------GL383525.1
f486a5a44493d2e6bf72bf95ae898e3c------------JH806574.2
f7ee47af8d462cd9aeb6d40de99acb36------------JH806585.1
fa5fa49d281fc855dd1076c4f51bd8dc------------KE332498.1
faa48b73103366d1da02065870a58bda------------GL383543.1
faae4c952e9c38254538e1853b786276------------GL582976.1
fc93038463f9660e139435537ef53a5c------------KB663604.1
fdeb8db11e8544a638179a592c051331------------JH636061.1
fef0bc815f4826ea408515d8ec74ca80------------JH636057.1
ff7c4316cb69a8d571bd7ef85c1a10e4------------JH720455.1
+ +This table indicates that while most contigs contain the same data, there are several with **sequence differences between the references**. Among those are **Chromosome 3**, **Chromosome Y**, and the **Mitochondrial Contig**. + +Anecdotally the changes are for bases for which there was low confidence, with those low confidence bases masked out to be the `IUPAC` symbol for any base. However, _there does not seem to be a detailed comparison readily available (i.e. there's no proof that this is true)_. + +Therefore, when doing comparisons across the four reference versions for each of these contigs, some care should be taken. + + +## Additional Information +Some further details can be found on [this DNAnexus wiki page](https://wiki.dnanexus.com/scientific-notes/human-genome) as of 2019/01/30. + +[Back to Top](#0) + +
diff --git a/docs/funcotator/forum_info/forum_post_tutorial.md b/docs/funcotator/forum_info/forum_post_tutorial.md new file mode 100644 index 00000000000..b3b01835ab8 --- /dev/null +++ b/docs/funcotator/forum_info/forum_post_tutorial.md @@ -0,0 +1,635 @@ + +## 0 - Introduction +This page explains what **Funcotator** is and how to run it. + + +### 0.1 - Table of Contents +0. [0.0 Introduction](#0) + 1. [0.1 Table of Contents](#0.1) +1. [1.0 Funcotator Background Information](#1) + 1. [1.1 Data Sources](#1.1) + 1. [1.1.1 Data Source Folders](#1.1.1) + 2. [1.1.2 Pre-Packaged Data Sources](#1.1.2) + 1. [1.1.2.1 Downloading Pre-Packaged Data Sources](#1.1.2.1) + 2. [1.1.2.2 gnomAD](#1.1.2.2) + 3. [1.1.3 Data Source Downloader Tool](#1.1.3) + 4. [1.1.4 Disabling Data Sourcesl](#1.1.4) + 5. [1.1.5 User-Defined Data Sources](#1.1.5) + 1. [1.1.5.1 Configuration File Format](#1.1.5.1) + 1. [1.1.5.1.1 Simple XSV Config File Example](#1.1.5.1.1) + 2. [1.1.5.1.2 Locatable XSV Config File Example](#1.1.5.1.2) + 2. [1.1.5.2 Cloud Data Sources](#1.1.5.2) + 6. [1.1.6 Data Source Versioning](#1.1.6) + 2. [1.2 Input Variant Data Formats](#1.2) + 3. [1.3 Output](#1.3) + 1. [1.3.1 Output Data Formats](#1.3.1) + 1. [1.3.1.1 VCF Format](#1.3.1.1) + 2. [1.3.1.2 MAF Format](#1.3.1.2) + 2. [1.3.2 Annotations for Pre-Packaged Data Sources](#1.3.2) + 1. [1.3.2.1 Gencode Annotation Specification](#1.3.2.1) + 4. [1.4 Reference Genome Versions](#1.4) + 5. [1.5 Comparisons with Oncotator](#1.5) + 1. [1.5.1 Funcotator / Oncotator Feature Comparison](#1.5.1) + 2. [1.5.2 Oncotator Bugs Compared With Funcotator](#1.5.2) +2. [2.0 Tutorial](#2) + 0. [2.0 Requirements](#2.0) + 1. [2.1 Running Funcotator in the GATK With Base Options](#2.1) + 2. [2.2 Optional Parameters](#2.2) + 1. [2.2.1 - --ignore-filtered-variants](#2.2.1) + 2. [2.2.2 - --transcript-selection-mode](#2.2.2) + 3. [2.2.1 - --transcript-list](#2.2.3) + 4. [2.2.2 - --annotation-default](#2.2.4) + 5. [2.2.1 - --annotation-override](#2.2.5) + 6. [2.2.2 - --allow-hg19-gencode-b37-contig-matching](#2.2.6) +3. [3.0 Known Issues](#3) +4. [4.0 Github](#4) +5. [5.0 Tool Documentation](#5) + +---- + + +## 1 - Funcotator Background Information + +Funcotator (**FUNC**tional ann**OTATOR**) analyzes given variants for their function (as retrieved from a set of data sources) and produces the analysis in a specified output file. + +This tool allows a user to add their own annotations to variants based on a set of data sources. Each data source can be customized to annotate a variant based on several matching criteria. This allows a user to create their own custom annotations easily, without modifying any Java code. + +An example Funcotator workflow based on the GATK Best Practices Somatic Pipeline is as follows: +![](https://us.v-cdn.net/5019796/uploads/editor/rt/fapszh63zvd0.png "") + + +### 1.1 - Data Sources +Data sources are expected to be in folders that are specified as input arguments. While multiple data source folders can be specified, **no two data sources can have the same name**. + + +### 1.1.1 - Data Source Folders + +In each main data source folder, there should be sub-directories for each individual data source, with further sub-directories for a specific reference (e.g. _hg19_, _hg38_, etc.). In the reference-specific data source directory, there is a configuration file detailing information about the data source and how to match it to a variant. This configuration file is required. + +An example of a data source directory is the following: +``` + dataSourcesFolder/ + Data_Source_1/ + hg19 + data_source_1.config + data_source_1.data.file.one + data_source_1.data.file.two + data_source_1.data.file.three + ... + hg38 + data_source_1.config + data_source_1.data.file.one + data_source_1.data.file.two + data_source_1.data.file.three + ... + Data_Source_2/ + hg19 + data_source_2.config + data_source_2.data.file.one + data_source_2.data.file.two + data_source_2.data.file.three + ... + hg38 + data_source_2.config + data_source_2.data.file.one + data_source_2.data.file.two + data_source_2.data.file.three + ... + ... +``` + +### 1.1.2 - Pre-Packaged Data Sources +The GATK includes two sets of pre-packaged data sources, allowing for [Funcotator](https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_funcotator_Funcotator.php "Funcotator") use without (much) additional configuration. +These data source packages correspond to the **germline** and **somatic** use cases. +Broadly speaking, if you have a **germline VCF**, the **germline data sources** are what you want to use to start with. +Conversely, if you have a somatic VCF, the somatic data sources are what you want to use to start with. + + +### 1.1.2.1 - Downloading Pre-Packaged Data Sources +Versioned gzip archives of data source files are provided here: +● FTP: [ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/funcotator/](ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/funcotator/) +● Google Cloud Bucket: [gs://broad-public-datasets/funcotator/](https://console.cloud.google.com/storage/browser/broad-public-datasets/funcotator) + + +### 1.1.2.2 - gnomAD +The pre-packaged data sources include gnomAD, a large database of known variants. gnomAD is split into two parts - one based on exome data, one based on whole genome data. +Due to the size of gnomAD, it cannot be included in the data sources package directly. Instead, the configuration data are present and point to a Google bucket in which +the gnomAD data reside. This will cause [Funcotator](https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_funcotator_Funcotator.php "Funcotator") to actively connect to that bucket when it is run. +For this reason, **gnomAD is disabled by default**. +To enable gnomAD, simply change directories to your data sources directory and untar the gnomAD tar.gz files: +``` +cd DATA_SOURCES_DIR +tar -zxf gnomAD_exome.tar.gz +tar -zxf gnomAD_genome.tar.gz +``` + +Because [Funcotator](https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_funcotator_Funcotator.php "Funcotator") will query the Internet when gnomAD is enabled, performance will be impacted by the machine's Internet connection speed. +If this degradation is significant, you can localize gnomAD to the machine running [Funcotator](https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_funcotator_Funcotator.php "Funcotator") to improve performance (however due to the size of gnomAD this may be impractical). + + +### 1.1.3 - Data Source Downloader Tool +To improve ease-of-use of [Funcotator](https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_funcotator_Funcotator.php "Funcotator"), there is a tool to download the pre-packaged data sources to the user's machine. +This tool is the **[FuncotatorDataSourceDownloader](https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_funcotator_FuncotatorDataSourceDownloader.php "FuncotatorDataSourceDownloader")** and can be run to retrieve the pre-packaged data sources from the google bucket and localize them to the machine on which it is run. +Briefly: +For **somatic** data sources: +``` +./gatk FuncotatorDataSourceDownloader --somatic --validate-integrity --extract-after-download +``` +For **germline** data sources: +``` +./gatk FuncotatorDataSourceDownloader --germline --validate-integrity --extract-after-download +``` + + +### 1.1.4 - Disabling Data Sources +A data source can be disabled by removing the folder containing the configuration file for that source. This can be done on a per-reference basis. If the entire data source should be disabled, the entire top-level data source folder can be removed. + + +### 1.1.5 - User-Defined Data Sources +Users can define their own data sources by creating a new correctly-formatted data source sub-directory in the main data sources folder. In this sub-directory, the user must create an additional folder for the reference for which the data source is valid. If the data source is valid for multiple references, then multiple reference folders should be created. Inside each reference folder, the user should place the file(s) containing the data for the data source. Additionally the user **must** create a _configuration file_ containing metadata about the data source. + +There are several formats allowed for data sources: + + + + + + + + +
Data Format ClassData Source Description
simpleXSVSeparated value table (e.g. CSV), keyed off Gene Name OR Transcript ID
locatableXSVSeparated value table (e.g. CSV), keyed off a genome location
gencodeClass for GENCODE data files (gtf format)
cosmicClass for COSMIC data
vcfClass for Variant Call Format (VCF) files
+ +Two of the most useful are arbitrarily separated value (XSV) files, such as comma-separated value (CSV), tab-separated value (TSV). These files contain a table of data that can be matched to a variant by _gene name_, _transcript ID_, or _genome position_. In the case of _gene name_ and _transcript ID_, one column must contain the gene name or transcript ID for each row's data. + +* For gene name, when a variant is annotated with a gene name that exactly matches an entry in the gene name column for a row, that row's other fields will be added as annotations to the variant. +* For transcript ID, when a variant is annotated with a transcript ID that exactly matches an entry in the transcript ID column for a row, that row's other fields will be added as annotations to the variant. +* For genome position, one column must contain the contig ID, another column must contain the start position (1-based, inclusive), and a column must contain the stop position (1-based, inclusive). The start and stop columns may be the same column. When a variant is annotated with a genome position that overlaps an entry in the three genome position columns for a row, that row's other fields will be added as annotations to the variant. + + +#### 1.1.5.1 - Configuration File Format +The configuration file is a standard Java properties-style configuration file with key-value pairs. This file name **must end in .config**. + + +##### 1.1.5.1.1 - Simple XSV + +The following is an example of a Locatable XSV configuration file (for the Familial Cancer Genes data source): + +``` +name = Familial_Cancer_Genes +version = 20110905 +src_file = Familial_Cancer_Genes.no_dupes.tsv +origin_location = oncotator_v1_ds_April052016.tar.gz +preprocessing_script = UNKNOWN + +# Whether this data source is for the b37 reference. +# Required and defaults to false. +isB37DataSource = false + +# Supported types: +# simpleXSV -- Arbitrary separated value table (e.g. CSV), keyed off Gene Name OR Transcript ID +# locatableXSV -- Arbitrary separated value table (e.g. CSV), keyed off a genome location +# gencode -- Custom datasource class for GENCODE +# cosmic -- Custom datasource class for COSMIC +# vcf -- Custom datasource class for Variant Call Format (VCF) files +type = simpleXSV + +# Required field for GENCODE files. +# Path to the FASTA file from which to load the sequences for GENCODE transcripts: +gencode_fasta_path = + +# Required field for GENCODE files. +# NCBI build version (either hg19 or hg38): +ncbi_build_version = + +# Required field for simpleXSV files. +# Valid values: +# GENE_NAME +# TRANSCRIPT_ID +xsv_key = GENE_NAME + +# Required field for simpleXSV files. +# The 0-based index of the column containing the key on which to match +xsv_key_column = 2 + +# Required field for simpleXSV AND locatableXSV files. +# The delimiter by which to split the XSV file into columns. +xsv_delimiter = \t + +# Required field for simpleXSV files. +# Whether to permissively match the number of columns in the header and data rows +# Valid values: +# true +# false +xsv_permissive_cols = true + +# Required field for locatableXSV files. +# The Name or 0-based index of the column containing the contig for each row +contig_column = + +# Required field for locatableXSV files. +# The Name or 0-based index of the column containing the start position for each row +start_column = + +# Required field for locatableXSV files. +# The Name or 0-based index of the column containing the end position for each row +end_column = +``` + + +##### 1.1.5.1.2 - Locatable XSV + +The following is an example of a Locatable XSV configuration file (for the ORegAnno data source): + +``` +name = Oreganno +version = 20160119 +src_file = oreganno.tsv +origin_location = http://www.oreganno.org/dump/ORegAnno_Combined_2016.01.19.tsv +preprocessing_script = getOreganno.py + +# Whether this data source is for the b37 reference. +# Required and defaults to false. +isB37DataSource = false + +# Supported types: +# simpleXSV -- Arbitrary separated value table (e.g. CSV), keyed off Gene Name OR Transcript ID +# locatableXSV -- Arbitrary separated value table (e.g. CSV), keyed off a genome location +# gencode -- Custom datasource class for GENCODE +# cosmic -- Custom datasource class for COSMIC +# vcf -- Custom datasource class for Variant Call Format (VCF) files +type = locatableXSV + +# Required field for GENCODE files. +# Path to the FASTA file from which to load the sequences for GENCODE transcripts: +gencode_fasta_path = + +# Required field for GENCODE files. +# NCBI build version (either hg19 or hg38): +ncbi_build_version = + +# Required field for simpleXSV files. +# Valid values: +# GENE_NAME +# TRANSCRIPT_ID +xsv_key = + +# Required field for simpleXSV files. +# The 0-based index of the column containing the key on which to match +xsv_key_column = + +# Required field for simpleXSV AND locatableXSV files. +# The delimiter by which to split the XSV file into columns. +xsv_delimiter = \t + +# Required field for simpleXSV files. +# Whether to permissively match the number of columns in the header and data rows +# Valid values: +# true +# false +xsv_permissive_cols = true + +# Required field for locatableXSV files. +# The Name or 0-based index of the column containing the contig for each row +contig_column = 1 + +# Required field for locatableXSV files. +# The Name or 0-based index of the column containing the start position for each row +start_column = 2 + +# Required field for locatableXSV files. +# The Name or 0-based index of the column containing the end position for each row +end_column = 3 +``` + + +#### 1.1.5.2 - Cloud Data Sources + +Funcotator allows for data sources with source files that live on the cloud, enabling users to annotate with data sources that are not physically present on the machines running Funcotator. +To create a data source based on the cloud, create a configuration file for that data source and put the cloud URL in as the src_file property (see [Configuration File Format](#1.1.5.1) for details). +E.g.: +``` + ... +src_file = gs://broad-references/hg19/v0/1000G_phase1.snps.high_confidence.b37.vcf.gz +... +``` + + + +### 1.1.6 - Data Source Versioning +Each release of the data sources contains a version number. Newer versions of Funcotator require minimum versions of data sources in order to run. If a new version of Funcotator is run with an older version of the data sources, an error will be thrown prompting the user to download a new release of the data sources. + +Similarly newer releases of the data source packages are not reverse compatible with older versions of Funcotator. However, in this case Funcotator may or may not throw an error or warning. + +To ensure compatibility when upgrading Funcotator, always download the latest data sources release. Similarly, when updating data sources make sure to update Funcotator to the latest version. + + +### 1.2 - Input Variant Data Formats +Currently Funcotator can only accept input variants in the form of a [_VCF_](https://samtools.github.io/hts-specs/VCFv4.2.pdf "_VCF_") file. + + +### 1.3 - Output + + +### 1.3.1 - Output Data Formats +Funcotator supports output in both [_VCF_](https://samtools.github.io/hts-specs/VCFv4.2.pdf "_VCF_") format and [_MAF_ ](https://wiki.nci.nih.gov/display/TCGA/Mutation+Annotation+Format+(MAF)+Specification "_MAF_ ") format. + + +#### 1.3.1.1 - VCF Output +[VCF](https://samtools.github.io/hts-specs/VCFv4.2.pdf "VCF") files will contain the annotations for each variant allele as part of a **custom _INFO_ tag** - `FUNCOTATION`. This custom tag will contain a pipe-separated (|) list of annotations for each alternate allele on a given line of the VCF. The VCF header will contain an INFO field comment line for the FUNCOTATION data describing the field name for each value in the pipe-separated list. For variants with multiple alternate alleles, the INFO field will contain multiple lists of annotations (each list separated by a comma), the order of which corresponds to the alternate allele being annotated. + +For example: + +``` +#fileformat=VCFv4.2 +... + #INFO= +... +#CHROM POS ID REF ALT QUAL FILTER INFO +chr19 8914955 . C A 40 . FUNCOTATION=No Value|broad.mit.edu +``` + +In this example, the variant has one alternate allele (_A_) with two fields (_\_dbSNP_Val_Status_ and _Center_). The values of the fields are: + + + + +
Field NameField Value
dbSNP_Val_StatusNo Value
Centerbroad.mit.edu
+ +For multiple alternate alleles: +``` +#fileformat=VCFv4.2 +... + #INFO= +... +#CHROM POS ID REF ALT QUAL FILTER INFO +chr7 273846 . C A,G 40 . FUNCOTATION=No Value|broad.mit.edu,Big Value Here|brandeis.edu +``` + +In this example, the variant has one alternate allele (_A_) with two fields (_\_dbSNP_Val_Status_ and _Center_). The values of the fields are: + + + + + + +
Alternate AlleleField NameField Value
AdbSNP_Val_StatusNo Value
ACenterbroad.mit.edu
GdbSNP_Val_StatusBig Value Here
GCenterbrandeis.edu
+ +This formatting is the result of limitations in the VCF file specification. + + +#### 1.3.1.2 - MAF Output +The [_MAF_ ](https://wiki.nci.nih.gov/display/TCGA/Mutation+Annotation+Format+(MAF)+Specification "_MAF_ ") format used in Funcotator is an extension of the standard TCGA MAF. It is based on the MAF format specified for Oncotator [here under Output Format](https://portals.broadinstitute.org/oncotator/help/ "here under Output Format"). While the actual columns can vary (due to different data sources being used to create annotations), columns 1-67 will generally be the same. + +In the case of a variant with multiple alternate alleles, each alternate allele will be written to a separate line in the MAF file. + + +### 1.3.2 - Annotations for Pre-Packaged Data Sources +The pre-packaged data sources will create a set of baseline, or default annotations for an input data set. +Most of these data sources copy and paste values from their source files into the output of Funcotator to create annotations. In this sense they are trivial data sources. + + +#### 1.3.2.1 - Gencode Annotation Specification +Funcotator performs some processing on the input data to create the Gencode annotations. Gencode is currently required, so Funcotator will create these annotations for all input variants. +See [this forum post](https://gatkforums.broadinstitute.org/dsde/discussion/23389/funcotator-annotation-specifications "this forum post") for the specification of Gencode annotations in Funcotator. + + +#### 1.4 - Reference Genome Versions +The two currently supported genomes for annotations **out of the box** are **hg19** and **hg38**. This is due to the pre-packaged Gencode data sources being for those two references. **Any reference genome with published Gencode data sources can be used.** + + +#### 1.4.1 - hg19 vs b37 Reference +The Broad Institute uses an alternate hg19 reference known as b37 for our sequencing. UCSC uses the baseline hg19 reference. These references are similar but different. + +Due to the Gencode data source being published by UCSC, the data sources all use the hg19 reference for hg19 data (as opposed to b37). Funcotator detects when user data is from the b37 reference and forces the use of the hg19 data sources in this case. The user is warned when this occurs. Generally speaking this is OK, but due to the differences in the sequence data it is possible that some erroneous data will be created. + +This effect has not yet been quantified, but in most cases should not be appreciable. For details, see [this forum post](https://gatkforums.broadinstitute.org/dsde/discussion/23390/grch37-hg19-b37-humang1kv37-human-reference-discrepancies "this forum post"). + + +#### 1.5 - Comparisons with Oncotator +Oncotator is an older functional annotation tool developed by The Broad Institute. Funcotator and Oncotator are fundamentally different tools with some similarities. Some comparison highlights between Oncotator and Funcotator are in the following two tables: + + +#### 1.5.1 - Funcotator / Oncotator Feature Comparison + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
FuncotatorOncotatorNotes
Override values for annotationsYesYes
Default values for annotationsYesYes
VCF inputYesYes
VCF outputYesYesAnnotation format b/w Funcotator and Oncotator differ.
MAF inputNoYes
MAF outputYesYes
TSV/maflite inputNoYes
Simple TSV outputNoYes
Removing datasources does not require developerYesYes
hg38 supportYesNo
Cloud datasourcesYesNoAll data sources supported
Transcript override listYesYes
Default config speed somatic (muts/min) (hg19)
Default config speed germline (muts/min) (hg19)A very long time....
Default config speed somatic (muts/min) (hg38)N/A
Default config speed germline (muts/min) (hg38)N/A
DocumentationTutorial; Specifications forum post; inclusion in workshop materialsMinimal support in forum
ManuscriptPlannedYes
HGVS supportNoYes
BigWig datasource supportNoLinux only
Seg file input/outputNoYes
Transcript modes: canonical and most deleterious effectYesYes
Transcript mode: ALLYesNo
Exclude annotations/columns on CLIYesYes
Automated datasource download toolYesNo
Automated tool for creating datasourcesNoYes
Web applicationNoYesUses old version of Oncotator and datasources
Config file to specify CLI argumentsYesNoGATK built-in command line arguments file
Simple MAF to VCF NoYes
Or
VCF to MAF conversion
Inferring ONPsNoYes (Not recommended)Mutect2 infers ONPs when calling variants.  This is not the job of a functional annotator.
Ignores filtered input variantsYesYes
Mitochondrial amino acid sequence renderingYesNo
gnomAD annotationsYes (cloud support)Not recommendedv2.1 support for hg19
V2.0.2 support for hg38 liftover coming soon
Must be manually enabled
UniProt ID annotationsYesYes
Other UniProt annotations (e.g. AAxform)NoYes
Custom fields: t_alt_count; t_ref_count; etcMAF Output OnlyYes
“other_transcripts” annotationYesYes
Reference context annotationsYesYes
COSMIC annotationsYesYes
UCSC ID annotationsYesYesIn Funcotator UCSC ID is part of the HGNC data source.
RefSeq ID annotationsYesYes
+ + +#### 1.5.2 - Oncotator Bugs Compared With Funcotator + + + + + + + + + + + + + + + +
Fixed in FuncotatorFixed in OncotatorNotes
Collapsing ONP counts into one numberN/ANo
Variants resulting in protein changes that do not overlap the variant codon itself are not rendered properlyYesNo
Appris ranking not properly sortedYesNo
Using protein-coding status of gene for sorting (instead of transcript)YesNo
De Novo Start in UTRs not properly annotatedYesNo
Protein changes for Frame-Shift Insertions on the Negative strand incorrectly renderedYesNo
MNP End positions incorrectly reportedYesNo
MNPs on the Negative strand have incorrect cDNA/codon/protein changesYesNo
For Negative strand indels; cDNA string is incorrectYesNo
Negative strand splice site detection boundary check for indels is incorrectYesNo
Inconsistent number of bases in reported reference context annotation for indelsYesNo
5’ Flanking variants are reported with an incorrect transcript chosen for Canonical modeYesNo
Variants overlapping both introns and exons or transcript boundaries are not rendered properlyNoNoFuncotator produces a ‘CANNOT_DETERMINE’ variant classification and minimal populated annotations.
+ + +## 2 - Tutorial + + +### 2.0 - Requirements +1. Java 1.8 +2. A functioning GATK4 jar +3. Reference genome (fasta files) with fai and dict files. Human references can be downloaded as part of the [GATK resource bundle](https://software.broadinstitute.org/gatk/download/bundle "GATK resource bundle"). Other references can be used but must be provided by the user. +4. A local copy of the [Funcotator data sources](#1.1 "Funcotator data sources") +5. A VCF file containing variants to annotate. + + +### 2.1 - Running Funcotator in the GATK With Base Options + +Open a command line and navigate to your GATK directory. +``` +cd ~/gatk +``` + +At this point you should choose your output format. There are [two output format choices](#1.3 "two output format choices"), one of which must be specified. + +Additionally, you must specify a reference version. This reference version is used verbatim to determine which data sources to use for annotations. That is, specifying `hg19` will cause Funcotator to look in the `/hg19` folder for data sources to use. + +A VCF instantiation of the Funcotator tool looks like this: +``` +./gatk Funcotator --variant variants.vcf --reference Homo_sapiens_assembly19.fasta --ref-version hg19 --data-sources-path funcotator_dataSources.v1.2.20180329 --output variants.funcotated.vcf --output-file-format VCF +``` + +A MAF instantiation of the Funcotator tool looks like this: +``` +./gatk Funcotator --variant variants.vcf --reference Homo_sapiens_assembly19.fasta --ref-version hg19 --data-sources-path funcotator_dataSources.v1.2.20180329 --output variants.funcotated.maf --output-file-format MAF +``` + + +### 2.2 - Optional Parameters + + +#### 2.2.1 - --ignore-filtered-variants +This flag controls whether Funcotator will annotate filtered variants. By **default, this flag is set to true.** +To annotate filtered variants, run Funcotator with this flag set to false: + +``` +./gatk Funcotator --variant variants.vcf --reference Homo_sapiens_assembly19.fasta --ref-version hg19 --data-sources-path funcotator_dataSources.v1.2.20180329 --output variants.funcotated.maf --output-file-format MAF --ignore-filtered-variants false +``` + + +#### 2.2.2 - --transcript-selection-mode +This parameter determines how the primary annotated transcript is determined. The two modes for this parameter are **BEST_EFFECT**, **CANONICAL**, and **ALL**. By **default, Funcotator uses the CANONICAL** transcript selection mode. + +The explanations and rules governing the two transcript selection modes are as follows: + +_**BEST_EFFECT**_ +Select a transcript to be reported with details with priority on effect according to the folowing list of selection criteria: +* Choose the transcript that is on the custom list specified by the user. If no list was specified, treat as if no transcripts were on the list (tie). +* In case of tie, choose the transcript that yields the variant classification highest on the variant classification rank list (see below). +* If still a tie, choose the transcript with highest level of curation. Note that this means lower number is better for level (see below). +* If still a tie, choose the transcript with the best appris annotation (see below). +* If still a tie, choose the transcript with the longest transcript sequence length. +* If still a tie, choose the first transcript, alphabetically. + +_**CANONICAL**_ + +Select a transcript to be reported with details with priority on canonical order according to the folowing list of selection criteria: + +* Choose the transcript that is on the custom list specified by the user. If no list was specified, treat as if all transcripts were on the list (tie). +* In case of tie, choose the transcript with highest level of curation. Note that this means lower number is better for level (see below). +* If still a tie, choose the transcript that yields the variant classification highest on the variant classification rank list (see below). +* If still a tie, choose the transcript with the best appris annotation (see below). +* If still a tie, choose the transcript with the longest transcript sequence length. +* If still a tie, choose the first transcript, alphabetically. + +_**ALL**_ +Same as CANONICAL, but indicates that no transcripts should be dropped. Render all overlapping transcripts. + + +#### 2.2.3 - --transcript-list +This parameter will restrict the reported/annotated transcripts to only include those on the given list of transcript IDs. This list can be given as the path to a file containing one transcript ID per line OR this parameter can be given multiple times each time specifying a transcript ID. + +_When specifying transcript IDs, **transcript version numbers will be ignored**._ + +Using a manually specified set of transcripts for the transcript list: +``` +./gatk Funcotator --variant variants.vcf --reference Homo_sapiens_assembly19.fasta --ref-version hg19 --data-sources-path funcotator_dataSources.v1.2.20180329 --output variants.funcotated.maf --output-file-format MAF --transcript-list TRANSCRIPT_ID1 --transcript-list TRANSCRIPT_ID2 +``` + +Using an equivalent transcript file: +``` +./gatk Funcotator --variant variants.vcf --reference Homo_sapiens_assembly19.fasta --ref-version hg19 --data-sources-path funcotator_dataSources.v1.2.20180329 --output variants.funcotated.maf --output-file-format MAF --transcript-list transcriptFile.txt +``` + +Contents of `transcriptFile.txt`: +``` +TRANSCRIPT_ID1 +TRANSCRIPT_ID2 +``` + + +#### 2.2.4 - --annotation-default +This parameter specifies a default value for an annotation. This default value for this annotation will be used for any annotated variant. However if this annotation would be added by Funcotator to this variant, the Funcotator value will overwrite this default. + +To specify this annotation default, the value on the command line takes the format: +`ANNOTATION_FIELD:value` + +For example, to set the _Center_ annotation to _broad.mit.edu_: +``` +./gatk Funcotator --variant variants.vcf --reference Homo_sapiens_assembly19.fasta --ref-version hg19 --data-sources-path funcotator_dataSources.v1.2.20180329 --output variants.funcotated.maf --output-file-format MAF --annotation-default Center:broad.mit.edu +``` + +It is valid to provide both the`--annotation-default` and `--annotation-override` arguments to Funcotator, however the behavior of specifying an annotation-default and an annotation-overrid for the _same annotation field_ is undefined. + + +#### 2.2.5 - --annotation-override +This parameter specifies an override value for an annotation. If the annotation were to be added to a variant by a data source, the value for that annotation would be replaced with the value specified in the annotation override. If the annotation would not be added by a data source it is added to the output with the given value. + +To specify this annotation default, the value on the command line takes the format: +`ANNOTATION_FIELD:value` + +For example, to override the _NCBI_Build_ annotation to _HG19_: +``` +./gatk Funcotator --variant variants.vcf --reference Homo_sapiens_assembly19.fasta --ref-version hg19 --data-sources-path funcotator_dataSources.v1.2.20180329 --output variants.funcotated.maf --output-file-format MAF --annotation-override NCBI_Build:HG19 +``` + +It is valid to provide both the `--annotation-override` and `--annotation-default` arguments to Funcotator, however the behavior of specifying an annotation-override and an annotation-default for the _same annotation field_ is undefined. + + +#### 2.2.6 - --allow-hg19-gencode-b37-contig-matching +This flag will cause hg19 contig names to match b37 contig names, allowing a set of variants created on an hg19 reference to match a b37 reference and visa-versa. + +hg19 was created by UCSC. b37 was created by the Genome Reference Consortium. In practice these references are _very_ similar but have small differences in certain bases, as well as a different naming convention for chromosomal contigs (`chr1` in hg19 vs `1` in b37). In 99.9% of cases the results will be identical, however for certain genomic regions the results will differ. + +This flag **defaults to _true_.** + +To run Funcotator without this hg19/b37 matching: +``` +./gatk Funcotator --variant variants.vcf --reference Homo_sapiens_assembly19.fasta --ref-version hg19 --data-sources-path funcotator_dataSources.v1.2.20180329 --output variants.funcotated.maf --output-file-format MAF --allow-hg19-gencode-b37-contig-matching false +``` + + +## 3 - Known Issues +The current list of known open issues can be found on the GATK github page [here](https://github.com/broadinstitute/gatk/labels/Funcotator). + + +## 4 - Github +Funcotator is developed as part of GATK. The GATK github page is [here](https://github.com/broadinstitute/gatk/). + + +## 5 - Tool Documentation +Tool documentation is written in the source code for Funcotator to better explain the options for running and some details of its features. +The tool documentation for Funcotator is [here](https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_funcotator_Funcotator.php). + +---- + +[Back to Top](#0) + +