Contents
Introduction
Anno is short for "annotation", it is used for annotate variants. Our goal is to provide abundant information for genetic variants promptly. Some annotation software output only one type of annotation, while Anno will provide annotations to all transcripts of a gene. Anno support various file format: VCF file, plain file, plink association output file and epacts file.
Quick tutorial
You have an input file in VCF format, and your goal is to annotate it using refFlat genes database. Then you just need the following command:
anno -i input.vcf -o output.vcf -r hs37d5.fa -g refFlat_hg19.txt.gz -p priority.txt -c codon.txt
Required files:
hs37d5.fa Reference genome in NCBI build 37 (you also need to download hs37d5.fai )
refFlat_hg19.txt.gz Gene database in refFlat format (from UCSC website). You can also use Gencode version 7 or Gencode version 11.
codon.txt Human codon table.
priority.txt Priority file, which determines wich annotation type is more important
Outputs:
Annotated VCF will be named output.vcf. Annotations comes in two tags: ANNO and ANNOFULL. The first tag ANNO showed the most important annotation types (determined by priority file); the second tag ANNOFULL showed detailed annotations. Let's see one example annotation:
ANNO=Nonsynonymous:GENE1|GENE3;ANNOFULL=GENE1/CODING_GENE:+:Nonsynonymous(CCT/Pro/P->CAT/His/H:Base3/30:Codon1/10:Exon1/5):Normal_Splice_Site:Exon|GENE3/CODING_GENE:-:Nonsynonymous(AGG/Arg/R->ATG/Met/M:Base30/30:Codon10/10:Exon5/5):Normal_Splice_Site:Exon|GENE2/NON_CODING_GENE:+:Upstream
ANNO tag displayed the most important variant type is Nonsynonymous and that happened at GENE1 and GENE3; ANNOFULL tag is the full set of annotation and it consists of two parts, for GENE1 and GENE3 respectively. The first part is for GENE1:
GENE1/CODING_GENE:+:Nonsynonymous(CCT/Pro/P->CAT/His/H:Base3/30:Codon1/10:Exon1/5):Normal_Splice_Site:Exon
The format can be explained by sections, and we use annotation for GENE1 as an example:
- GENE1 : gene name
- CODING_GENE : transcript name
- + : forward strand
- Nonsynonymous, Normal_Splice_Site, Exon : various annotation types
- CCT/Pro/P->CAT/His/H : Proline to Histidine mutation
- Base3/30 : mutation happens on the 3rd base of the total 10 bases
- Codon1/10 : mutation happens on the 1st codon of the total 10 codons
- Exon1/5 : mutation happens on the 1st exon of the total 5 exons
Console information:
When anno finished annotation successfully, you will see it output necessary information as below. You will find basic summary of reference genome, ts/tv ratio, total number of variants and so on.
The following parameters are available. Ones with "[]" are in effect:
Available Options
Required Parameters : -i [input.vcf], -o [output.vcf]
Gene Annotations :
-g [/net/fantasia/home/zhanxw/anno/resources/refFlat_hg19.txt.gz]
-r [/net/fantasia/home/zhanxw/anno/resources/hs37d5.fa]
--inputFormat [], --checkReference, -f [], -p []
-c [], -u [], -d [], --se [], --si []
Other Annotations : --genomeScore [], --bed [], --tabix []
Auxillary Functions : --outputFormat [], --indexOutput
Use default priority file: /net/fantasia/home/zhanxw/anno/priority.txt
Use default codon file: /net/fantasia/home/zhanxw/anno/codon.txt
Load reference genome /net/fantasia/home/zhanxw/anno/resources/hs37d5.fa...
DONE: 86 chromosomes and 3137454505 bases are loaded.
Load codon file /net/fantasia/home/zhanxw/anno/codon.txt...
DONE: codon file loaded.
Load priority file /net/fantasia/home/zhanxw/anno/priority.txt...
DONE: 25 priority annotation types loaded.
Load gene file /net/fantasia/home/zhanxw/anno/resources/refFlat_hg19.txt.gz...
DONE: 39173 gene loaded.
DONE: Generated frequency of each annotype type in [ output.vcf.anno.frq ].
DONE: Generated frequency of each highest priority annotation type in [ output.vcf.top.anno.frq ].
Ts/Tv ratio: 0.666667
Ts observed: 4 times; Tv observed: 6 times.
DONE: Generated frequency of each base change in [ output.vcf.base.frq ].
DONE: Generated frequency of each codon change in [ output.vcf.codon.frq ].
DONE: Generated frequency of indel length in [ output.vcf.indel.frq ].
DONE: 59 varaints are annotated.
DONE: Generated annotation output in [ output.vcf ].
Annotation succeed!
Where to Find It
Anno code is hosted online anno. You can download the source and compile (type 'make release').
For CSG internal users, the compiled executable file is at: /net/fantasia/home/zhanxw/anno/executable/anno
The source code is located at:/net/fantasia/home/zhanxw/anno You can type 'make release' to compile your own executable file. Type "make test1" or "make test2" will demonstrate the command line to annotate example VCF files.
Usage
Command line
After you obtain the anno executable (either by compiling the source code or by downloading the pre-compiled binary file), you will find the executable file under executable/anno.
Here is the anno help page by invoking anno without any command line arguments:
some_linux_host > executable/anno
..............................................
... G(ene) A(nnotation) ...
... Xiaowei Zhan, Goncalo Abecasis ...
... zhanxw@umich.edu ...
... Dec 2012 ...
................................................
Required Parameters
-i : Specify input VCF file
-o : Specify output VCF file
Gene Annotation Parameters
-g : Specify gene file
-r : Specify reference genome position
--inputFormat : Specify format (default: vcf). "-f plain " will use fir
st 4 columns as chrom, pos, ref, alt
--checkReference : Check whether reference alleles matches genome reference
-f : Specify gene file format (default: refFlat, other optio
ns: knownGene, refGene)
-p : Specify priority of annotations
-c : Specify codon file (default: codon.txt)
-u : Specify upstream range (default: 50)
-d : Specify downstream range (default: 50)
--se : Specify splice into extron range (default: 3)
--si : Specify splice into intron range (default: 8)
--outputFormat : Specify predefined annotation words (default or epact)
Other Annotation Tools
--genomeScore : Specify the folder of genome score (e.g. GERP=dirGerp/,
SIFT=dirSift/)
--bed : Specify the bed file and tag (e.g. ONTARGET1=a1.bed,ONT
ARGET2=a2.bed)
--tabix : Specify the tabix file and tag (e.g. abc.txt.gz(chrom=1
,pos=7,ref=3,alt=4,SIFT=7,PolyPhen=10)
Please specify input file
Input files
anno runs on the input VCF file specified on the command-line using flag '-i'.
Additionally, you need to specify gene file using flag '-g'. You can use the default refFlat file (using HG19 genome build): /net/fantasia/home/zhanxw/anno/refFlat_hg19.txt.gz
Parameters
Some of the command line parameters are described here, but most are self explanatory.
- Reference genome file
-r Specify a FASTA format reference genome file.
Specify -r option enable anno to give more detailed information, for example, instead of annotating a variant as exon, it will tell you which codon, which exon that variant locates, whether its synonymous/non-synonymous and etc.
Anno requires Fasta index file and that will save running memory and speed up annotation. You can use "samtools faidx XXX.fa" to generate Fasta index file.
For example, you can specify Fasta file of the whole genome and use "-r /data/local/ref/karma.ref/human.g1k.v37.fa"
- Gene file format
Currently, anno support gene file in refFlat format. A prepared list of all gene obtained of UCSC website is: "/net/fantasia/home/zhanxw/anno/refFlat_hg19.txt.gz" . To use that file, you use flag -g.
As anno support refFlat file by default, you can use refFlat format without specify gene format flag -f.
To use knownGene or refGene format, you need to specify both -g and -f flat to tell anno which gene file and which format it is. For example, -g /net/fantasia/home/zhanxw/anno/knownGene.txt.gz -f knownGene.
- Codon file
Codon file can tell the relationship between triplet codons and amino acids. A default file is located in: /net/fantasia/home/zhanxw/anno/codon.txt. If you have special codon file, you can specify that using flag -c, otherwise, anno will use the default codon file:
default codon file
# DNA codon table. Stop codon is coded as: Stp, O, Stop
#Codon AminoAcid Letter FullName
AAA Lys K Lysine
AAC Asn N Asparagine
AAG Lys K Lysine
AAT Asn N Asparagine
ACA Thr T Threonine
...
- Annotation ranges
-u how far apart from 5'-end of the gene are counted as upstream
-d how far apart from 3'-end of the gene are counted as downstream
The -u and -d parameters define the range of upstream and downstream.
-se how far apart from 5'-end of the gene are counted as upstream
-si how far apart from 3'-end of the gene are counted as upstream
The -se and -si defines the splice region. By default, 3 bases into the exon and 8 bases into the introns are defined as splicing sites. If mutations happen in these regions, we will annotate it as "Normal_Splice_Site" unless the mutations happens in the traditionally important "GU...AG" region in the intron, in that case, we will "Essential_Splice_Site" as annotation.
- Annotate by range
--bed : Specify the bed file and tag (e.g. ONTARGET1=a1.bed,ONTARGET2=a2.bed)
BED file is commonly used to represent range. Here you will check if certain genome position is contained in one or more ranges specified in the BED format. An example BED file, example.bed, is as follows:
1 10 20 a
1 15 40 b
There are two ranges: range a is from chromsome 1 position 10 (inclusive) to chromosome 1 position 20 (exclusive); range b is from chromsome 1 position 15 (inclusive) to chromosome 1 position 40 (exclusive).
When use --bed ONTARGET=example.bed as parameter, the output may look like this: ONTARGET=a,b in the VCF INFO field.
- Annotate by genome score
--genomeScore : Specify the folder of genome score (e.g. GERP=dirGerp/,SIFT=dirSift/)
Genome score is a special binary file format (designed by Hyun) storing per-position genome score, such as GERP score, SIFT score. You will need to pre-process those scores in a directory. To annotate genome score, use --genomeScore TAG_NAME=DIRECTORY. In VCF output file, you will have TAG_NAME=some_value in the INFO field. In other output format, you will see a separate column.
- Annotate by tabix input
--tabix : Specify the tabix file and tag (e.g. abc.txt.gz(chrom=1,pos=7,ref=3,alt=4,SIFT=7,PolyPhen=10)
Tabix file can be used in annotation. Here we required you provide bgzipped, indexed file. In above example, we use abc.txt.gz as input, and column 1,7,3,4 as chromosome, position, reference allele, alternative allele. We also use column 7 as SIFT score and column 10 as PolyPhen score. Similar syntax as previous, you will get outputs similar to 'SIFT=0.110;PolyPhen=0.00' in the VCF INFO field. NOTE, in bash, please use quote around parenthesis, otherwise parenthesis is not correctly treated. A working example is as follows:
--tabix '/net/fantasia/home/hmkang/bin/annovar/humandb/hg19_ljb_all.txt.gz(chrom=1,pos=2,ref=4,alt=5,mySift=6,mySC=7,myPP2=8,myPC=9)'
Example
anno can annotate a VCF file and also output statistics of 4 frequency table: annotation type; base change; codon change; indel size. More details will be given below.
Built-in example
In example/ folder, you can see test.vcf, which is a toy example. You can invoke anno using the following command line:
cd example; ./anno -i test.vcf -r test.fa -g test.gene.txt -c ../codon.txt -o test.out.vcf
Sample outputs are listed below:
\1) Annotated VCF file, test.out.vcf
#VCF_test
#from http://www.sph.umich.edu/csg/liyanmin/vcfCodingSnps/Tutorial.shtml
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA12891 NA12892 NA12878
1 3 . A G 50 depth=20 ANNO=GENE1/CODING_GENE:+:Exon:Utr5:Normal_Splice_Site|GENE3/CODING_GENE:-:Exon:Utr3:Normal_Splice_Site|GENE2/NON_CODING_GENE:+:Upstream GT:GQ:GD 1/0:31:12 0/0:28:14
1 5 . C A 50 depth=20 ANNO=GENE1/CODING_GENE:+:Exon:Nonsynonymous(CCT/Pro/P->CAT/His/H:Base3/30:Codon1/10:Exon1/5):Normal_Splice_Site|GENE3/CODING_GENE:-:Exon:Nonsynonymous(AGG/Arg/R->ATG/Met/M:Base30/30:Codon10/10:Exon5/5):Normal_Splice_Site|GENE2/NON_CODING_GENE:+:Upstream GT:GQ:GD 1/0:31:12 0/0:28:14
...
The annotation results are stored in the INFO column after ANN= tag. The annotation format is defined as following:
"|" separates different transcripts, e.g. in the first line, chromosome 1 position 3, there are 3 annotations: "GENE1/CODING_GENE:+:Exon:Utr5:Normal_Splice_Site" and "GENE3/CODING_GENE:-:Exon:Utr3:Normal_Splice_Site" and "GENE2/NON_CODING_GENE:+:Upstream"
":" separates within gene annotation in the following order: gene, strand, exon/intron, details.
\2) Statistics files:
Four frequency table will be generated after annotation. For example:
test.out.vcf.anno.frq
Stop_Loss 1
Utr5 2
Utr3 2
CodonRegion 2
CodonGain 2
Frameshift 2
Synonymous 3
StructuralVariation 3
Noncoding 3
Nonsynonymous 4
Deletion 6
Upstream 6
Insertion 6
Essential_Splice_Site 8
Downstream 8
Intron 12
Exon 21
Normal_Splice_Site 25
test.out.vcf.base.frq
A->G 1
T->C 1
T->G 2
A->C 2
C->A 5
test.out.vcf.codon.frq
Arg->Met 1
Pro->Thr 1
Arg->Arg 1
Pro->His 1
Gly->Gly 1
Pro->Pro 1
Stp->Tyr 1
Leu->Val 1
test.out.vcf.indel.frq
1 1
-4 1
3 1
-3 1
Contact
Questions and requests should be sent to Xiaowei Zhan (zhanxw@umich.edu) or Goncalo Abecasis (goncalo@umich.edu)
Author sincerly appreicate Yanming Li for his wonderful tutorial on gene annotation software vcfCodingSnps, and Hyun Ming Kang for his code related to genome scores and his consistent suggestions and feedbacks.