[SnpEff] SnpEff 사용방법2 VCF annotation, SnpEff ANN field, Sequence Ontology term 설명

안녕하세요 한주현 입니다.

오늘은 SnpEff 로 VCF annotation하는 간단한 명령어와 annotation된 VCF의 컬럼에서 ANN field 중 annotation, Annotation_Impact 컬럼에 대해 알아보겠습니다.

SnpEff 설치 방법에 대해 찾으신다면 다음 링크를 참고해주세요

http://korbillgates.tistory.com/61

SnpEff 사용방법1 - 다운로드 및 설치, vcf annotation

위의 링크에서도 알아봤듯이 VCF annotation 하는 명령어를 간단히 리뷰해보도록 하겠습니다.

SnpEff로 VCF annotation 하는 방법

java -jar snpEff.jar hg19 in.vcf > out.vcf

java 1.7이상 에서 작동하며 라인이 많으면 많은 메모리가 필요할 수 있습니다.

SnpEff로 annotated 된 VCF를 관찰해볼까요?

Annotation 하기 전 VCF

#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  NA00001
20      14370   .       G       A       29      PASS    NS=3;DP=14;AF=0.5;DB;H2 GT:GQ:DP:HQ     0|0:48:1:51,51

Annotation 결과 VCF

##SnpEffVersion="4.1g (build 2015-05-17), by Pablo Cingolani"
##SnpEffCmd="SnpEff  hg19 test.vcf "
##INFO=<ID=ANN,Number=.,Type=String,Description="Functional annotations: 'Allele | Annotation | Annotation_Impact | Gene_Name | Gene_ID | Feature_Type | Feature_ID | Transcript_BioType | Rank | HGVS.c | HGVS.p | cDNA.pos / cDNA.length | CDS.pos / CDS.length | AA.pos / AA.length | Distance | ERRORS / WARNINGS / INFO' ">
##INFO=<ID=LOF,Number=.,Type=String,Description="Predicted loss of function effects for this variant. Format: 'Gene_Name | Gene_ID | Number_of_transcripts_in_gene | Percent_of_transcripts_affected' ">
##INFO=<ID=NMD,Number=.,Type=String,Description="Predicted nonsense mediated decay effects for this variant. Format: 'Gene_Name | Gene_ID | Number_of_transcripts_in_gene | Percent_of_transcripts_affected' ">
#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  NA00001
20      14370   .       G       A       29.0    PASS    NS=3;DP=14;AF=0.5;DB;H2;ANN=A|intergenic_region|MODIFIER|DEFB125|DEFB125|intergenic_region|DEFB125|||||||||     GT:GQ:DP:HQ     0|0:48:1:51,51

Annotation을 진행한 VCF를 보시면 굵은글씨로 나타낸 ANN field가 생긴 것을 확인 할 수 있습니다.

ANN field의 자세한 설명은 다음 링크를 참조:

http://snpeff.sourceforge.net/SnpEff_manual.html#ann

ANN field는 pipe 문자 ( | )로 구분되어있습니다.

예시에서 보면 MODERATE로 나온것은 파이프로 문자를 나눴을 때 3번째 위치한 것으로 ##INFO를 참고하여 보았을때 Annotation_Impact입니다.

Allele	Annotation	Annotation_Impact	Gene_Name	Gene_ID	... 후략
A	intergenic_region	MODIFIER	DEFB125	DEFB125	... 후략

Annotation_Impact는 "HIGH", "MODERATE", "LOW", "MODIFIER" 로 총 4 종류가 있으며,

Sequence Ontology (SO) term을 기준으로 SnpEff가 4종류로 나눈것입니다.

아래 표는 Annotation_Impact와 그 의미를 정리해놓은 표로 다음 링크를 참고하였습니다

http://snpeff.sourceforge.net/SnpEff_manual.html

Impact	Meaning	Example
HIGH	The variant is assumed to have high (disruptive) impact in the protein, probably causing protein truncation, loss of function or triggering nonsense mediated decay.	stop_gained, frameshift_variant
MODERATE	A non-disruptive variant that might change protein effectiveness.	missense_variant, inframe_deletion
LOW	Assumed to be mostly harmless or unlikely to change protein behavior.	synonymous_variant
MODIFIER	Usually non-coding variants or variants affecting non-coding genes, where predictions are difficult or there is no evidence of impact.	exon_variant, downstream_gene_variant

SnpEff는 vcf의 position을 기준으로 ANN field의 Annotation 컬럼에 SO term 값을 넣고, 이 값을 기준으로 Annotation_Impact를 분류하여 넣습니다.

다음 링크의 6~8 페이지에 SO term에 해당하는 Putative Impact 분류가 있습니다.

http://snpeff.sourceforge.net/VCFannotationformat_v1.0.pdf

또는 snpeff manual page에서도 아래와 같이 표가 정리되어있습니다.

Effect Seq. Ontology	Effect Classic	Note & Example	Impact
coding_sequence_variant	CDS	The variant hits a CDS.	MODIFIER
chromosome	CHROMOSOME_LARGE DELETION	A large part (over 1% or 1,000,000 bases) of the chromosome was deleted.	HIGH
duplication	CHROMOSOME_LARGE_DUPLICATION	Duplication of a large chromoome segment (over 1% or 1,000,000 bases).	LOW
inversion	CHROMOSOME_LARGE_INVERSION	Inversion of a large chromoome segment (over 1% or 1,000,000 bases).	MODERATE
coding_sequence_variant	CODON_CHANGE	One or many codons are changed e.g.: An MNP of size multiple of 3	MODERATE
inframe_insertion	CODON_INSERTION	One or many codons are inserted e.g.: An insert multiple of three in a codon boundary	MODERATE
disruptive_inframe_insertion	CODON_CHANGE_PLUS CODON_INSERTION	One codon is changed and one or many codons are inserted e.g.: An insert of size multiple of three, not at codon boundary	MODERATE
inframe_deletion	CODON_DELETION	One or many codons are deleted e.g.: A deletion multiple of three at codon boundary	MODERATE
disruptive_inframe_deletion	CODON_CHANGE_PLUS CODON_DELETION	One codon is changed and one or more codons are deleted e.g.: A deletion of size multiple of three, not at codon boundary	MODERATE
downstream_gene_variant	DOWNSTREAM	Downstream of a gene (default length: 5K bases)	MODIFIER
exon_variant	EXON	The variant hits an exon (from a non-coding transcript) or a retained intron.	MODIFIER
exon_loss_variant	EXON_DELETED	A deletion removes the whole exon.	HIGH
exon_loss_variant	EXON_DELETED_PARTIAL	Deletion affecting part of an exon.	HIGH
duplication	EXON_DUPLICATION	Duplication of an exon.	HIGH
duplication	EXON_DUPLICATION_PARTIAL	Duplication affecting part of an exon.	HIGH
inversion	EXON_INVERSION	Inversion of an exon.	HIGH
inversion	EXON_INVERSION_PARTIAL	Inversion affecting part of an exon.	HIGH
frameshift_variant	FRAME_SHIFT	Insertion or deletion causes a frame shift e.g.: An indel size is not multple of 3	HIGH
gene_variant	GENE	The variant hits a gene.	MODIFIER
feature_ablation	GENE_DELETED	Deletion of a gene.	HIGH
duplication	GENE_DUPLICATION	Duplication of a gene.	HIGH
gene_fusion	GENE_FUSION	Fusion of two genes.	HIGH
gene_fusion	GENE_FUSION_HALF	Fusion of one gene and an intergenic region.	HIGH
bidirectional_gene_fusion	GENE_FUSION_REVERESE	Fusion of two genes in opposite directions.	HIGH
rearranged_at_DNA_level	GENE_REARRANGEMENT	Rearrengment affecting one or more genes.	HIGH
intergenic_region	INTERGENIC	The variant is in an intergenic region	MODIFIER
conserved_intergenic_variant	INTERGENIC_CONSERVED	The variant is in a highly conserved intergenic region	MODIFIER
intragenic_variant	INTRAGENIC	The variant hits a gene, but no transcripts within the gene	MODIFIER
intron_variant	INTRON	Variant hits and intron. Technically, hits no exon in the transcript.	MODIFIER
conserved_intron_variant	INTRON_CONSERVED	The variant is in a highly conserved intronic region	MODIFIER
miRNA	MICRO_RNA	Variant affects an miRNA	MODIFIER
missense_variant	NON_SYNONYMOUS_CODING	Variant causes a codon that produces a different amino acid e.g.: Tgg/Cgg, W/R	MODERATE
initiator_codon_variant	NON_SYNONYMOUS_START	Variant causes start codon to be mutated into another start codon (the new codon produces a different AA). e.g.: Atg/Ctg, M/L (ATG and CTG can be START codons)	LOW
stop_retained_variant	NON_SYNONYMOUS_STOP	Variant causes stop codon to be mutated into another stop codon (the new codon produces a different AA). e.g.: Atg/Ctg, M/L (ATG and CTG can be START codons)	LOW
protein_protein_contact	PROTEIN_PROTEIN_INTERACTION_LOCUS	Protein-Protein interacion loci.	HIGH
structural_interaction_variant	PROTEIN_STRUCTURAL_INTERACTION_LOCUS	Within protein interacion loci (e.g. two AA that are in contact within the same protein, prossibly helping structural conformation).	HIGH
rare_amino_acid_variant	RARE_AMINO_ACID	The variant hits a rare amino acid thus is likely to produce protein loss of function	HIGH
splice_acceptor_variant	SPLICE_SITE_ACCEPTOR	The variant hits a splice acceptor site (defined as two bases before exon start, except for the first exon).	HIGH
splice_donor_variant	SPLICE_SITE_DONOR	The variant hits a Splice donor site (defined as two bases after coding exon end, except for the last exon).	HIGH
splice_region_variant	SPLICE_SITE_REGION	A sequence variant in which a change has occurred within the region of the splice site, either within 1-3 bases of the exon or 3-8 bases of the intron.	LOW
splice_region_variant	SPLICE_SITE_BRANCH	A varaint affective putative (Lariat) branch point, located in the intron.	LOW
splice_region_variant	SPLICE_SITE_BRANCH_U12	A varaint affective putative (Lariat) branch point from U12 splicing machinery, located in the intron.	MODERATE
stop_lost	STOP_LOST	Variant causes stop codon to be mutated into a non-stop codon e.g.: Tga/Cga, */R	HIGH
5_prime_UTR_premature start_codon_gain_variant	START_GAINED	A variant in 5'UTR region produces a three base sequence that can be a START codon.	LOW
start_lost	START_LOST	Variant causes start codon to be mutated into a non-start codon. e.g.: aTg/aGg, M/R	HIGH
stop_gained	STOP_GAINED	Variant causes a STOP codon e.g.: Cag/Tag, Q/*	HIGH
synonymous_variant	SYNONYMOUS_CODING	Variant causes a codon that produces the same amino acid e.g.: Ttg/Ctg, L/L	LOW
start_retained	SYNONYMOUS_START	Variant causes start codon to be mutated into another start codon. e.g.: Ttg/Ctg, L/L (TTG and CTG can be START codons)	LOW
stop_retained_variant	SYNONYMOUS_STOP	Variant causes stop codon to be mutated into another stop codon. e.g.: taA/taG, */*	LOW
transcript_variant	TRANSCRIPT	The variant hits a transcript.	MODIFIER
feature_ablation	TRANSCRIPT_DELETED	Deletion of a transcript.	HIGH
regulatory_region_variant	REGULATION	The variant hits a known regulatory feature (non-coding).	MODIFIER
upstream_gene_variant	UPSTREAM	Upstream of a gene (default length: 5K bases)	MODIFIER
3_prime_UTR_variant	UTR_3_PRIME	Variant hits 3'UTR region	MODIFIER
3_prime_UTR_truncation + exon_loss	UTR_3_DELETED	The variant deletes an exon which is in the 3'UTR of the transcript	MODERATE
5_prime_UTR_variant	UTR_5_PRIME	Variant hits 5'UTR region	MODIFIER
5_prime_UTR_truncation + exon_loss_variant	UTR_5_DELETED	The variant deletes an exon which is in the 5'UTR of the transcript	MODERATE
sequence_feature + exon_loss_variant	NEXT_PROT	A 'NextProt' based annotation. Details are provided in the 'feature type' sub-field (ANN), or in the effect details (EFF).	MODERATE

참고로 다음은 Sequence Ontology의 페이지:

http://www.sequenceontology.org/

예시 position에서 ANN field의 SnpEff Annotation은 intergenic_region으로 붙어있고,

SnpEff의 table 기준에 의하면 MODIFIER로 나와있습니다.

이상으로 SnpEff로 annotation 하는 방법과 ANN field의 Sequence Ontology term에 대해 알아봤습니다.

그럼 다음시간에 만나요~~~~