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1. External Summary Affinity Signals Elements Signals Browser Reference Gene 0 Click to toggle Ensemble Gene Ve 0 Click to toggle Known Gene 0 Click to toggle Small RNA 0 Glick to toggle MicroRNA Target From TargetScan z 0 Click to toggle Long Non coding RNA 0 Click to toggle HapMap ef Consensus 0 Click to toggle GENCODE V12 Annotation 0 Click to toggle GENCODE V12 2wayconspseudos 0 Click to toggle GENCODE V12 PolyAs 0 Click to toggle GENCODE V12 Long Noncoding RNAs 0 Click fo toggle GENCODE V12 tTRNAS 0 Click to toggle 2 5 LD Signals First top variant in a LD block of leading SNV will be reported in the table viewer here user can check other significant variants on same LD 16 19 Vanant Binding GWAS3D benomic LD External Summary Affinity Signals Elements Signals Browser SNPID POS LOCUS FINAL P LEADSNP ORIG m3132958 632297901 Chori 1 5e 196 rs3129941 1 378 rs4959027 632383050 6p21 32 1 99e 195 rs3129941 1 37 rm3132959 632298942 C orf10 1 1e 194 rs3129941 1 37 showing 1 to 4 of 4 entries 2 6 External Browser GWAS3D provides some useful external browser for annotating and ranking the genetic variant especially for Non coding variant 2 6 1 GWASrap QuickRap Genetic variants representation annotation and prioritization in the post GWAS era Cell Res 2012 doi 10 1038 cr 2012 106 2 6 2 Regulomedb Annotation of functional variation in personal genomes using RegulomeDB2. Pouzf _kKnown T G 11 77529 gt 3 224098 Factor Name Pout known Factor Info Poueti_ 4 known Oct 1_transfac_M007136 Relative Affected Position 15 E 5 10 15 20 Wablogo 3 3 Genotype Ti Strand Start 32516991 End 32519013 Score 5172 92 Pvalue 1 4016E 04 2 3 GWAS3D Signals Cell type specific chromatin dynamic data are collected from literature ENCODE project and other resources GWAS3D can capture those cell specific signals for investigated variant 2 3 1 Long Range Interactions 5C ChIA PET Hi C Report the long range interactions signals of target variant located data are curated from literature based on 5C ChIA PET and Hi C techniques 2 3 2 Gene Promoters RefGene User Defined Region Report the gene promoter signals of target variant located mapping is based on user defined up downstream of RefGene TSS 2 3 3 Enhancers H3K4me1 H3K27ac P300 DHS Report the putative enhancer signals of target variant located data are collected from ENCODE ChIP seq of TFs Histone modification and DNase Seq H3K4me1 H3K27ac P300 DHS 2 3 4 Insulators CTCF Report the putative insulator signals of target variant located data are collected from ENCODE ChIP seg of TF CTCF 2 3 5 ChromHMM Markers Promoter Enhancer Insulator Report the putative chromatin markers signals of target variant located data are predicted by ChromHMM 2 3 6 Conservation Regions GERP Elements Report the conservative elements of target var
3. ee N A UN A LG mr aor 3 S A eS VF Z Fees FER biz W Af ON H J Ss ao ee ST E en S di AN AS ZS ZS S 33 8 OD AU we g SS A SGo OD P Eo So o s h 6 Q Ce g A gt T ZZ oF F GRR ESE o Xos ZC GES amp amp eS Sg Ss 1 2 Several information can be easily read from the plotting from the outer to inner there are Significant regulatory variants and distal interaction regions genes and genomic location chromosome number and distal interaction indicator line For example following figure shown 8 19 GWAS SNP rs9267551 is detected as a significant regulatory variant a this variant located on the 5 UTR region of DDAH2 d in chromosome 6 c One of important regulatory features for this variant which can be viewed from the plotting is the region of rs9267551 located has a long range interaction signal to another locus near VWA Z b interactive elements with significant regulatory variant will start with I_ the red line indicated this signal e and the intensity of interaction is represented by wideness 2 WW y D o N A gt BZ oO Bg 8 CD a ke 2 e 2 y NY w di KS SS e ee SF ye pes eg NS A ees FF FT Aw ws Mae Se NO Star Ne ff A NW cop e i Bee NEU Ek EMMT2 g 6 EHMT2 i STK19 Gelee 8 2 GWAS3D signal statistics Right overview panel will summary the GWAS3D result which indicate the number of significant variants detected the number of variants have been detec
4. reported as significant effect in current GWAS 2 1 3 LD plot Hapmap LD information of this variant for investigated population 12 19 Variant Binding GWAS3D benomic LD External summary Affinity Signals Elements Signals Browser General Information rsid rs28490179 Min Build ID Snp Class Single Base Het Avg Het Se 0 Sub By Hapmap Ancestral Allele T Unique Ctg ID 224514666 Unique Chr 6 Unique Pos Bp 32519005 Gene Annotation intergenic RefGene undefined Published GWAS Reference Click to toggle PUBMEDID 20018961 1 42e 13 DBGAP 217 1 42e 13 LD Plot Population CEU 1 0 5 o2 915 8038 32 588 955 32 4682 088 J2 535 200 32 008 353 Chromosome amp Position 2 2 Binding Affinity GWAS3D will report the significant binding affinity changes for different alleles of target variant by scanning defined factor motifs on variant locus top 5 factors will be reported with detailed factor and binding information For example following figure shown a known motif of TF Pou2f2 has a significant binding affinity change between allele T and allele G in the 15th position of that motif The score and P value indicate the degree and probability of affinity change which will be used for GWAS3D prioritization 13 19 Variant Binding GWAS3D benomic LO External Summary Affinity Signals Elements Signals Browser Significant binding affinity change of selected factors have been detected by GWAS3D 5 Click fo toggle
5. usage and function of GWAS3D In order to access the public site please visit http jjwanglab org gwas3d Please check the site for the most up to date versions of the user manual Features 1 Detecting regulatory Non coding human genetic variants specially 2 Wide spread cell types supporting 3 Cell type specific chromatin state data including signals of distal interaction enhancer promoter insulator and predicted functional elements etc 4 73 ENCODE TRANSFAC Jasper motifs of TF families including hundreds of known and novel motifs 5 Enhanced regulatory variants prioritization 6 Comprehensive variants annotation 7 Multiply association SNV list formats support 8 Useful user defined parameters nice visualization and usability 4 19 Create New Run To perform a new run for your GWAS association result or SNV list please follow 1 Enter the name of the investigated study 2 Specify your E mail Address to retrieve your job a notification will be sent to your assigned mailbox Please specify the name of your study Doo O O Please specify your E mail Address to retrieve your job fs 3 Select an input format for GWAS result GWAS3D currently support four different formats including Plink like format VCF like format single SNP Id and genomic coordinates Before association file is inputted please notice that our system is based on the latest homo species genome assembly version hg19 GRCh37 and dbSNP 13
6. 5 19 ciation P value Wd Without GWAS Association Statistics Pvalue Cutoff vgl 6 Select SNP data set and population HapMap Al SNP Data Set HapMap HI II pm CEU European American CEPH Population 4 AW African ancestry in Southwest USA el KEEN CHE Han Chinese in Beijing China CHD Chinese in Metropolitan Denver Colora 1000 Genomes Pilot 1 SNP Data Set 1000 Genomes Pilot 1 Le EUR European se EI AFR African Population ASN East Asian AMR Ad Mixed American 7 Define a cutoff for haplotype checking The RSquare value of linkage disequilibrium will be restricted between 0 6 to 1 with leading SNP Varant Linkage Option Linkage Disequilibrium Cutoff R 8 Select a investigated cell type Specify the target cell type related to the GWAS traits or your objective Please select without cell type restriction when you don t make sure the phenotype information or no matched cell type Cell Type Option 1GM12878 HIE BE l e HeLa 53 specific Cell Type upon MCF 7 GM06990 CD4 9 Select TF motifs used as binding affinity scanning Support mutiple selection Regulatory Elements Option ENCODE TF Familay Motifs use Cii for mutiple selection in fe clearall select all 10 Only scan know motifs Only consider the know motifs of selected factors Known Motifs E Using Known Motifs for Scanning 11 Define TF binding site P value The P value cuto
7. 7 The input variants coordinates should be consistent with hg19 if have While the SNP identification is no special restriction about version we will convert SNPs to dbSNP 137 automatically It is encouraged to use association data with P value as input variant list without association P value is acceptable Input File Format Help GWASSD supports following input format please use VCF format if you want to input allele information Plink like format Chromosome dbSNP Id Position P value VCF like format Chromosome Position SNP Ild RefAllele Alt Allele P value Variants Coordinates Chromosome Position P value Single SNP Id dbSNP Id P value We accept user provided regions for variants inclusion Specific Region Chromosome Start End Variants should be separated by newline fields should be separated by tab or comma A example is available by clicking the example link 4 Choose input text or upload a input file Input Format EE Ei Upload Association SNPs File 50M islets AIS Se Paste Association SNPs List example Plink like example VCF like examole dbSNP examole Only genomic coordinates re 5 Inout data without association P value or define P value cutoff Select if your data is pure SNV list The P value cutoff refers to the maximal P value cutoff variants with P value larger than the cutoff will be discarded Investigated population HapMap Ltd for computing the synthetic association
8. GWAS3D User Manual v1 3 Table of contents Introduchon o oo cece ec ec ccc ececececeececececeeeeeeaecececuceeaeaeaececeeeaeaeaecececueueaeaeaeseseeneneaeaeaeseeueneeeataeaeeneass 3 SYSTEM REQUIFEMOENTS c0ccccccccccsesescsececeeececesecesedacesevecectsaceseseeesedeceseescscseresceecseeeencaess 4 Welcome eege 4 STE 4 Create New Run ME 5 GWAS3D Result Overview a ananannnnunnnnnnnnannnnnnnnnnrnrnrnnrnrannnrnrarnnrnrannnrnrannnrnrnnnnrnrnrrnrnrnrnnrnne 8 GWASSD Result Details 11 Download Heeult ccc ececeececececeeeeeecececeeeeaeaecececeeaeaeaesececueneaeaeaeseeeeneaeaeaeseeeseeevaeaeaesnenss 18 Retrieve Jobs 19 2 19 Introduction Interpreting noncoding common phenotypically associated variants is an indispensable step to understand molecular mechanism of complex traits 1 The Encyclopedia of DNA Elements ENCODE project identified a comprehensive map of functional elements in active chromatin states by advanced techniques such as ChIP seq DNase seq bisulfate sequencing chromosome conformation capture etc 2 Recent work showed that the associated SNPs detected by genome wide association study GWAS are significantly enriched in those regulatory regions including many transcriptional factor binding sites TFBSs histone modification marked regions Dnase hypersensitive sites DHSs and expression quantitative trait loci eQTLs 3 Also those regulatory elements can engage in long range looping interactions to exert elabor
9. Genome Res 2012 doi 10 1101 gr 137323 112 2 6 3 UCSC ENCODE Browser An integrated encyclopedia of DNA elements in the human genome Nature 2012 6 489 7414 57 74 Variant Binding GWAS3D benomic LO External Summary Affinity Signals Elements Signals Browser Annotate Prioritize Current SNV on GWASrap QuickRap Rank Annotate Current SHV on ENCODE elements Requlomedb query Annotate Current SHV on UCSC ENCODE Genome Browser 17 19 Download Result All significant information detected by GWAS3D can be downloaded from the download tab of GWAS3D PLOT panel User can download the significant variants information one per LD all significant variants information and GWASSD plotting in this tab The GWAS3D download file contain following information CHRPOS SNP ID GENOTYPE LOCUS FINAL PVALUE LEAD SNP LEADSNP PVALUE RSQUARE SIGNALS Overview Download Significant variants information One per LOY All significant variants information GWAS3D plotting 18 19 Retrieve Jobs There are three ways to retrieve your submit job in GWAS3D 1 Received by E mail Please fill right E mail address for the notification in the input page Po Please specify your E mail Address to retrieve your job 2 Check from a fixed link GWASS3D provides a encrypted link for retrieving your job GWAS3D Jobs GWAS3D You can retrieve your job frong hitp jjwanglab org qwas3d Z3dhczNkLTEZNTY20DEwMzk SGE Queue Job
10. ID Job Name Run Time s Status 3 Check from workspace cookies in client browser GWASS3D provides a cookies mechanism with your used web browser it will help you manage all of your submit jobs 1329 1328 1326 1325 1324 1323 1322 1321 1320 1317 1316 1315 1314 gwas3d gwasid gwasid gwas3d gwas3d gwasid gwasi3d gwas3d gwasid gwas3d gwas3d gwas3d gwas3d 12 28 15 15 12 28 12 10 12 27 19 51 12 27 19 44 12 27 19 08 12127 18 45 1227 18 17 12 27 18 12 12 27 15 43 12 27 13 00 12 27 11 51 12 27 11 21 12 27 11 03 19 19
11. ate gene regulation Genetic variation in those noncoding regions may affect the function of cis acting elements or distal interactions and finally contribute to complex phenotype 4 Therefore combinatory analysis of GWAS data and profile of functional elements to capture regulatory variants in a particular disease trait is needed GWAS3D http jjwanglab org gwas3d systematically compute the probability of genetics variants affecting regulatory pathways and underlying disease trait associations by integrating chromatin state functional genomics sequence motif and conservation information when given GWAS data or variant list We first collected and curated genome wide chromosome conformation 5C Hi C ChlIA PET data enhancer insulator promoter marks H3K4me1 H3K27ac P300 CTCF DHS and chromHMM predicted functional elements in 16 different cell types Using those regulatory regions we mapped genetic variants to them and evaluated the binding affinity changes of regulatory factors by scanning 73 ENCODE motifs Finally we combined original GWAS signal risk haplotype binding affinity significance and conservation information to prioritize the genetic variants GWAS3D also provided comprehensive annotations and visualizations to help users interpreting the results Main Functions e Identify the most probable functional variant associated with interested trait in given risk loci e Prioritize the leading variants when given a full list
12. ff of putative TF binding site scanning Strict setting may reduce the false positive Binding Site P value x 10 12 Define promoter range The range of promoter region should be restricted between 100 10000 upstream and 50 5000 downstream of TSS Promoter Range Up Down vi 500 f 13 Input a user costumed region if wanted If defined system only consider the variants in those regions Specific Region example 14 Define maximal variants and interactions could be displayed in the plotting Plotting Option Plotting Variant Size 30 Plotting Interaction Size After preparing the parameters please make sure all required information is filled Then click the submit button the job will be submitting to web server 7 19 GWAS3D Result Overview 1 Circos style regulatory variants visualization Entering your workspace by clicking the finished job system will first display a Circos style graph with some interactive attributes 1 1 Circos style plotting for variants visualization displays broad horizontal area genome widely Top variants with highest regulatory signals and distal interaction regions are displayed in the outer circle The genes or genomic locations connected to respective SNVs will be showed in the inner circle rss13115 PRRC2ZA 6 BAG6 OSP69 S 40d 61 E 085 5 T S DFWWOL 6T grdt E Si 1589 Buch 1 sl 00 kees 7 OA e a gv he ee AN
13. formation in table Significant variants are sorted by their final P value a and some variants will be highlighted with orange background if final P value original P value gt 10E5 b However some variants can rank at the top area although the final P value is below than original P value since those variants are significant enough in GWAS association test The selected variant will be marked by red background c Variant with different GWAS3D signals can be represented by color markers which indicate different function elements or chromatin status d including Leading variant Significant TFBS affinity distal interaction promoter region of a gene putative enhancer region CTCF binding region regulatory region annotated by ChromHMM and GERP conservation element e ME cH search UE E E SNPID POS LOCUS FINAL_P LEADSNP ORIG_P R2 STATUS e a aA 8 n e rs613115 631620020 DAZe zt 1 28e8 124 rs3134603 6 32126002 PPT2 1298 113 3360 116 1 rs4 146582 rs1015166 6 32816958 TAP1 6 32798731 TAP 3 626 110 6 9e 107 r3101942 rs1015166 3 09e 110 0 813 6 66e 107 4 D Leading variant aa Significant TFBS affinity Mapping on distal interaction EJ Mapping on promoter region of a gene E Mapping on putative enhancer region Mapping on CTCF binding region E Mapping on regulatory region annotated by ChromHMM D Mapping on GERP conservation element e 1 2 Table operation Table can be searched a adju
14. iant located data are predicted by rejected substitution of GERP 14 19 Vanant Binding GWAS3ID Genomic LD External Summary Affinity Signals Elements Signals Browser Long Range Interactions 5C ChHIA PET Hi C 4 Click to toggle K562 chr6 32128373 32148684 chr6 32116233 32128353 K562 chr6 32128373 321480684 chr6 32093775 32107518 K562 chr 32128373 32148684 chro 3271547 7 7 32168255 K562 chr6 32128373 32148684 chr6 32934756 32956220 Gene Promoters RefGene User Defined Region Enhancers H3K4me1 H3K2fac P300 DHS 0 Insulators CTCFHO ChromHMM Markers Promoter Enhancer Insulator 1 Click to toggle K562 chr 3214606022 32148222 Conservation Regions GERP Elements 1 Click fo toggle chr6o 32147316 32148173 2 4 Genomic Elements Comprehensive genomic mapping annotations are provided to indicate which functional element connect to target variant 2 4 1 Reference Gene Gene annotation from NCBI Refseq 2 4 2 Ensemble Gene Gene annotation from Ensemble 2 4 3 Known Gene Gene annotation from UCSC 2 4 4 Small RNA SNORNA and miRNA annotations from UCSC 2 4 5 MicroRNA Target TargetScan generated miRNA target site predictions 2 4 6 Long Non coding RNA Human long non coding RNA from re annotated microarray studies 2 4 7 HapMap eQTL 15 19 HapMap eQTL consensus 2 4 8 ENCODE transcripts and functional elements ENCODE gene annotation Variant Binding GWAS3D genomic LD
15. of GWAS result e Evaluate the deleteriousness of genetic variants affecting the gene regulation without any prior effect e Annotate genetic variant from regulatory perspective l Ward L D and Kellis M 2012 Interpreting noncoding genetic variation in complex traits and human disease Nat Biotechnol 30 1095 1106 2 Dunham I Kundaje A Aldred S F Collins P J Davis C A Doyle F Epstein C B Frietze S Harrow J Kaul R et al 2012 An integrated encyclopedia of DNA elements in the human genome Nature 489 57 74 3 Schaub M A Boyle A P Kundaje A Batzoglou S and Snyder M 2012 Linking disease associations with regulatory information in the human genome Genome Res 22 1748 1759 4 Sanyal A Lajoie B R Jain G and Dekker J 2012 The long range interaction landscape of gene promoters Nature 489 109 113 3 19 System Requirements GWASS3D is best accessed using the Google Chrome web browser It has been tested to work with Mozilla Firefox and Safari and Internet Explorer 9 Not all functions are available with Internet Explorer 8 due to a lack of HTML5 support by IE It don t support the old version of IE under 8 Since GWAS3D uses many JavaScript features and libraries and will display batch of dataset in one web page it has some requirements about the hardware configuration Recommend configuration two cores CPU and 2G memory Welcome This document aims to introduce the
16. sted entries b scrolled c paged d under user intervention Show 50 C b entries Search f a SNPID POS LOCUS FINAL_P LEADSNP ORIG_P R2 status rs984778 6 32400088 6p21 32 5 Oe 200 183129871 1 15e 299 0 96 HEHE rs3135377 6 32385399 6p21 32 4 21e 197 rs3129941 1 37e 197 0 871 H rs28490179 6 32519005 6p21 32 6 08e 191 183129860 8 62e 192 0 864 Ke rs6857 19 45392254 PVRL2 6 73e 160 rs2075650 1 75e 157 0 862 E Ec rs2050188 6 32339897 CBort10 1850 144 rs6903608 1550 144 0 803 rs2647044 6 32667910 6p21 32 1 35e 139 rs2647044 5 18e 142 1 152272593 6 31601344 PRRC2A 4 38e 138 183130617 6 29e 135 1 E rs1044506 6 32172065 NOTCH4 6 2e 129 183132946 4 92e 126 0 893 HEHE 8 rs2227956 6 31778272 HSPAIL 1 98e 128 rs9267649 3 11e 126 0 954 a D rs1573646 6 32731624 HLA DOQB2 5 41e 101 182301271 2386 100 0 851 Showing 1 to 50 of 360 entries 11 19 2 Significant regulatory variants information panel 6 related information about selected significant variant can be checked from different tabs of right panel including Variant Summary Binding Affinity GWAS3D Signals Genomic Elements LD Signals and External Browsers Variant Binding GWAS3D Genomic LD External Summary Affinity Signals Elements Signals Browser 2 1 Variant summary 2 1 1 General information Report the variant basic information for target SNP such as allele frequency SNP attributes 2 1 2 Published GWAS Reference Report the reference or publication if this SNP is
17. ted having significant TFBS affinities the number of variants have been detected affecting the long range interaction the number of variants have direct effect by GWAS leading SNPs and the number of variants have indirect effect by high LD of GWAS leading SNPs Oveniew Download GWASSD Statistics Based on the K562 Cell Line and CEU Population GWASS3D detected 360 variants have regulatory signals affinity change affects promoter activity or distal interaction in this study 360 variants have been detected having significant TFBS affinities changes 322 Variants have been detected affecting the long range interaction D I variants have direct effect by GWAS leading SNPs 62 variants have indirect effect by high LD of GWAS leading SNPs 3 Panel switch 9 19 GWASS3D is a one stop web page for switching the current panel to GWAS3D details panel please click the Go To Variants Details button a or move mouse to the left hovering bar for selection b following figure shown GWAS3D PLOT p ee Eeer Go To Vanants Details GWASS3D INFO 10 19 GWASSD Result Details 1 Significant regulatory variants table viewer GWAS3D provides a table showing significant regulatory variants information detecting by GWAS3D algorithm In this table only one top variant in a LD block of leading SNV will be reported but user can check other significant variants on same LD from LD signal tab of right information panel 1 1 Variant in
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