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1. m cm cmm 7 se em os Sn vox a 2 mmm a mg gm EEUU Ben 2 5 ee ee 4 SH m B HE u E In bar chart generation part Z score is the statistical value in PAGE calculation mean value is the mean of the value of all entries in the row Mean change is mean minus standard deviation which presents the change of expression when comparing to the whole row background While user can set two color values for up regulation terms and down regulation terms GO Abundance Chart amp Choose category and row Biological Process Cellular Component Mollecular Function Row 1 Row 2 Advanced Parameter Settings Bar value 9 Z score Mean Mean change Bar style 9 Glass Bar O Filled Bar 3D Bar Cylinder Bar Color Value gt 0 Value lt 0 HEX format only default As mentioned before Z score which is bigger than O or smaller than O will be presented using different colors which set by user de Z score of GO term But if you choose mean value they are in the same color since all mean is bigger than O o E 1 c o z MSE E GE s o 5 829 g TF g2 P PEEEEEESEEEEEEEBBEES SG SESEEEGEFEEEEEEEEEEEEESE GO accession C How to use BLASTAID tool The BLASTAID tool is not an analysis tool but an associated one used mainly for two purposes 1 Transfer your IDs which are not available to agriGO to available ones 2 use blast search to a2. and Sorghum genome data is compiled from phytozome Grape genome data is compiled from Genoscope Medicago genome data is from Medicago truncatula sequencing resources Maize genome data is from MaizeSequence org Castor bean genome data is from Castor Bean Genome Database Brachypodium distachyon genome data is from Ensembl Bovien genome data is from Bovine Genome Database Silkworm genome data is from SilkDB M grisea genome data is from Magnaporthe grisea Database affymetrixmetrix CSV files and array sequences are from NetAffx 5 How to use tools provided by agriGO Quick introduction to analysis procedure 1 Choose tool and set parameters You should choose one tool to go forward At the right side several frames containing annotation text are interactive The content will change depending on exact parameters you chose You can make the help frames show or hidden by using HELP buttons at top right of the page Show or hide help frames we HELP ON OFF 1 Select analysis tool Parametric Analysis of Gene Set Enrichment PAGE N select one tool help frames 2 Select the species 9 Supported species Customed annotation 2 Submit your job and perform analysis After submitting your job the agriGO will pre check the validity of your upload data If your job is submitted successfully a job ID will be given Since the analysis process could take a while you may close the waiting page and use the job ID to
3. GO 0005886 GO 0009651 GO 0004708 24 011 at GO 000813 GO 0009631 GO 0006970 GO 0005739 GO 0045271 267093 at GO 0015085 GO 0030026 GO 0009631 GO 0006816 GO 0006882 GO 0009705 GO 0015369 GO 0009651 256882 at GO 0005575 GO 0009631 GO 0003723 GO 0000166 259570 at G0 0009631 GO 0009737 GO 0010286 GO 0005575 GO 0006970 GO 0009414 GO 0003674 245288 at GO 0042127 GO 0009733 GO 0009735 GO 0003700 GO 0005634 60 0009631 GO 0003713 265480 at 60 0009737 G0 0005773 GO 0009631 GO 0003674 GO 0005886 GO 0042631 24 095 at GO 0009737 GO 0009631 GO 0009361 GO 0005575 G0 0003674 GO 0009414 In graphical result part user can choose one or two rows to draw the image If two rows are selected a third color system purple colors will be used in demonstrating those terms have different regulation direction in two rows Graphical Results Choose category and row No more than 2 rows 9 Biological Process Cellular Component Mollecular Function Cl Row 1 U Row 2 The following example presents two rows in one graph You can check the annotation diagram below the result There are three color systems red means up regulated terms blue means down regulated and purple presents the term is regulated in different direction in tow rows And if the term has same regulated direction in both rows it will have double borders In the box r1 1e 10 means the q value of the term in row1 is 1e 10 and zs presents Z score L m mms nm m
4. annotated reference The default parameter is using suggested backgrounds For each species agriGO will give all possible the background types To those species without a relatively completed profile backgrounds from neighbored organisms are suggested Users can select based on their practical need otherwise use customized reference 3 Select reference select bg ref type Suggested backgrounds Q Customized reference Example Customized annotated reference choose from the suggested backgrounds More than one job Try BATCH MODE In the case that you do not want any of suggested pre computated background you can use customized reference instead NOTE IDs in reference list should from the same species that one selected above for query list Q suggested backgrounds 9 Customized reference Example Q Customized annotated reference 248964 at 250299 at 246018 at 257083 s at 2567653 at uf species that you selected 249910 at above for query list 257925 at 260227 at 261037 sat More than one job Try BATCH MODE Also you can use any IDs if you choose customized annotated reference mode however the price is to attach with GO accession to obtain such freedom suggested backgrounds Customized reference Example 9 Customized annotated reference D008150 O 0006468 O 0016301 D0D06499 T paste you IDs with O 0005439 O 0004569 Pd GO accession here O 0008150 O 0
5. check the work later Please note that results of your jobs will be stored on our server for THREE DAYS After 3 days all information of the job will be deleted If you want elongation contact me Your job has been submitted successfully Job ID 952249782 The process could take a while this page will redirect to results if it is done You can save the job ID to check it at any time within three days Parsing query list availability Done Mapping GO annotation Done Generating output files Done All done Redirect to results page 3 Explore results The agriGO provides different ways to browse results of different tools Some of them are flexible but you may need some specific setting to make them to castor to your own demands And detailed introduction to these tools in the manual in the following will help you to achieve it A How to use Singular Enrichment Analysis SEA analysis SEA is a traditional and widely used method It is simple to use and simple to understand User only needs to prepare a list of gene probe names and enrichment GO terms will be found out after statistical test from pre calculated background or customized one STEP 1 To use SEA analysis you should firstly select the type of your query list either single names or names with GO accession If you choose using supported species in agriGO you only need to provide a list of sequence identifiers It Should be noted that you would better sel
6. qoem 1e 107 XPRESSED DURING 1 growth stapes BEST Arabidopsis thaliana protein nateh is BLP EARLY LIGHT INDUCIBLE ROTEL m T ail aia p yll Bering TAIRSATASIAEREO I Has 240 Blast h s bo 25 ppan i dd geger Arcee i Bacteria E Hetara 0 Fuld ma Us Vinapes 0 Other Eukaryotes 55 poarte NCBI BLink probes endasg LTE LOW TEMPERATURE D4DUCEO 30 Balengi to the dehydrin Grote faery mhh enntara Fighhy corbi GO DOL 2J0 membrane bi o Fen riiui Chet amp amp regetiraely amp camened ss Par dedueess he me Ge Yi and hune Ach degen LTI2 acd LTE GO DO00T37 responas to Abscusc acid stimulus aracesass Sfexpredders enne Eragin talergsor Lecabed in enembranes MANA up dea ape nite dapin id abiere Bond LOM 25M at DSDL cold acchmation fle 117 WE DLS 30 UTS Pu TIONS N eelesular funere unkeemwn IRSOLVED IN in 5 prorcesdes LOCATED IN meme es e ESSED DA 146 peni structures EXPREESSED DURE arent eee CONTADOS mero DOHAI ss Dihin Imergro 1P COL reponi Co wahr daprivelon BEST Aratedopdes thalana peoten match dt CORS COLD REGULATED 47 TAZR AT1G0440 1 Has 2237 Blast hots to 32 LEE specus Anchae D Bactena dy Heera BID Fang 5 Plans 2155 Viruses 0 Other Ewwaryobes 11 seurre MCI find M prebu enda 051 Puce oS Sve Hee 1j uOPalyecas iracaferase succede irt hae nceodes a geom wth guerede amp eee amer doen waty SUS SUCROSE ZrNTHRASE 1 us Fue TIONS DN uDPglyeceayBranaferie ac
7. which is achieved using PAGE method is also available Furthermore we have BLASTAID tool for ID transfer or annotation And search as well as download function is accessible The agriGO can give out rich outputs like graphical result bar chart result and hierical tree which composing a comprehensive understanding of biological meaning of user s input data What is SEA analysis SEA analysis means Singular enrichment analysis which is tranditional but widely used SEA analysis is designed to identify enriched Gene Ontology GO terms in a list of microarray probe sets or gene identifiers Finding enriched GO terms corresponds to finding enriched biological facts and term enrichment level is judged by comparing query list to a background population from which the query list is derived Which statistics method should choose in SEA tool When the input list is compared with the previously computed background or is a subset of reference list choose hypergeometric or fisher for latter only when your query number is quite small When the input list has few or no intersections with the reference list the Chi square tests are more appropriate What is PAGE analysis PAGE is Parametric Analysis of Gene Set Enrichment Kim et 2005 BMC Bioinfomatics PAGE method is using Central Limit Theorem in statistics this method is simple and efficient Different to SEA it takes expression level into account and can deal with a long list of genes
8. 006468 O 0016301 D006499 o 0006499 Or upload file Limited to 4MB More than one job Try BATCH MODE You can paste direct or upload your file for latter please make sure the file no bigger than 4MB STEP 3 The advanced options are optional but quite important These options are default hidden and need to one click to make them visible In SEA analysis there are three statistical test methods hypergeometric chi square and fisher test When the input query list is compared with the previously computed background or is a subset of reference list choose hypergeometric or fisher When both of your query list number and reference list number are quite small you may better choose fisher test When the input query list has few or no intersections with the reference list the Chi square tests are more appropriate Next you can choose method to do the multi test adjustment Seven adjustment methods are available here including Yekutieli FDR under dependency Bonferroni Hochberg Hochberg FDR Hommel Holm False Discovery Rate Though would suggest perform adjustment test you truly can turn off it and use no adjust While you choose no adjust then you may set significant level below higher Terms under the cutoff of the significant level will be highlighted and emphasized in analysis results and it will affect your test output Minimum number of mapping entries means that GO annotations that do not appear in at least the selec
9. 2 GO 004858T2 amp 60 0010286 AT3 18620 Ze 110 GO 00082T0 AT4G3884D Ze 25 GO 0009T33 GO 0003409 GO 00036T4 GO 0005366 INGA Medtrdz100950 INGA Medtrdzg100980 INGA Medt r4 101390 INGA Medt rd2z098440 INGA Medtrdzg101410 INGA Medt r4g098300 Ll D How to use search tool Search tools in agriGO are easy to understand and use Here are some tips 1 Unless you contact me and ask for elongation the job ID is available within 3 days 2 There is a short cut at top right corner for job searching 3 In advance search you have to define the species firstly 4 You can either search single one or a list no more than 100 of sequence identifiers 5 Input IDs are case insensitive but will be agriGO s format in the output Search analysis result Advance search Select species Arabidopsis thaliana Submit identifier Search 6 FAQ What is agriGO The agriGO is designed to automate the job for experimental biologists to identify enriched Gene Ontology GO terms in a list of microarray probe sets or gene identifiers with or without expression information and it is also a GO related database The agriGO specially focus on agricultural species What is GO The Gene Ontology GO project provides a controlled vocabulary to describe gene and gene product attributes in any organism The GO project is a collaborative effort to address the need for consistent descriptions of gene products in different
10. Minimum number of mapping entries contains two rows First row is ID second row is GO accession For 10 he our 60 0008150 GO 0006468 3 GO 0016301 Gene ontology type GO 0006455 i i GO 0006699 T j i z 9 Complete GO O Plant GO slim at G00005499 GO 0004565 Customized GO annotation Or upload file OK now you can click submit to start analysis now You may explore output of analysis results in the following part of manual Parametric Analysis of Gene Set Enrichment PAGE Result Results generated by PAGE analysis have many similar points to SEA analysis thus it is suggested to browse SEA result introduction part firstly And only unique features to PAGE results will be explained Since PAGE tool can analysis several rows at one time and terms in each row will be calculated each row has its significant GO terms Number of significant GO terms for each row is listed in the brief summary part Analysis Brief Summary Job ID 484660634 Useful within 3 days Species Arabidopsis thaliana Annotated ID number 9584 Ga Download Significant GO terms row 1 175 E Details Significant GO terms row 2 60 Details Total GO terms 1532 ee Download The number of terms is determined by the row you selected which is colored by red A simple colorful model named CM for short is available The color used in the CM is same to the color used in graphical result in which red color system means up regulate
11. User Manual Zhou Du adugduzhou gmail com Version 1 0 1 What is agriGO The agriGO is designed to automate the job for experimental biologists to identify enriched Gene Ontology GO terms in a list of microarray probe sets or gene identifiers with or without expression information and it is also a GO related database The agriGO specially focus on agricultural species 2 Why use agriGO The agriGO provides heavy support to agricultural species Not only limited to SEA analysis GSEA which is achieved using PAGE method is also available Furthermore we have BLASTAID tool for ID transfer or annotation And search as well as download function is accessible The agriGO can give out rich outputs like graphical result bar chart result and hierical tree which composing a comprehensive understanding of biological meaning of user s input data 3 What data argiGO contains We currently support on 35 species including 280 datatypes Please check the data statistics page for detail information We will continue adding more species and datatypes 4 How agriGO prepare its data Raw GO annotation data is generated using BLAST Pfam InterproScan by agriGO or obtained from B2G FAR center or from Gene Ontology Arabidopsis genome data is from TAIR Rice TIGR genome data is from Rice Genome Annotation Project Rice KOME data is from KOME database Rice Gramene data is from Gramene center Populus genome data is collected from JGI Soybean
12. all GO terms in the similar type table or just the data User can select terms to draw graphical result or create bar chart Please note that the parameters used in graph or chart generating is fetched from your cookie and your cookie will be set or changed when you generate graphical results or GO abundant chart which has been mentioned in part 2 and 3 While it will make you a bit trouble if you would like adjust the images created here to redo the part 2 or 3 work once more to change the settings Click the checkbox left to GO term can select all GO terms at one time You can Browse in tree traversing mode _o Browse all GO terms Download Or select from following significant terms to Draw graphical results Create bar chart gih O60 term Ontology Description Number in input list NumberinBG Ref p value co 0009409 P response to cold 18 233 1 60 13 L160 0009266 response to temperature stimulus 19 343 1 2e6 11 1G0 0009415 response to water 14 164 2 38 11 L160 0009414 response to water daprvahan 13 155 1 5e 10 You can click the GO name to collapse extend ontology terms in tree traversing mode A bottom that can make all significant selected or not is available and those selected terms can be used in drawing graphical results or to create bar chart Please note that at least one significant term should be included in graph generation otherwise the graphical result will be some kind blank and meaningless C
13. d and blue means down regulated And each block present the term s Z score for the row You can select row s and term s to generate further images Select terms to Draw graphical results Create bar chart gh GO Information CM C Row 1 O Row 2 No GO Term Onto Number Description 1 2 7 score Mean qvalue Z score Mean qvalue LlGoo 00090531 P 11 cold acclimation 0 08 71 16 160 0009408 1 15 4 L160 0009415 13 43 1G0 0009266 13 32 L160 0009414 12 4 1G0 0010119 i1 75 1G0 0009737 11 34 1G0 0015931 9 0 6 8 gt 1Go 0008550 9 1 z 10 G0 0010118 8 7 5 1 11 16G0 0007523 8 5 5 E S iia m a response ta cold a response to water response to temperature stimulus response to water deprivation regulation of stomatal movement s a Li a Lh BJ d response te absgsic acid stimulus nudeobase nucleoside nucleotide and nude acid transport D 0 response to stress stomatal movement n 5 5 s Oo n 0 OO ono uu circadian rhythm The term s detailed information is generated if you click the number This page may be a bit simple because it is quite possible that there are too many entries mapping to the GO ou are browsing GO 0009631 11 cold acclimation 239516 at GO 0005737 GO 0016020 GO 0009737 GO 0015629 GO 0005634 GO 0009631 GO 0003779 GO 0009414 262 84 at GO 0009941 GO 0005983 G0 0009570 G0 0009631 GO 0009610 GO 0005739 GO 0050521 GO 0010353 223646 at GO 0005737 GO 0009814 GO 0009531 GO 0000165
14. databases The GO collaborators are developing three structured controlled vocabularies ontologies that describe gene products in terms of their associated biological processes cellular components and molecular functions in a species independent manner There are three separate aspects to this effort first we write and maintain the ontologies themselves second we make cross links between the ontologies and the genes and gene products in the collaborating databases and third we develop tools that facilitate the creation maintainence and use of ontologies Definition from http www geneontology org What is updated in agriGO compare with EasyGO The agriGO is a successor of EasyGO and it go further 1 We create new website interface The database structure and scripts of agriGO are redesigned Both page loading speed and analysis speed of agriGO now are improved because of the change 2 The agriGO service is especially focus on agricultural species It supports species is extended to 35 including as much as 280 datatypes 3 We added new analysis tools for new agriGO such as PAGE analysis and BLASTAID tool 4 The result output and information are richer compare with EasyGO 5 The agriGO could also work as a GO database with search and download service What are the unique features of agriGO compare with other GO webserver database The agriGO provides heavy support to agricultural species Not only limited to SEA analysis GSEA
15. ds are available here including Yekutieli FDR under dependency Bonferroni Hochberg Hochberg FDR Hommel Holm False Discovery Rate Though would suggest perform adjustment test you truly can turn off it and use no adjust While you choose no adjust then you may set significant level below higher Terms under the cutoff of the significant level will be highlighted and emphasized in analysis results and it will affect your test output Minimum number of mapping entries means that GO annotations that do not appear in at least the selected number of entries will not be shown In other word higher you set the number more entries needed to make one GO term appear in the analysis result Gene ontology type Plant GO slim is a cut down version of the GO ontologies containing a subset of the terms in the whole GO for plant If you can also upload your own customized GO annotation file once your identifiers are not accepted directly by agriGO The file s size is limited to 4MB 3 Advanced options optional Email notification Multi test adjustment method Yekutieli FDR under dependency Significance Level Select one method to do the multi test adjusbment No adjustment iz not suggested if you have many lines in your query list Use 0 05 es smallar significant level if you do not parfarm adjust If you choose customized annotation you vill use your awn GO x annotation data Please make sure your request in the Format that
16. ect species and check all allowed ID types of corresponding species then submit your IDs Only allowed IDs are suitable to be analysis in this type mode And you can mix your IDs from different types Just ensure they are allowed IDs 2 Select the species select query list type 9 Supported species Customed annotation select species of your Query list Example 2257280 at 2238570 at 2853875 at 267261 at 247851 at 25998516 at 259426 at 248337 at d 245560 at list in this textarea 261749 at 266119 at No your ID Try BLAST4ID 245734 at If you choose customized mode you are no long limited by agriGO owned species any more You can use any IDs you have but only be noticed IDs should attach with GO accession 2 Select the species n if you chodse customized 9 Customed annotation mode yo should provide list of names and corresponding Query list Example GO accessions GoO 0008150 GO 0006468 GO 0016301 GO 00064958 60 0006499 60 0005499 GO 0004568 GoO 0008150 GO 0006468 GO 0016301 GO 00064958 GO 00064958 Mo your ID Try BLAST4ID Cet eh OK theoretically you can just click Submit to perform analysis now and simply skip following steps Nevertheless if you want set more advanced parameters then keep on reading this manual STEP 2 Now you can set the background or reference There three types suggested backgrounds customized reference and customized
17. graph rank direction and font size The result format means which output format you preferred The rank direction is used to define the direction in your output for instance the direction in the example image is top to bottom And the font size is self evident that user can set smaller size if there are many nodes in their result Graphical Results 4 Select Category Biological Process Cellular Component C Mollecular Function Advanced Parameter Settings Graphic result format 9 PNG CO PDF OJ3PEG OGIF svo Graph rank direction Top to Bottom Left to Right O Bottom to Top Right to Left Graph font size pt O7 Os 9 O10 O11 O12 Gansa mag Click the generate image bottom after you set all parameters well The graphical result will be presented according to your own settings The graphical result is a GO hieratical image containing all statistically significant terms These nodes in the image are classified into ten levels which are associated with corresponding specific colors The smaller of the term s q value the more significant statistically and the node s color is darker and redder Note q value here means that the value of the multiple test adjusted p value Inside the box of the significant terms the information includes GO term q value GO description item number mapping the GO in the query list and background and total number of query list and background But when those term whose q value is
18. gy Arabidopsis genome data is from TAIR Rice TIGR genome data is from Rice Genome Annotation Project Rice KOME data is from KOME database Rice Gramene data is from Gramene center Populus genome data is collected from JGI Soybean and Sorghum genome data is compiled from phytozome Grape genome data is compiled from Genoscope Medicago genome data is from Medicago truncatula sequencing resources Maize genome data is from MaizeSequence org Castor bean genome data is from Castor Bean Genome Database Brachypodium distachyon genome data is from Ensembl Bovien genome data is from Bovine Genome Database Silkworm genome data is from SilkDB M grisea genome data is from Magnaporthe grisea Database affymetrixmetrix CSV files and array sequences are from NetAffx How often does agriGO update Normally we will update our database every 3 months but if we will update agriGO if some important data source is newly available Improvement and updating to agriGO tools are irregulated Can check result from old version by new agriGO Sorry but no Because we reconstructed the database and redesigned the website organization analysis result from EasyGO is not supported in agriGO How to make agriGO add new customized datatype User can contact the agriGO administrator by email adugduzhou gmail com to discuss more details
19. he species for your query data Please make sure that identifiers in your input should be one of datatypes inside the right information table If your identifiers are not stored in agriGO there is another two ways one is provided your own GO annotation file the other is to use our BLASTAID service allowed 15 types in Arabidopsis TAIR lezus name AT3G34250 ACLS TAIR object name AT3OZaZ250 1 GenBank ID amp APESOZ31 1 DOSJ1D B B11514 1 EMBL ID CAA19168 1 UniPrat Swiss Preeot ID BIM1_ARATH UniProt Tremi CQSLK91 ARATH el I RefSeq Peptide ID NP 554434 2 Select the species Poe tat ea dei ATH Genome Arey aris 267626_ a Affyrmmetrix 8K Genome Array GEPL71 15878_at Arabidopsis thaliana Operon Array v3 Maeyerowitz GEFL2810 4021026_01 Agilent 3 Oligo Microarray GPLZ871 amp 84 POSESA QARZTE array Yale University GPLSEB 3243009 Ha your ID Try BLASTAID STEP 2 In PAGE analysis user should pay more attention to input data As presented in the following image as least two rows must be provided The first row is sequence identifiers and followings are numerical value The numerical value is fold change FC or log2 transformed FC value latter preferred of the identifiers expression under different condition If you do not have expression data then SEA may be the alternative choice In agriGO s example there are 3 rows in this example First row is ATH1 probeset name the second row is expre
20. higher than the cutoff set by the user only GO information will be given in the box To better understand the graphical result investigation of the annotation diagram is suggested If user chooses PNG or JPG or GIF result format linkage to the term s detail is available by clicking those blocks Col Oe Ee amwnabocia PA d Part 3 The terms selected here are children terms of root one or called secondary level terms or significant terms of secondary level terms Thus the bar chart gives user a brief portray since the GO terms are relatively general description Similar to the procedure of graphical result user should specified their parameters before create the GO abundance chart User can try these setting to obtain favorite view of the chart bar Note the setting you used will be recorded in your cookie and these settings will be default ones in your future jobs In other word you may try several times and make your last attempt as your own features GO Abundance Chart Select Category Biological Process O Cellular Component Mollecular Function Advanced Parameter Settings Bar style 9 Glass Bar OFilled Bar O 3D Bar Cylinder Bar Query bar color LASCFF Bg Ref bar color 66FF33 HEX format only default B X legend content 9 GO annotation GO accession font 14 ulli I illi X legend rotation 300 270 to 315 is suggested F Generate hanh Here the bar chart is using glass bar st
21. lick on the number will lead you to term s detail information Y ou can click to collapse extend ontology terms Or check all significant terms ON OFF Select terms to Draw graphical results 2 Create bar chart jj 3 GO 0008150 biological process 8 60 0032502 developmental process q value 0 54 Num 17 GO 0050789 regulation of biological process q value 0 7 Num 23 GO 0000003 reproduction q value 0 84 Num 5 GO 0016043 cellular component organization q value 0 84 Num 7 GO 0065007 biological regulation q value 0 71 Num 25 GO 0032501 multicellular organismal process q value 0 77 Num 13 GO 00099587 cellular process q value 0 6 Num 68 The term s detail page is as following The agriGO will give all entries can be annotated to the term besides a brief summary And for each entry the annotation includes GO terms GO source description Job ID 452654537 GU Accession GO 0COS403 Ontulogy Bologecal Process Description response to cold Aneobsbed Tobal member in query list 18 155 Anmoteated Total namlbser im backgeeumd redeszences 233 22478 hanse G0 terms GO pume Description Direct deacripiben og teanxler From target piene codeg BLIP EARLY LIGHT INDUKTABLE PROTEIN ehleregehyl bedeg Encoded an early kgbi idua prove EARL PROPRES DUCABLE PROTEIN ELIP1 FUNCTIONS IN eleraphyll binding INVOLVED IN response to cald EXPRESSED Ih 21 plant a 25432 ae Shou a
22. nnotate your sequences with GO To use BLASTAID user should set target database at first and then E value cutoff The program should be correctly selected based on sequence types of user s input and target database Generally speaking all array sequences are nucleotide and other genome sequences are protein The process may take a long while thus the E mail address should be given for the Email notification 2 Set parameters Target database Arabidopsis TAIR pep eg AT3G34250 1 E value cutoff Choose a program BLASTP BLASTN OBLASTX TBLASTN TBLASTX Email address required PO The result interface is a bit simple but enough for usage There are 88 entities of total 129 matched Download Downloadable text result query_nane target name evalue G accessions INGA Medtrdz101000 AT5G23180 1 0 0 GO 0019825 6GO 0042761 0005783 GO 0010345 INGA Medtrdgi00910 AT3G28T00 0 0 GO 000557T5 GO 000367T4 GO 0008150 IMGA Medtrdg101300 AT3G TEGU Je 34 GO 00055T5 60 00036T4 ING Medtrdg 88220 AT4G3884D 2e 24 GO 0008733 60 00089409 Go 00036T4 GO 0005386 INGA Medtrdg101140 ATS 65005 le O6 GO 0005575 0003150 0003676 ING Medtrdg 98500 AT2 16750 4e 142 GO 000486T4 GO 0006468 GO 0012505 GO 0005524 AT1GT62 00 1 amp 18 GO 00035T4 60 00452T1 0008150 ATS gt 02820 5e 45 GO 0005634 0006974 0048478 GO 00082T0 GO 00036T6 GO 000TO4 AT4G 10130 1e 35 GO 00055T5 GO 00310T2 GO 0006457 AI4G 38580 je 86
23. probesets PAGE use a two tailed test to count Z score and the caculation of p value will be if Z score gt 0 p value is 2 1 x if Z score lt 0 p value is 2 x What is BLASTAID The BLASTAID tool is not an analysis tool but an associated one used mainly for two purposes 1 Transfer your IDs which are not available to agriGO to available ones 2 use blast search to annotate your sequences with GO Which tool should choose It will depend on what data you have If you only have a list of identifiers or only interested about them SEA will be your choice And if you like take expression data into count and would like compare several dateset then you may try PAGE The BLASTAID is only an associated tool use it if you really need it Why graphical chart image does not display on my PC The bar chart result need flash player to browse correctly And you may need different tool to display different format graphical result for example Adobe reader SVG brower Contact me if you install related tool but still can not see the results How many datatypes are supported by agriGO We currently support on 35 species including 280 datatypes Please check the data statistics page for detail information We will continue adding more species and datatypes How agriGO obtains its data source Raw GO annotation data is generated using BLAST Pfam InterproScan by agriGO or obtained from B2G FAR center or from Gene Ontolo
24. r of mapping entries ontologies containing a subset of the berms in the whole GO for plant 5 v Email If you provide a mail address a notification will be send when the analysis iz completed with the link te the results Gene ontology type Complete GO Plant GO slim Email notification Greeting You can now click submit to perform the analysis You can always get interactive help from the right help frames and a detailed tutorial in this manual if you still have any question then contact me directly In the following we will discuss the outputs of the SEA analysis Singular Enrichment Analysis SEA Results Part 1 A brief summary of your job will be given The job ID is useful within 3 days A file containing all entities in the query list that can be annotated by GO associated with descriptions is able to download Analysis Brief Summary Job ID 252249782 Useful within 3 days Species Arabigopsis thaliana Background Reference Affymetrix ATH1 Genome Array GPL198 Annotated number in query list 158 ee Download Annotated number in background reference 22479 Significant GO terms 23 l Details Part 2 In this part you can browse the hieratical graph result Note that the graphical result was generated as separate graphs for each of the three GO categories namely biological process molecular function and cellular component After select the category uses can specified their favorite output format
25. ssion fold change FC value of cold treatment to CK cold CK after half hour Third row is expression FC of cold CK after 24 hour cold treatment Only 600 probesets are in the quick example for the fast load of the HTML page To obtain a full view of PAGE method you can download the full example file and explore the following analysis procedure 3 Submit your data You choose Examplal There are 3 rows in this example First raw ig ATH1 prabeset name the second row ia expression fold change FC value of cold treatment to CK cold CK after half hour Third row is expression FC af cold CK after 24 hour cold treatment Only 300 probesets are in the ligt Far fast load af the HTML page Te obtain a full view of PAGE method you can download the Example fila and explore the analysis procedure Query list Quick Examplad Full Example 244906 aE 1 23 I3453B8 satj l 73 244535 ak 1 08 z44940D ak 1 07 244944 sg t 44550 at D 77 244968_al 0 72 223569 _ of 0 58 2449 70_ak 1 36 244979 ak 0 98 Zz44sEQO akb 0 97 244581 _at 0 82 z549 22 ak z Ia45983 sakt l 03 2448084 at 1 10 z442385 ak 1 16 244986 aE 1104 Or upload file 1 1 9 34 C m Cb Beh onm ges oe First row must belidentifiers M following are numerical value iB C UR ub D oe UD es hi a D EID RO UN CD Ud P UJ L6 CA LJ 8 da oe C3 C L3 Ui E E CE E E E Pao rr eri P STEP 3 Next you can choose method to do the multi test adjustment Seven adjustment metho
26. ted number of entries will not be shown In other word higher you set the number more entries needed to make one GO term appear in the analysis result Gene ontology type Plant GO slim is a cut down version of the GO ontologies containing a subset of the terms in the whole GO for plant Last if you provide a mail address a notification will be send when the analysis is completed with the link to the results Providing a email address is optional to SEA analysis because it is very fast 4 Advanced options optional Statistical test method Hypergeometric Statistical test When tha input list is compared with the previously compubed Background aris a subset of reference list choose hypargeomoatric or fisher for latter only whan your query number is Multi test adjustment method quite small When the input list haz few or no intersections with the reference list tha Chi square tests are more appropriate Multi test adjustment method Choose method to do the multi test adjustment If you choose no adjust then you may xat significant level higher Yekutieli FOR under dependency Significance Level 0 05 r Significance Level Terms under the cutoff will be highlighted and emphasized in analysis results Minimum number of mapping entries GO Annotations that do not appear in atleast the selected number of entries will not be shown Y Gane ontology type Plant GO slim is a cut down version of the GO Minimum numbe
27. troty ducred e dyrsthase barr IN Gn ccomacg fencer to cold p Amicia 4 preseases LOCATED DH celular cemponent unknewn EXIMESSED IN 31 plant structured EXPRESSED Outlined 14 gn 248958 at racc Piscina ts Hacen 5 8 3 CONTAINS Interfre OOMAI a Sucre gynthase nant and er aeobaerena merPre E PEDE 2BIO Soerose ayra babe CEEDOOMITO reaennsa t in ODS Ghycomyl transferase aeoue 1 LeterPra DPROO1196 BEST Arabidapsas thaligea protein abri ig SUSE UDP glycesyk an a eeu den iin E pick i ant gg EJ Sucre pynthise Drarbierase Dranstenemg gressu groups TAIRIAT3GAEISI 1 Has 5253 Blast hats to 5257 probiers in m s Archse 181 Bacteria 2052 Meteo 87 Fungi dd Plans SES Virngpas Oy Ceher Euvaryabeg L652 source ROB pre endasg COOP eld ake demain probem 1 RRA Ending deubla aranded OMA hnding udes acd besdesg dus d DA A Berd Encoded a cold shock damar piattini aoho is oad aechmanen by Blocking the secsdary aIrodture el mk a E DHA duplex u um faciktunas rans atc at celd temperature M stes domain protein 2 tesora FUNCTE CAS 1h dessble stranded DMA bi B How to use Parametric Analysis of Gene Set Enrichment PAGE PAGE method is argued by Kim BMC Bioinformatics 2005 6 144 Using Central Limit Theorem in statistics this method is simple and efficient Different to SEA it takes expression level into account and can deal with a long list of genes probesets STEP 1 Firstly you should choose t
28. yle default colors GO annotation as X legend 14px font and 300 for X legend rotation Here are some tips 1 Have a glance of all four bar styles and select one you like 2 Use HEX format to define colors and there is a website we already suggested 3 If you prefer GO annotation as X legend content you may use smaller font once there are too many words 4 270 to 315 is suggested for X legend rotation in which 270 means vertical and 315 means 45 degree slope and you can try other number which may satisfy your taste but seems somehow strange to me The bar chart is created based on scripts from Open Flash Chart It is powerful You can drag borders to resize and adjust the image size and ratio And bars are accessible to term s detailed information A Save as Image bottom is existed but only useful when you are using FireFox browser and if you can also use your Print Screen bottom on your keyboard or other tools to download this image Drag chart border to resize or click input list bar to check details Feature sets are saved Saversiimacelal You might need FireFox to achieve it click to check detail Input list Background Reference drag border to resize the image GO annotation Part 4 In this part detailed information is given All GO significant terms will presented in the following table And you can browse the GO terms using tree traversing mode we will discussed it later or can browse
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