PRIMEGENSw3 User Manual
Contents
1. Designed by Garima Kushwaha Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia Figure 1 PRIMEGENSw3 page for choosing between different utilities to design primers or probes Cover CpG Island Primer design covering CpG Island is one of the unique features of PRIMEGENS v2 which can be used to study methylation patterns of various oncogenes and tumor suppressor genes This feature designs primers for genes that have CpG islands present in close proximity to their respective TSSs Primers can be designed to amplify genes whose expressions are suspected to be influenced by nearby CpG islands Detailed description for working of this utility is present on website as Details link in front of the link to this utility 1 User Manual PRIMEGENSw3 2010 Around TSS Primer design covering TSS is a feature of PRIMEGENS which helps the designed primers to cover the region around a transcription start site TSS of any gene To cover specific region around the TSS of any gene the user is only required to provide gene symbols for which the primer design is required PRIMEGENS is capable of extracting their respective TSSs from the UCSC Genome Database currently for March 2006 assembly Detailed description for working of this utility is present on website as Details link in front of the link to this utility Around max cut sit region PRIMEGENS can2. TAATTOC EAA TOLA Baie TH THA T AAL TOG 8 TOA TETCTICCTOGAGTE l MAGN TTA T TATTICA Tit TIGA ADETE ThA TLAAC TOG TRATTOTTGGCA Tat TIONG GLC TETT CAC iG TGC AC TTC TTT OTE TO GT CaS TTT TCC TT Be Eai eee a h e e ijl Fal m i jen pa p l l E l E Fal Bal a n R F Fa plm solt 1 125 plai t 112871 Giyada PeSPE2 18 11D Glyaaee TMk LUGE Citi meN IMFI Glee LIET Giydi 17 plaadi TN L036 Gladi mei 1184 Glymaege ITN LTL Glai A 1171 Glymaedge 7778 iTi Glada TA 1 Glyseroli yeasts 1138 Glad 1g 1750 10a Gly 1p 1750 1 83 olmadigim 1 36 plyaji 1095 Glymad 1p 1754 1 153 Glyaad 1p8 1754 LUTE Gyan 10 1758 101 Gly 1p 170 1155 Glyead 1p 1754 1 82 blyad 19 1780 1 133 Glynd p20 7 250 Glyua gaan 11145 lynai Lph360 2 06 Glynd 1030 1006 Gyan Ipai 20116 Glyaad Lgl L1H giyasi 130 7H Glynd 1gh400 178 laad igda 251 Glymad Laat A Glai Lee 21117 Ghymad Lghabae 1117 lynai Lpe200 7121 GLyua gaat I Glynd 169 20136 Lymn e 141381 Glynd 1300 2133 Glyd ae E Glyaad 130 2035 h Glynad 1030 1095 3 Failed sequences Named as name of the query file followed by query_failed txt This file contains input query sequences in fasta format for which primer design is failed That is no primer pair found in the given constraints User can use this file for primer design using PRIMEGENS again with different primer design par
3. Database Support References Download User Manual Copyright Acknowledgement Contact Us Designed by Garima Kushwaha Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia Figure 14 b Confirmation pop up on refreshing page Step3 Result Visualization On PRIMEGENS succedssfull execution and primer or probe design best results are shown in a form of table on web page with all information about each designed primers or probes as shown in Figure 15 a Double clicking on any row of this table or in other words each designed primer record visualize the position of both left and right primer on its corresponding query sequence as shown in Figure 15 b Also name and links to all output files generated by PRIMEGENS are shown for user to see the results in their browser or right click and download them to their computer All these files are still in the same directory as was provided in the link High throughput Primer and Probe Design University of Missouri Columbia mm Home Design Results Algorithm e SSPD Best primers e PSPD Double click on any primer pair result in the following table to visualize their position e FSPD on its corresponding sequence e Check Left Left Left Left Right Right Bind Query name Left Primer Sequence primer Start primer __ primer Right primer Sequence primer Start primer inding ae L h primer Tm GC ae L h Pri Specificity position Lengt content
4. PRIMEGENS run is taking too long this http primegens_org Garima cagi bin 24894 can be bookmarked or copied to access the result files Also Cemiele me cienme the user can provide their email address for PRIMEGENS to email this link to directory having result files of the current PRIMEGENS run for future use Only E mail address SendEmail Please refresh this page to see the updated results Implementation Database Support References Download User Manual Click on this link to access i results This link Copyright Acknowledgement Contact Us Designed by Garima Kushwaha will show all the input and Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia output files for the current process Figure 14 a Next page after hitting View Status button on last page shown by Figure13 11 User Manual PRIMEGENSw3 2010 PRIMEGENS w3 be Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home Design Results Algorithm PRIMEGENSv2 is still running Please refresh this page to see the updated results refresh lf the current amp To display this page Firefox must send information that will repeat any action such as a search or copied to access the result files Also eee mec the user can p order confirmation that was performed earlier EGENS run for future use Only Cancel Implementation
5. ae aa Probe Sequence start end Binding 811529022 0000 0099 GCACGAGAACCGCTCCTCTGGTCCGTGGCCATGGCGTCGTCCATGCTCTCCGCTACCACCGTGCCACTCCAGCAGGGGGGCGG Specificity 811529022 0500 0599 CGGCATCGACCTCGTCATCGAGGGCACCGGCGTCTTCGTCGACCGCGAGGGCGCGGGGAAGCACATCCAGGCGGGGGCCAAG eon Design 11529022 1000 1099 AGGTGAACCAGGCGTTCCGCGACGCCGCGGCCAACGAGCTCACGGGCATCCTCGAGGTCTGCGACGTGCCGCTCGTGTCCGTC 22334604 0000 0099 GACGACGTTGCGCAGAGAATCCCCAAGCAAACAAACAGGGTAGAGCGTgaggagaggagaggagaggagaggagagg TGGGTCTGt hea 22334604 0500 0599 TGAGAAGCTGAAGAAGGTGCTAGAGGTGTACGAGGCACGCCTGACCAAGTGCAAGTACCTTGCTGGAGACTTCCTCAGCCTCG References 811417968 0000 0099 TTTTTTACCAAATCCGGACAGTCTTGAGCTCTCTCAGTCTCAGCATTTCAGTTCCCAGATTCACAGAGCCCCAGCCCCACAAGT A Download User lt 7 Manual Following are the result files available after running PRIMEGENS QueryFormat1_ZeaMays fa Query file QueryFormat1_ZeaMays fa_probe txt Probe design results Copyright Acknowledgement Contact Us Designed by Garima Kushwaha Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia Figure 16 Probe Design Result Visualization Input File Format PRIMEGENS support following query file format 13 User Manual PRIMEGENSw3 2010 1 FASTA format with gene names Following figure shows one sample file for this file format First shows file with sequence and the other shows without sequence gt gi 2276989 16 516 Glycine max dehydri
6. as follows Step 1 Upload Input files For Regular Primer Design To design primers and probes PRIMEGENS require two types of inputs One is the query file having the sequence for which primers probes need to be designed and the database file having all the other sequence that are present in the PCR reaction Sequences in database file are the sequences to which PRIMEGENS will check for any potential cross hybridization and thereby select primer probe that are specific to the sequence of interest from sequence mixture On PRIMEGENSws web server user can upload the query sequence PCR template file and their own custom database file sequence mixture in PCR or use available genomes supported by PRIMEGENS PRIMEGENSws do also provide different sample data for both query and database sequences for users to test primer probe design using PRIMEGENS algorithms As per their selection the corresponding upload or selection box gets activated for the user to provide respective option 2 User Manual PRIMEGENSw3 2010 If any of these files query or database file is not uploaded by the user before hitting submit button the program will exit giving the error message as Query file has not been uploaded or Database file has not been uploaded Figure 2 a c shows the webserver page having various options for input files required by PRIMEGENSw3 Figure2 b shows the available sample query options different query formats on webser
7. for Around TSS utility Here the query file is Gene symbol list file The gene symbols are taken from lists provided by UCSC Genome Browser s gene list Other than uploading gene symbol list file and corresponding genome it also requires special parameters i e Length of sequence upstream of TSS and Length of sequence downstream of 4 User Manual PRIMEGENSw3 2010 TSS to pick query sequence around TSS Both of these parameter values have been assigned with some default values for testing purpose P RI M EG E N S W3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Input Files Upload Query file Use Sample File Human gene symbols View Sample files e PSPD e FSPD Upload your own file Browse e Check Binding Specificity Select Genome e Probe Design Only Use Sample Genome Sample genome Available Genomes None Implementation Database Support References Special Parameters Download User Manual Length of sequence upstream of TSS 100 default 100 Length of sequence downstream of TSS 100 default 100 Next Reset Figure 4 Input file page for Around TSS Utility For Around max cut site region Design Figure 5 shows the input file page for Around max cut site region utility Here the query file is same as for regular primer design Other than uploading query file and database it also requires special parameters which are Number of Cut sites Cut s
8. her http SSPD is running It might take few minutes to hours to Update PSPD FSPD Check Binding Specificity rimegens org Garima cgi bin 8485 After the completion of primer design PRIMEGENS Click View Status button to check PRIMEGENS run status If PRIMEGENS fun is complete clicking this button will automatically rediredct user to result page ony View Status Probe Design a Only ees Return to our home page Implementation Database Support References Download User Manual Copyright Acknowledgement Contact Us Designed by Garima Kushwaha Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia Figure13 Page after running PRIMEGENS Figure 14 a shows the page that come on on hitting View Status button on page shown in Figure 13 if PRIMEGENS run is not finished Figure14 b shows confirmation pop up that shows after pressing refresh button on its next page PRIMEGENS is still running for the job submitted This absolutely safe to press Resend without loosing design results and keep refreshing to check the PRIMEGENS completion It takes few minutes for sample data for testing purpose P RI M EG E N S W3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home Design Results Algorithm e SSPD PRIMEGENSv2 is still running e PSPD e FSPD e Check refresh Binding Specificity lf the current
9. position Lengt E Probe Design S a CGACATGGTCAAGGTCATCT 1145 20 58 5 50 AGTCATTTCGGGGTGAATCC 1313 20 Only nas Implementation ai AAACAGGGTAGAGCGTgagga 32 21 61 2 52 AAGTTGATGGGCACGATCTC 199 20 Database Support References m T368 CACAAGTAGCGAAAtggcag 77 20 59 5 50 ACACTGTTGTCCCTGTTCTCCT 244 22 Download User Manual die GITTGCATIGTTTGCGCTCC 14 20 63 9 50 TAACAAGTTGGCAACCCCCT 164 20 E 4 i Following are the result files available after running PRIMEGENS QueryFormat1_ZeaMays fa Query file QueryFormat1 ZeaMays fa_query failed txt sequences with failed primer design QueryFormat1 ZeaMays fa_query primer list txt alternate primers for each sequence QueryFormat1 ZeaMays fa_primer_xls best primer sequences Figure 15 a Primer Design result visualization 12 User Manual PRIMEGENSws3 2010 H ome Design Results Algorithm Best primers Double click on any primer pair result in the following table to visualize their position on its corresponding sequence Left Left Left Left Right Right Bindina Query name Left Primer Sequence primer Start primer _ primer Right primer Sequence primer Start primer a iti pearl primer Tm GC kat iti Lenath P Specificity position Leng conten position Leng 7511523022 CGACATGGTCAAGGTCATCT 1145 20 585 50 AGTCATTTCGGGGTGAATCC 1313 2 Only si Sor ener TE ers AAACAGGGTAGAGCGTgagga 32 21 612 52 AAGTTGATGGGCACGATCTC 199 20 Databas
10. 1521989 Zea mays CL7600_1 RNA sequence gb AY110635 qi 21215225 ug Ze 1326 Len 1437 11338551 947056E04 x1 947 2 week shoot from Barkan lab Zea mays COMA aRMA sequence gb BG462326 gi 13384639 PSI5112058 MZCCL10155E09 g Maize Endosperm cDNA Library Zea mays cDNA mRNA sequence gb 0450819 gi 67022070 gt 11527744 Zea mays POOOT amp 406 BANA sequence gb AYO4SO0 gi 21207958 ug Zm 1336 len 1306 P427581382 2M BROO17EG13 f J BFb Zea mays cOWA 3 mRNA sequence clone end 3 gb QV174574 qi 76936032 PS11526595 Zea mays PC0078926 mRNA sequence qb A 106023 gu 21209107 ug 2m 1334 Len 1194 5522334098 Zea mays clone ELOINO424F12 d ARMA sequence gb BTO 18489 gi 54653270 ug Ze 1337 len 1728 511524811 Zea mays PCO123922 mRNA sequence gb AY107813 qi 1210891 ug Ze 1338 len 730 211325644 Z010 09212 A 7010 AAFC ECOAC Fusarium graminearum com silk Zea mays cDNA clone Zelo 09al 5525104934 MZCCL10063E08 g Haize Endosperm cDNA Library Zea mays cDNA mRNA sequence gb C0443843 gi 67015094 S 11526474 Zea mays PO0G99943 mRNA sequence gb AY106150 gi 21209228 ug 2m 344 len 1572 gt 525115823 MACCL10210C10 g Haize Endospere cONA Library Zea mays COMA BAMA sequence gb C0454086 gi 67026237 S279 3705 ZH _BFOO198F22 f ZM BFD Zea mays cDNA 3 mRNA sequence clone _end 3 gb 0V515676 gi 78087283 si 5756 Zea mays PCOGT7Z26J0 BANA Sequence obsAY IOGEAR gi 21709046 g 7e 1350 ajz 3 FASTA format with chromosome position That is que
11. 48 ilyas Lg 70 mi yin Lg37 768 mil yes LQST 768 2 ai yin LgS9265 1 mip yin Lgs98S5 1 lym lgl L mil ymadlg4 1856 2 sO Lymad L916 1 OL ymad1g45 748 1 iL ymad gas 748 2 GL ymad2ge2 74a 1 mGLymad2ge3460 L mG Lynas go3408 2 aL ye 2geS678 1 p p p p p p pll l 2 Alternate primer pairs detailed LEFT PRIMER TOO TGAGGAGGGAC TGAAAG AAGAGTOTGAAGCCTGOCGAT CASGAGAACGGCCAAAOARG COAG TACAATCOCCAGACAA AATOASOGCATGCCAATE TC CTTCGCCACTCC TATCAAGC GTGAATOTCC TAAGGGGCAA TOOOTTTATCCCAGTTCCAG GICTTCAAAAGGGATOOCAA CCGCAGAGAAGNGGACAAAC CAATTTCCATATCCCAACGG ATCC COCAGGAAAAAAGAGA AGG TEOG TSC TETCAAAGTC GCAAC TE TCCGTTGAAL TCC TOCAGAGASCATOG TTCAG GOCAAC TG TCAGAAACAGTA GC TOGCAATCAATACAGGGT ACATAGACGC TOCAAACGTG TCACACAGAGAATTACOCGG GACCCAGCTCAAAGACAAGC GCACTGATTTTCACGCAGAA AGCACGAGC TOGATITOTIT AGCACGAGC TGGATTTGTTT TTCATAAAS TC GGG TaGA6C RIGHT_PRIMER TGAAAACCCAAAAAAC TOCG CCATATECTCCAAATCCOCT AAAGGG TOC TOATCAAL TOG TOCACTOTC TTC TGGAG TG GOTCAGCC TTA TOGARABA GOA TACCOAACOTCOTTOT ACAATOCOCACAAGACTA TOA COTCTOC TOC TCAGA TOG TC ATOACGGAG TTGO TGGAGAL AGGGTAAAGCAACAGAGCGA TATAGGCCTOGATT TGACGC GOGTCTATSCCTATGOCTTC AACAGCAGCAAATE TTGCAL ASGGOOTTGTOTTIGTTIMA AGU TOCOGG TRANG TETCTTT CACTIC TOCAGAGGCAGACC COCAAACCTOCTTICAACA TT CCATARCAGOSATCOCAGIT CACCATTICAAAOCOCAG TT COAG CATOOC AAAGAT ATCASTGGCATCATGCTTCA TSCACTGICTTCC TGGAG TG TGCACTOTC TTCC TGGAG TG GTO TOAACAGCGGATAGCAA Named as name of the
12. 7068 SOX14 cross SRY box 14 chr 55532248 55533847 50X17 cross SRY box 17 chr20 62149423 62151022 SOX18 right SRY box 18 chr3 1829123 16 182913915 SOX2 right sex determining region Y box 2 chr13 94159780 94161379 50X21 right SRY box 21 chrX 139412718 139414317 SOX3 right SRY sex determining region Y box 3 chr16 971009 972608 SOX8 cross SRY sex determining region Y box 8 chrl 7 67627956 67629555 50X9 cross transcription factor 50X9 chr2 171279307 171280906 SP5 cross 5p5 transcription factor chr 166490976 166492575 T right transcription factor T chr2 161979366 161980965 TBR1 left T box brain 1 chr2 161980766 161982365 TBR1 right T box brain 1 chr22 18123426 18125025 TBX1 cross T box 1 isoform C chr17 56831239 56832838 TBX2 cross T box 2 For database file PRIMEGENS support two types of format 1 Single type database i e one file containing all sequences in Fasta format eg Glycine max database 2 Genome type database whole genome in multiple files i e one file per chromosome Currently PRIMEGENS allow user to upload only single type database i e a single file with file size 10MB Web server provides in house database for various model organisms which user can select In case user wants to use genome for any other organism they can contact PRIMEGENS developer with this request to for support In query file user can input the nucleotide sequence for each query sequence or can just give gene names o
13. AAAATGTTAA GTAA gt gi 4838146 81 563 Glycine max seed maturation protein PM36 PM36 mRNA complete cds ATGGCATCCCATAGGCAAAGC TATGAAGCTGGTCAAAC TAAGGGCCGAAC TGAGGAAAAGACGAACCAGA CGATGGGCAATATTGGAGAGAAGGC TCAAGC TGCAAAGGAGAAGACCCAGGAAATGGCCCAAGC TGCAAA GGAGAAGACCCAACAAACAGCCCAAGC TGCCAAGGACAAGACTTGCGACAC TTCCCAAGCGGCAAAGGAG AAGACCCAACAGAATACAGGAGC TGC TCAACAAAAGACC TCAGAGA TGGGCCAGTCCACGAAGGAATCGG gt TC2 17759 gt TC2 17788 gt TC2 17789 gt TC2 17986 2 FASTA format with gene names and functional description That is gene names and description without query nucleotide sequence The database type must be single type database in this case 14 User Manual PRIMEGENSw3 2010 PS11529068 Zea mays PCOS0862 WANA sequence gb AY103556 gi 21206634 ug Ze 1314 Len 966 P511526431 Zea mays PCO120598 aRNA sequence gb AY106199 gi 21209271 ug Za 1316 len 1504 D 11522057 Zea mays CL7 amp 39 1 aRMA sequence gb AY110567 gi 21214976 ug Za 1319 len 1734 11527763 Zea mays POOGE2E17 wANA sequence gbsAY104861 gi 21207939 ug 2m 1321 len 1190 411527081 Zea mays PCOLO1093 BANA sequence gb AY105543 gi 21208621 ug Ze 1322 Len 2424 027574516 ZH BOG LESP1 r ZH BFb Zea mays COMA 5 mRNA sequence clone end gb DVIG7708 qi 76919424 22333859 Zea mays Clone ELO1NO519806 d RMA sequence gb BTO 18720 gi 54653509 ug Ze 1324 len 2099 511529028 Zea mays PCOO92990 ANA sequence gb AY103596 qi 21206674 ugeZu 1325 len 1220 51
14. PRIMEGENSw3 2010 PRIMEGENSw3 User Manual PRIMEGENSws is Web Server version of PRIMEGENS program to automate high throughput primer and probe design It provides three separate utilities to select targeted regions of interests from genome for PCR amplification long with its regular primer design process PRIMEGENSws3 s different utilities for primer and probe design are Regular Primer Design Cover CpG Island Around TSS Around max cut sit region eS Figure 1 shows the webpage showing different options for the user choos for primer or probe design for these utilities P RI M EG N S W3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Design primers randomly over query sequence Home Regular primer design based on all parameters provided by the user Details Algorithm implementation Design primers covering nearest CpG island for any gene of interest me Cover CpG Island This utility is supported only for HUGO gene symbols using Human Details Database Support CpG islands and TSS list provided by UCSC genome browser References Design primers round TSS for gene names present in gene list Download User Around TSS provided by USCS genome browser Details MENNEN This utility is supported for various available genomes of model organisms Design primers around maximum restriction enzyme Around max oksia aginn cut site region on a giver query sequence Details
15. PSPD Parameters for Probe Design ONLY e FSPD e e Check Probe BLAST hit Binding Max cc Specificity e Probe D rie Min GC Probe Design Only Probe Length Probe Period Implementation Database Support Probe Region References Unique Download User Manual Parameters required by PRIMEGENS algorithm Email for notification optional print Result Display detailed v Number of Primers Allowed Hybridization in results E Maximum possible Amplicon Size Minimum Oligo size print Sequence in Bisulfite Mode print Number of CpG sites in primer 2 University OT WViISSOUr ColumpIa Figure 12 Page for setting parameters specific to PRIN dk Run_PRIMEGENS GENS when algorithm type as Probe Design is After filling up all these parameter forms user should hit RUN PRIMEGENS for the final run of the primer design program User can hit RUN PRIMEGENS without putting any value on this page and PRIMEGENS will design primers using all default parameters 10 User Manual PRIMEGENSw3 2010 After running PRIMEGENS server will show the link to find the output files Figure13 shows the page with the link that comes after PRIMEGENS starts running P RI M G E N S W3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home Thank You for using PRIMEGENS Algorithm All your input and outfiles will be available
16. Then for Primer3 parameters user can provide specific desired characteristics of the primer that can be used by a third party program Primer3 to design primers For example melting temperature primer length etc 3 Parameters required for Fragment Specific Primer Design FSPD program These parameters are used by PRIMEGENS only when it has to design primers using FSPD algorithm Here user can provide parameters for primer design only if they opted for algorithm type as FSPD 4 Parameters required for Probe Specific Primer Design PSPD program 6 User Manual PRIMEGENSw3 2010 These parameters are used by PRIMEGENS only when it has to design primers using PSPD algorithm Here user can provide parameters for primer design only if they opted for algorithm type as PSPD 5 Parameters for Probe Design These parameters are used by PRIMEGENS only when it has to design only sequence specific probes Here user can provide parameters for probe design only if they opted for algorithm type as Probe Design Next page gives the Il t Figure 6 12 below show the input parameter pages of PRIMEGENS tool Here user an a provide PRIMEGENS their own values or just run PRIMEGENS using all default values FIQUre f atin shows one of the help pop ups available for each parameter by clicking the questionmark symbol in front of each PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Co
17. also be used to search for regions with the maximum enzyme digestion sites cut sites within each query sequence and design primers around these cut sites This ensures the presence of cut sites in the PCR product and is very useful in Methylation specific PCR Detailed description for working of this utility is present on website as Details link in front of the link to this utility For each of these utilities PRIMEGENSw3 has a simple sequence of operations which consist of two basic steps 1 Uploading data files PCR templates file for primer design and optional database for cross hybridization check 2 Primer design specifications which consist of setting various design parameters for example Primer3 parameters BLAST parameters for cross hybridization check etc and 3 Program execution and result visualizations t allows user to select three different algorithms for primer design in each of its utility They are 1 Sequence specific Primer Design SSPD allowing primer design for any random DNA sequence 2 Fragment specific Primer Design FSPD allowing multiple primer pair design distributed uniformly across target sequence for investigating large sequences 3 Probe specific Primer Design PSPD allowing users to design target sequence specific probes and associated primers pairs In addition to this it can also be used to design sequence specific probes Using web server version of PRIMEGENS software is a three step process
18. ameters In addition to these three files PSPD generate an additional output file 1 Gene specific fragment only PSPD Named as name of the query file followed by query_failed txt This file is generated only during Probe specific primer design PSPD This file contains gene specific fragment probe for each input query sequence that PSPD find using global alignment of query sequence with the database sequences These are the gene specific fragments that PSPD ultimately use to design primers for their corresponding query sequence This file could be useful for microarray probe design The primer pair designed for each query sequence as designed to amplify these gene specific probes This is a normal FASTA formatted file 18 User Manual
19. d User e e Primer num Ns accepted MERIEN size range opt size fault Primer start Primer liberal base e Primer first base index e i e PE dafasit codon position Primer max Primer self end p Primer self anv p Figure 7 Page for setting Primer3 parameters for primer design After setting all Primer3 parameters and clicking Next button PRIMEGENS asks to set BLAST parameters Figure 8 shows the page to set BLAST parameters PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home eons a Algorithm a aaa ee l e SSPD e PSPD Parameters required by BLAST program e FSPD e Check Binding Gap Open Penalty Specificity Word size e Gap Extension penalty eC e Probe Design Only Implementation Next Back Database Support References Download User Manual Copyright Acknowledgement Contact Us Designed by Garima Kushwaha Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia Figure 8 Page for setting BLAST parameters After setting all BLAST parameters and clicking Next button PRIMEGENS asks to set parameters specific to PRIMEGENS Figure 9 12 shows the page to set these parameters g User Manual PRIMEGENSws3 2010 PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home nto a n set to a default va
20. e Support References Download User Manual gt 11431057 os GTTTGCATTGTTTGCGCTCC 14 20 63 9 50 TAACAAGTTGGCAACCCCCT 164 20 E m r gt S11417968 Zea mays heat shock protein 26 HSP26 mRNA complete cds nuclear gene for plastid product cds p 91 813 gb L28712 gi 453669 ug Zm 11 len 946 TITTTTACCAAATCCGGACAGTCTTGAGCTCTCTCAGTCTCAGCATTTCAGTTCCCAGATTCACAGAGCCCCAGCCEAGA gctccgttcgcgattgccggccggctctccccCGTT GCGCGCCTCCCGGTCCGCGCCTGGAGGCCGGCGC ACGGGTTTGCGTCGTCCGGGAGAGCCCGCTCGCTCGCCGTGGCATCCGCGGCGCAGGAGAACAGGGACAACAGTE LC GC GGCAACAGGCAGCAGGGCAACGCCGTGCAGCGCCGCCCGCGCCGCGCGACA GCGCTCGACATCTCCCCATCCCCGTTCGGATTAGTTGACCCGATGTCGCCGATGCGGACGATGCGGCAGATGCTGGA CACGATGGACAGGCTGTTCGACGACGCCGTGGGGTTCCCCATGGGGACACGGAGGTCCCCGGCGACCACCGGCGA CGTACGCCTGCCGTGGGACATCGTGGAGGACGAGAAGGAGGTGAAGATGCGGATCGACATGCCGGGCCTCGCGCG CGACGAGGTGAAGGTGATGGTGGAGGACGACACGCTGGTCATCCGAGGGGAGCACAAGAAGGAGGAGGGCGCGG AGGGCGGCAGCGGCGGCGACGGCGACGGGTGGTGGAAGCAGCGCAGCGTGAGCTCCTACGACATGCGGCTGGCG CTGCCGGATGAGTGCGACAAGAGCAAGGTGCGGGCCGAGCTCAAGAACGGCGTGCTCCTCGTCACCGTGCCCAAGA CCGAGGTGGAGCGCAAGGTCATCGACGTGCAGGTCCAGTAGACAAACTGAGCTGTCACGACGGGTCTTTGTGTAGCA CTAGCGTGGGTTGGGCTCTTCGCGCCGACCGTATCAGTACTCGTGACCCAACGAGTGGAATAATGTGAATAAATAAAC GCTTCAATTCTGCTATTCC Figure 15 b Visualizing Primer position in query sequence P RI M EG E N S w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home Algorithm Design Results
21. fite Mode 2 print Number of CpG sites in primer Back Run_PRIMEGENS Copyright Acknowledgement Contact Us Figure 10 Page for setting parameters specific to PRIMEGENS when algorithm type as FSPD is chosen 9 User Manual PRIMEGENSw3 2010 PRIMEGENS w3 High throughput Primer and Probe Design Input Parameters Home rae All parameters nave been set to a default 310e Algorithm e SSPD e PSPD Parameters for Probe specific Primer Dedign PSPD e FSPD e Check Binding Min probe length Specificity Probe Design Only Min filter length jefault Max similarity Parameters required by PRIMEGENS algorithm Implementation ao Email for notification optional Database Support 2 nee print Result Display A detailed v Download User Number of Primers E Manual Allowed Hybridization in results Maximum possible Amplicon Size e Minimum Oligo size 2 default 15 print Sequence in Bisulfite Mode print Number of CpG sites in primer 2 University of Missouri Columbia Back Run_PRIMEGENS Copyright Acknowledgement Contact Us Digital Biology Laboratory Press RUN PRIMEGENS button for final execution of PRIMEGENS Figure 11 Page for setting parameters specific to PRIMEGENS when algorithm type as PSPD is chosen Mign tnrougnput Primer ana Prope Vesign Input Parameters Home All parameters have been set to a default value Algorithm e SSPD e
22. ites and Length of the Cut site region to pick query sequence around region with maximum of those cut sites All these parameter values have been assigned with some default values for testing purpose 5 User Manual PRIMEGENSw3 2010 PRIMEGENS w3 be Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Input Files Upload Query file Use Sample File Format 1 with sequence Upload your own file Browse Upload Database file Use Sample File View Upload your own file Available Genomes None Cut Site Information Number of Cut sites Cut Sites separated by comma eg COTT CGOG GO0C Length of Cut site Region Newt Reset Figure 5 Input file page for Around max cut site utility Step2 Input Parameters Next stage of PRIMEGENS server is to provide all input parameters for primer design All parameters have been set to some default values as standard parameters for best primer design Input parameters on this page of the server are divided into five sections as follows 1 Algorithm Type In this user can choose to design primers by three primer design algorithms supported by PRIMEGENS software or design just probes by choosing the last option SSPD has been selected by default 2 Parameters required for Blast and Primer3 program Here user can set parameters for MegaBLAST to look for cross hybridization of primers in database sequences provided by them
23. lue Algorithm All parameters have been set to a default value SSPD PSPD Parameters required by PRIMEGENS algorithm FSPD Check Binding Specificity Number of Primers default 10 Email for notification optional print Result Display detailed v OAM SURE Alowed Hybridization in results default Only l Maximum possible Amplicon Size default 9 Implementation Minimum Oligo size jefault 15 Database Support References Download User Manual print Sequence in Bisulfite Mode e print Number of CpG sites in primer 2 default 2 Note If uploaded sequences are in bisulfite mode Back Run_PRIMEGENS Copyright Acknowledgement Contact Us Figure 9 Page for setting parameters specific to PRIMEGENS PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia a Input Parameters es All parameters Nave Deen set a derault value Algorithm SSPD PSPD Parameters for Fragment specific Primer Dedign F SPD FSPD Check Fragment Length e default 50 Fragment Overlap p Binding Specificity Probe Design Only Parameters required by PRIMEGENS algorithm Implementation Email for notification optional Database Support print Result Display 2 detailed References Number of Primers default 1 Download User Manual Allowed Hybridization in results 2 Maximum possible Amplicon Size 2 Minimum Oligo size efault 15 print Sequence in Bisul
24. lumbia PEEN Input desae All parameters ve peen set to a aeta value Algorithm v SSPD PO e PSPD Design Algorithm a e FSPD SSPD Sequence Specific Primer Design FSPD Fragment Specific Primer Design e Check A Na aces Binding PSPD Probe Specific Primer Design OnlyProbeDesign te Specificity cPeidanieukisGwontotnbes fags Probe Design oa Only implementation Nextt a Database Support References Download User Manual Copyright Acknowledgement Contact Us Designed by Garima Kushwaha Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia Figure 6 Page for setting algorithm type 7 User Manual PRIMEGENSw3 2010 P RI M EG E N S W3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Input Parameters All parameters ave peen set toa lefa iit alue Home Algorithm Parameters required by Primer3 program to generate primers General Parameters Primer min Tm 4 Primer opt Tm Primer max Tm E Specificity aa PRIMER_MIN_TM Primer opt size e Primer max size e Probe Design fault 20 a lefault23 Only on J Minimum acceptable melting Primer min GC 2 Primer GC clamp c temperature Celsius for a primer oligo fault 40 fault0 Implementation Database Support Primer max Tm amp 8 Primer product opt Tm Primer product min Tm e References re meena ee Se Primer product Primer product Downloa
25. mple File View Mus musculus senome Database Support Saccharomyces cerevisiae Genome References Upload your own file Zebrafish Genome Download User Available Genomes None Specificity from sample files Details Manual u e Use S Cerevisae from available genomes if Format 3 Format 4 query sample is used Select Available Genomes option to Next Reset Note This website has been tested on Mozilla Firefox and Internet Explorer 8 activate the option to select any supported Figure 2 Input file page for Regular Design Utility database For Around CpG Design Figure 3 shows the input file page for Around TSS utility Here the query file is Gene symbol list file The gene symbols are taken from lists provided by UCSC Genome Browser s gene list P RI M E G E N S W3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Input Files Home Algorithm Upload Query file Use Sample File View Sample files Upload your own file Browse Binding Next Reset Specificity e Probe Design Only Implementation Database Support References Download User Manual Copyright Acknowledgement Contact Us Designed by Garima Kushwaha Digital Biology Laboratory MU Informatics Institute University of Missoun Columbia Figure 3 Input file page for Around CpG utility For Around TSS Design Figure 4 shows the input file page
26. n GmPM12 mRNA complete cds ATGGC TGAAGCACAAC TACGAGACCAGCATGGCAACCCTGTCCCACTCACCGATCAATACGGTAATCCGG TTATCTTAAC TGACGAGCGCGG TAATCCCGTCCAACTCAC TGGTGTCGC TACCACCGC TACCGGCACAGC AGGTTCTGGGTTTGGGTCC TATGG TACCGG TGC TTACGGTGG TGG TGCAAG TGCAACCACCGTTGCAGAT CTTTTGGCAACCCAACCAAGGAG TGGCAGGGAGGC TAGAGAGC TTCGTCGTTCCTCCAGTTCAAGCTCTA GCTCGTCTGAGGA TGA TGGGCAAGG TGGGAGGAGGAAGAAGGGAG TGAAGGA TAAAA TAAAAGAGAAAC T ACCAGGGG TAGGAGGAGGGAA TAATAATAAGGAGCATGCACACACAACAAC TGC TCCAACCACCGCCACT AACCACCCTGC TGATCAGCA TGAGAAGAAGGGCA TAA TGGAGAGGA TCAAAGAAAAATTGCC TGGCCACC ACACCCACTGA gt gi 6648967 65 838 Glycine max seed maturation protein PM26 PM26 mRNA complete cds ATGAGTCAGGAGCAGCCACGGCG TCCCAAAGGCCAAAACCCCATCAAATACGGCGACGTTTTTGICGICT CCGGCGACCTTGCACAGAAGCCCGTCGCACCGGAAGATGC TGCCATGATGCAAAGCGCGGAAAC TCGAGT GCTCGGACAAACCCAACCCGGCGGAGCAGC TTCCGTCATGCAATC TGCCGCCACCAGGAATGAACAGGCT GOCCTTGTCGGTCACCGGGACG TCACCGACGTTACCGGCGACCG TGGCGTCACAG TCACAGAAAC TAAAG TCCCTGGAAGACGCATTATAACCGAGGC TGTTGG TGGCCAGGTTGTGGAGCAG TATG TGGAGGCAAC TCC GGTTGAGGCAGGGC GAAGCAG TGCAATTAAGGAGAATGCCATAACAATAGGAGAGGCATTGGAGGCGACG GCACAGAC TG TGGG TCAGAAAGC GG TGGATCAGAGTGACGCC TTCGCGATTCAGGCGGCGGGAGGTGAGA GCAACGGGGAAG TAAACGTTATAACGCCGGGTGGAC TTGCGGC TATGGC TCAATCAGC TGC TGC TTATAA TGC TGAC TGCAAGC TTGACCAGGCCAAGG TCAAGC TCGCCGACATTTTGGCCGGAGCCACAGCCAAGTTG CCCOCGGACAAGGCCGCCACAC TGCAAGATGC TGAAGG TGTAGCGTG TGC TGAGG TGAGGAACAACCCTG ATGCCACCGCCACTCCCGGTGGCGTAGCCGC TTC TGTTGCGGC TGC TGC TAGGC TCAATG
27. query file followed by primers_list txt 17 User Manual PRIMEGENSw3 2010 This file contains alternate primer pairs for each input query sequences In case user wants to select alternate primer pairs this file provides multiple choices for selecting primer pairs for each query sequence This file also contains the similar information as that in first file for every alternate primers ail yee S002 10 1 Li TOG THASDAGIOAL TAAG 7 TOGTLAGOAGOGAL TARAG 2 ATOGTOADGADOGAC TAMA i CCGA TS Tk TTA TTC BS ATG TAG TOMA COCMAMAGIGS TO TETAT I COATTA EJ AAABGAGAC OL TANG LAA PI CARGRAAGOC TA TOLIKOG TE CGC AAM ANG TG TATA T siyi T l L KAATET TAAT 2 AAGTETAMSO TOCA TT I AWATOA TSC GAT 4 BORGTOTOAAGOC TOGA IT Si CGC Caan T Taal Ta 6 CETA GT AIGA Ti CRETE TG TTC GGATTTT bl GOATATSOCAGTSL TATT h TATTA TG RATAA 16 AGOGGA TT TA TG mil yaad l Em HEU RAA att Eh Cpa TAA TO Ca Ca Fi THA TT Ta al THAME TTT D MANADA ot LAMA Bb CARTI FNAM TIT di TOARTIGARAGCALOCTIT EI GC TAGS TETTA T Di GACCATTCGALCALTILCAT 1b GACCATTCGALCALTICCAT TOABAACLLABAARAL IUG ATAT TECALT IC TGR T TARAC CCAR TECG al TOALAGTTOGL AAC AMA TEC ATAT TEATA TOGT TOAAAAC TAAAAAME TECG CAAAAAGOTCA TCC MAA T ATALMALCOCIC TT ETO G BATT TAA TTC TT 1 ANAT TAATA TAT COATATECTOCAAATECCLT CCATARCTOCAMTOCCET SAT CAGCAC TOOLATE BAR LASDAL TOCCA GRIEG LATTAM T CATES CATA CTOCAMATOC CET COLAC TOCA TTEA CCRA TOCA itl TTA H CATES ATEMA
28. r chromosome position without their nucleotide sequence In case nucleotide sequence is not provided and gene name is given then database type should be single type database mentioned in database drop down menu or uploaded database sequence But if chromosome position is given database type should be genome type database where one file is present per chromosome Output format 16 User Manual PRIMEGENSw3 2010 Different number of and types of output files are generated by different design algorithm All three primer design algorithms SSPD Sequence Specific Primer Design FSPD Fragment Specific Primer Design and PSPD Probe Specific Primer Design generate three different types of output files as follows 1 Excel sheet best primer pair Named as name of the query file followed by primer xls This file contains best primer pair for each input query sequence along with other types of details as follows Column Name Description LEFT_PRIMER_GC CONTENT GC content of Left Forward primer RIGHT_PRIMER Right Reverse primer sequence PRODUCT _SIZE Product or amplicon size HYBRIDIZATION Number of hybridization for the primer in database Figure shows one sample of excel sheet output file generated by PRIMEGENS QUERY _MAPE sO LymaseTesbe7 18 L GL yma LOS L7i6 1 oi yin Loe 366 1 mG ymy Loss 3o6 2 mG Lynas Lgea756 OL ymad Lge 7630 Ol ymad 1ge93 18 OL ymad Lg 7 726 mOlymad 1938360 oil ymad 19360
29. ry file Use Sample File View Sample files owse Upload your own file Format 1 no sequence Format 2 with sequence Format 2 no sequence Format 3 with sequence Format 3 no sequence Format 4 with sequence Format no sequence mat used database must contain corresponding sequences d in no sequence query format for genomic database s check format from sample files Details Upload Database file Use Sample File View Upload your own file Browse_ Available Genomes None e Use S Cerevisae from available genomes if Format 3 Format 4 query sample is used Next Reset Note This website has been tested on Mozilla Firefox and Internet Explorer 8 3 User Manual PRIMEGENSw3 2010 PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Input Files Home Algorithm Arabidopsis TAIR9 cDNA g Arabidopsis TAIR9 CDS Upload Query file Maize CDS SPD Medicago CDS e d PD e Use Sample File E Rice all cDNA sp Upload your own file Rice all CDS j Sorghum all CDS pea te eh enc base must contain corresponding sequences sane i eget ape nce query format for genomic database Homo sapiens Genome Bisulfite Human Genome meee lt lt Anopheles gambiae Genome Only Caenorhabditis elegans Genome Upload Database file Drosophila Melanogaster Genome implementation a oo i DEER Use Sa
30. ry location on genome without its nucleotide sequence The database type must be genome like human genome in this case chri chri chri chri chr chrx chrx chrx chrl chri chrl chrii chrx chrx chrx chri chris chr chrl 42438529 42439129 chr chr9 chr7 chrs chri 143s 7117 143 7717 1499825 1500425 15 40446 1541046 1557122 1557722 1448383709 144838909 119942159 119942759 1199470 19 119947619 1199470 19 119947619 1645335 1645935 1645335 1645935 1645335 1645935 1645335 1645935 119912975 119913575 119912975 1199155 75 L199 12975 119913575 1645335 1645935 1645335 1645935 72699408 72700008 144838309 144838909 130354386 130354986 143 43 7308 143 43 7908 1448383 70 9 144838909 166699 1 1667591 4 FASTA format with chromosome position with functional description That is query location on genome and description without its nucleotide sequence The database type must be genome like human genome in this case 15 User Manual PRIMEGENSw3 2010 chr14 60182406 60184005 SIX1 right sine oculis homeobox homolog 1 chr2 45084347 45085946 SIX left sine oculis homeobox homolog 2 chr2 45085047 45086646 SIX2 cross sine oculis homeobox homolog 2 chr2 45022441 45024049 SIX3 right sine oculis homeobox homolog 3 chr14 60044975 60046574 SIX6 cross sine oculis homeobox homolog 6 chr20 253439 255038 SOX12 cross SRY sex determining region Y box 12 chr3 158965469 13896
31. ver Figure2 c shows the available genomes options on webserver a PRIMEGENS w3 High throughput Primer and Probe Design Home Algorithm Check Binding Specificity e Probe Design Only Implementation Database Support References Download User Manual PRIMEGENS w3 High throughput Primer and Probe Design Home Algorithm e SSPD e PSPD e FSPD e Check Binding Specificity e Probe Design Only Implementation Database Support References Download User Manual c Digital Biology Laboratory University of Missouri Columbia Input Files Upload Query file Select Upload you own Use Sample File Format 1 with sequence View Sample files Browse file option to activate Upload your own file the option for browsing f no sequence query format used database must contain corresponding sequences e Use gt chrX start end in no sequence query format for genomic database e Please check format from sample files Details your file Upload Database file Use Sample File View Upload your own file Browse Available Genomes None e Use S Cerevisae from available genomes if Format 3 Format 4 query sample is used C Jsa After uploading two Note This website has been tested on Mozilla Firef Seana corresponding files click next to move on to next page Digital Biology Laboratory University of Missouri Columbia Input Files Upload Que
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