User Manual
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1. Check Left Left Left Left Right Right Bindina Query name Left Primer Sequence primer Startprimer T primer Right primer Sequence primer Startprimer ae ition Length P Gc content ition Length P Specificity pos g pos g Probe Design S em CGACATGGTCAAGGTCATCT 1145 20 58 5 50 AGTCATTTCGGGGTGAATCC 1313 20 E Only id rcc AAACAGGGTAGAGCGTgagga 32 21 612 52 AAGTIGATGGGCACGATCTC 199 20 Database Support PM 2811417968 veferences Zea CACAAGIAGCGAAAtggcag if 20 59 5 50 ACACTGITGICCCTGIICICCT 244 22 Download User at Manual ecd GTTTGCATTGTTTGCGCTCC 14 20 639 50 TAACAAGTTGGCAACCCCCT 164 i p m TT zd 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 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 Check Left Left Left Left Right Right z Binding Query name Left Primer Sequence primer Start primer dacim primer Right primer Sequence X primer Start primer p Sp aie an position Length P GC content position Length priz UET CGACATGGTCAAGGTCATCT 1145 202. Maximum possible Amplicon Size e Minimum Oligo size e print Sequence in Bisulfite Mode e print Number of CpG sites in primer e efault 2 Back Run_PRIMEGENS Copyright Acknowledgement Contact Us Figure 10 Page for setting parameters specific to PRIMEGENS when algorithm type as FSPD is chosen PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia oad Input Parameters All parameters have been se o a detal Algorithm e SSPD PSPD Parameters for Probe specific Primer Dedign PSPD FSPD p E Check Min filter length efault Max similarity lefault C Binding Min probe length Specificity Probe Design Only Parameters required by PRIMEGENS algorithm Implementation l uM i i Email for notification optional Database Support print Result Display e detailed v Download User Number of Primers e Manual Allowed Hybridization in results e derauit l Maximum possible Amplicon Size e Minimum Oligo size e efault 15 print Sequence in Bisulfite Mode e print Number of CpG sites in primer e Back Run PRIMEGENS Copyright Acknowledgement Contact Us Figure 11 Page for setting parameters specific to PRIMEGENS when algorithm type as PSPD is chosen 9 User Manual PRIMEGENSw3 2010 AIgn tnrOugnput Primer ana rrope Vesign UniVersity OT IVISSOUIT GOIUITIDIG Home ydus A paramete
3. 585 50 AGICATITCGGGGIGAATCC 1313 20 7 Only Implementation fee AAACAGGGTAGAGCGT gagga 32 21 61 2 52 AAGTTGATGGGCACGATCTC 199 20 Database Support References Download User Manual in d GITIGCATIGITIGCGCICC 14 20 639 50 TAACAAGTTGGCAACCCCCT 164 20 4 Tm gt S11417968 Zea mays heat shock protein 26 HSP26 mRNA complete cds nuclear gene for plastid product Icds p 91 813 gb L28712 gi 453669 ug Zm 11 len 946 TITTTTACCAAATCCGGACAGTCTTGAGCTCTCTCAGTCTCAGCATTTCAGTICCCAGATTCACAGAGCCCCAGCCEAGA AGTAGCGAAAiggeaat gctccgttcgcgattgeceggcecggctctecec CGTIGCGCGCCTCCCGGTCCGCGCCTGGAGGCCGGCGC ACGGGTTTGCGTCGTCCGGGAGAGCCCGCTCGCTCGCCGTGGCATCCGCGGCGCAGGAGAACAGGGACAACAGTE es SGC GGCAACAGGCAGCAGGGCAACGCCGTGCAGCGCCGCCCGCGCCGCGCGACA GCGCTCGACATCTCCCCATCCCCGITCGGATTAGTTGACCCGATGTCGCCGATGCGGACGATGCGGCAGATGCTGGA CACGATGGACAGGCTGTTCGACGACGCCGTGGGGTTCCCCATGGGGACACGGAGGTCCCCGGCGACCACCGGCGA CGTACGCCTGCCGTGGGACATCGTGGAGGACGAGAAGGAGGTGAAGATGCGGATCGACATGCCGGGCCTCGCGCG CGACGAGGTGAAGGTGATGGTGGAGGACGACACGCTGGTCATCCGAGGGGAGCACAAGAAGGAGGAGGGCGCGG AGGGCGGCAGCGGCGGCGACGGCGACGGGTGGTGGAAGCAGCGCAGCGTGAGCTCCTACGACATGCGGCTGGCG CTGCCGGATGAGTGCGACAAGAGCAAGGTGCGGGCCGAGCTCAAGAACGGCGTGCTCCTCGTCACCGTGCCCAAGA CCGAGGTGGAGCGCAAGGTCATCGACGTGCAGGTCCAGTAGACAAACTGAGCTGTCACGACGGGTCTITGTGTAGCA CTAGCGTGGGTTGGGCTCTTCGCGCCGACCGTATCAGTACTCGTGACCCAACGAGTGGAATAATGTGAATAAATAAAC GCTTCAATTCTGCTATTCC Figure 15 b Visualizing Primer position in query sequence 12 Us
4. ATGCCACCGCCACTCCCGGTGGCGTAGCCOGC TTCTGTTGCGGC TGC TGC TAGGC TCAATGAAAATGTTAA GTAA 91 4838146 81 503 Glycine max seed maturation protein PM36 PM30 mRNA complete cds ATGGCATCCCATAGGCAAAGCTATGAAGC TGGTCAAAC TAAGGGCCGAAC TGAGGAAAAGACGAACCAGA CGATGGGCAATATTGGAGAGAAGGC TCAAGC TGCAAAGGAGAAGACCCAGGAAATGGCCCAAGC TGCAAA GGAGAAGACCCAACAAACAGCCCAAGC TGCCAAGGACAAGAC T TGCGACAC TTCCCAAGCGGCAAAGGAG AAGACCCAACAGAATACAGGAGCTGCTCAACAAAAGACCTCAGAGATGGGCCAGTCCACGAAGGAA TCGG 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 or 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
5. Binding mop nce query format for genomic database Homo sapiens Genome Bisulfite Human Genome meee elie Anopheles gambiae Genome Only Caenorhabditis elegans Genome Upload Database file Drosophila Melanogaster Genome Implementation Guinea Pig Genome Use Sample File View Mus musculus Genome Saccharomyces cerevisiae Genome Specificity from sample files Details Database Support References Upload your own fil Zebrafish Genome Download User Available Genomes None Manual Next Reset Note This website has been tested on Mozilla Firefox and Internet Explorer 8 Figure 2 Input file page for Regular Design Utility 3 User Manual PRIMEGENSw3 2010 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 e SSPD e PSPD e FSPD e Check Binding Specificity Use Sample File View Sample files Upload your own file Browse Next Reset e Probe Design Only Implementation Database Support References Download User Manual Copyright Acknowledgement Contact Us Designed by Garima Kushwaha
6. 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 16 User Manual
7. these parameters PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home ocala sian PR a Algorithm acid dc CC ena ee SP SP Parameters required by PRIMEGENS algorithm SP Email for notification optional e Check Binding print Result Display e detailed Specificity Number of Primers default 1C ixi E ME ioed Hybridization in results default 0 Only l Maximum possible Amplicon Size e default 9 Implementation Minimum Oligo size e lefault 15 Database Support References Download User Manual print Sequence in Bisulfite Mode e print Number of CpG sites in primer e Back Run PRIMEGENS Copyright Acknowledgement Contact Us Figure 9 Page for setting parameters specific to PRIMEGENS 8 User Manual PRIMEGENSws3 2010 PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Input Parameters Home All parameters have been set 3 default va Algorithm SSPD PSPD Parameters for Fragment specific Primer Dedign FSPD FSPD Chec k Fragment Length e lefault 5 Fragment Overlap e Binding Specificity Probe Design Only Parameters required by PRIMEGENS algorithm Implementation Email for notification optional Database Support print Result Display e detailed Referenc Number of Primers e Download User Manual Allowed Hybridization in results e
8. 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 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 TSS to pick query sequence around TSS Both of these parameter values have been assigned with some default values for testing purpose 4 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 Files Upload Query file Use Sample File Human gene symbols View Sample files Upload your own file Browse 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 regi
9. ENS can 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 PRIMEGENSwS 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 It 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 proc
10. FASTA format Following figure shows one sample file for this format gt gi 2276989 16 516 Glycine max dehydrin GmPM12 mRNA complete cds ATGGCTGAAGCACAAC TACGAGACCAGCATGGCAACCCTGTCCCACTCACCGATCAATACGGTAATCCGG TTATCTTAACTGACGAGCGCGGTAATCCCGTCCAACTCACTGGTGTCGCTACCACCGC TACCGGCACAGC AGGTTCTGGGTTTGGGTCCTATGGTACCGGTGCTTACGGTGGTGGTGCAAGTGCAACCACCGTTGCAGAT CTTTTGGCAACCCAACCAAGGAGTGGCAGGGAGGC TAGAGAGCTTCGTCGTTCCTCCAGTTCAAGCTCTA GCTCGTCTGAGGATGATGGGCAAGG TGGGAGGAGGAAGAAGGGAG TGAAGGA TAAAA TAAAAGAGAAAC T ACCAGGGGTAGGAGGAGGGAATAATAATAAGGAGCATGCACACACAACAACTGCTCCAACCACCGCCACT AACCACCCTGCTGATCAGCATGAGAAGAAGGGCA TAATGGAGAGGATCAAAGAAAAATTGCCTGGCCACC ACACCCACTGA gt gi 6648967 65 838 Glycine max seed maturation protein PM26 PM26 mRNA complete cds ATGAGTCAGGAGCAGCCACGGCGTCCCAAAGGCCAAAACCCCATCAAATACGGCGACGTTTTTGTCGTCT CCGGCGACCTTGCACAGAAGCCCGTCGCACCGGAAGATGC TGCCATGATGCAAAGCGCGGAAAC TCGAG T GCTCGGACAAACCCAACCCGGCGGAGCAGCTTCCGTCATGCAATCTGCCGCCACCAGGAATGAACAGGCT GGCCTTGTCGGTCACCGGGACGTCACCGACGTTACCGGCGACCGTGGCGTCACAG TCACAGAAAC TAAAG TCCCTGGAAGACGCATTATAACCGAGGC TGTTGGTGGCCAGGTTGTGGAGCAGTATG TGGAGGCAAC TCC GGTTGAGGCAGGGCGAAGCAGTGCAATTAAGGAGAATGCCATAACAATAGGAGAGGCATTGGAGGCGACG GCACAGACTGTGGGTCAGAAAGCGGTGGATCAGAGTGACGCCTTCGCGATTCAGGCGGCOGGAGGTGAGA GCAACGGGGAAGTAAACGTTATAACGCCGGGTGGAC TTGCGGCTATGGC TCAATCAGC TGC TGC TTATAA TGCTGACTGCAAGCTTGACCAGGCCAAGGTCAAGC TCGCCGACATTTTGGCCGGAGCCACAGCCAAGTTG CCCGCGGACAAGGCCGCCACAC TGCAAGATGC TGAAGGTGTAGCGTGTGCTGAGGTGAGGAACAACCCTG
11. G 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 If the current PRIMEGENS run is taking too long this http primegens org Garima cgi bin 24894 can be bookmarked or copied to access the result files Also memories 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 Copyright Acknowledgement Contact Us Designed by Garima Kushwaha Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia Figure 14 a Next page after hitting View Status button on last page shown by Figure13 PRI M EG E NS w3 Digital Biology Laboratory be 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 l Specificity lf the current F 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 meee the user can p order confirmation that was perfo
12. GGGUCAA TGGOTTTATCCCAGTTCCAG GTCTTCAAAAGGGATGOCAA CCGCAGAGAAGAGGACAAAC CAATTTCCATATCCCAACGG ATCCCCCAGGARAPRAAGAGA AGGTGOGTGCTGTCAAAGTC GCAACTCTCCGTTGAACTCC TGCAGAGANCATOGC TTC AG GCCAACTGTCAGAAACAGCA GCTGGCAATCAATACAGGGT ACATAGACGCTGCAAACGTG TCACACAGAGAATTACOCOG GACCCAGCTCARAGACAAGC GCACTGATTTTCACGCAGAA AGCACGAGC TGGATTTGTTT AGCACGAGC TGGATTTGTTT TTCATAAAS TC GGG TGGAGC RIGHT PRIMER TGAAAACCCARRAARCTCCG CCATATCCTCCAAATCCCCT AAAGGG TGCTGATCAACTGG TGCACTGTCTTCCTGGAGTO GGTCAGCCTTGATOGARAAA GaAGTACCOAACOTCOTTOT B AATOCCACAAORCCATOA CCTCTCCTGCTCAGATOGTC ATOACGGAGT TOGO TGOAGAC AGGGTAAACCAACAGAGCGA TATAGGCCTGGATTTGACGC GCGTCTATGCCTATGGCTTC AACAGCAGCAAATE TT GOAL ASGGOOTTGTOTTIGTT MA AGG TCCOGGTGAG TCTCTTT CACTTCTCCAGNGGCAGACC CCCAAALC TOC TTCAACATT CCATAACRGUAATCOLADOT CACCATTTCAAAGCCCAGTT CCARAAAGCATGGCAAAGAT ATCAGTGGCATCATGCTTCA TGCACTGTCTTCCTGGAGTO TGCACTGTCTTCCTGGAGTO GTGTGAACAGCOGATAGC AA MANE RO op ele ee ee d d d p ops ps Pu MuR g g PRIMEGENSw3 2010 2 Alternate primer pairs detailed Named as name of the query file followed by primers list txt 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 al pu 0506218 1 TGGTGAGGAGOCA
13. L TORAAG I RT TMG TAA a CCAM TE TGA TTA TT S ATG TSA TOR TOCAR STO TOTAT CGGAGTTTTTTGGOTTTTCA AAAS TOMGA CAADAARCC TA TT OCANGL I Cac BARR eS TG TOTAT Lae 1901793 1 Li Mis TOT CTGC AT 2 AGAGTGTGAACC TGCOATT Y MACGAGTOTGAAGCCTOCOAT 4 AGAGTGTGARGCCTGCCATT a COGCAGOGATTORDAARTAR 6 CC TGR TAS 7 CAGTGCTGGATTCGGATTTT E GOATATGGC AG TC TCATT 8 TATTGATG TODA TOAGOLCA 15 AGGGDATTTGDAGOGA TATOG Gl yng 1904358 1 Li CAADAGRA OCC Bataan zb COMDTACAATUOGCCAGAL AA 3 THA TTTGCCA 4 TAA TTT S ACAAGAGAAE G0 AAAA 6 CCAGTTGARCAGCACCCTTT 7 COAT IGA RLAGCALOCTIT EL COC TAGS Tp TGA Bb GRCCATTOGA CAL TIC CAT 16 GACCATTCGALCALTICCAT TOARARC LL ABAABAC ILOG ATCATC TRLAL TRE TOT TARAA CL ARARAAC TELG Tia WG TT AA AMATUC ATCATCTGCACTTCTCGGGT TAA CCAUAAAUAC TELS CAMARGGTCATCCGCAAAT ATACALACLCE IC TTE TOL G GATT PSS TTL AGER TL T ATCATCTGCAL TR TOG T COATATECTOCAMATOCCOT CCATARCCTCCAMTCCECT MR OAA TOCCA TATLI AAELLAGLAC TOCA LATI BACCAGCAC TOCCA TATI CCATARCCTCCAMATECCCT CCRCACTCCAM GA TTEA CETA TE CAAA SOGA TTEA CAAT OLATA EN TAATTCC TCOGCATTCCATC AMO TOC THA TC AC TGG TOGA RT T TEE TOGAGTG AMS ad TTT TETELA DOL E Pana Tol WATTTTTGGLA TA TTNG E TETTCCAMAAC M TT ACTTGCTTTTGTE TC AT CAA TTT CRT Ba EF RF RF PF e h ee 9 EE iE 4 pe ded dm BeE eR dex R3 Ful Pad Had Hu Ung Gu Fus Feg Feu olmadi T s E62 16 1 5 plynai TOS E62 18 11287 lysed 70586215 1 3B Gl yaad 70s 6218 IGE G
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 PRIMEGENSw3 s different utilities for primer and probe design are Regular Primer Design Cover CpG Island Around TSS Around max cut sit region ali udi Figure 1 shows the webpage showing different options for the user choos for primer or probe design for these utilities P RI M EG E N 9 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 AE 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 Detai
15. Tm e Pura pow e Primer min GC e Primer product opt Tm e Primer product opt size Primer first base index e auit Primer self anv o Digital Biology Laboratory University of Missouri Columbia Primer max Tm e Primer max size o Jefault 23 Primer GC clamp default O0 Primer product min Tm e Primer 4 j l num Ns accepted e Primer start codon position e Primer max 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 7 User Manual PRIMEGENSw3 2010 PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home ou cM MM EE Algorithm jet ae eae rome ie e SSPD e PSPD Parameters required by BLAST program F ce Word size 9 e Check Binding Gap Open Penalty e specificity Gap Extension penalty e 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
16. ault values Figure7 shows one of the help pop ups available for each parameter by clicking the questionmark symbol in front of each 6 User Manual PRIMEGENSws3 2010 PRIMEGENS w3 High throughput Primer and Probe Design Home Algorithm FSPD Check Binding Specificity Probe Design Only Implementation Database Support References Download User Manual Design Algorithm Input Parameters h h Iof parameters Nave Deen sg 1 default PSPD Probe Specific Primer Design Only Probe Design Digital Biology Laboratory University of Missouri Columbia E Next page gives the Hic user all parameter specifications used for primer probe design Copyright Acknowledgement Contact Us Designed by Ganma Kushwaha Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia Figure 6 Page for setting algorithm type PRIMEGENS w3 High throughput Primer and Probe Design Home Algorithm e SSPD PSPD FSPD Check Binding Specificity Probe Design Only Implementation Database Support References Download User Manual Input Parameters All parameters Parameters required by Primer3 program to generate primers General Parameters Primer min Tm e PRIMER MIN TM Minimum acceptable melting temperature Celsius for a primer oligo Primer max Tm V9 Primer product size range e Primer liberal base e Primer self end o Primer opt
17. database where one file is present per chromosome 14 User Manual PRIMEGENSw3 2010 Output format 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 HYBRIDIZATION Number of hybridization for the primer in database Figure shows one sample of excel sheet output file generated by PRIMEGENS QUERY NAME 61ymao078506218 1 mG Lyme LQ Lia 1 5lymy1954358 1 lymn19204350 2 lyna01g54756 jlymas1g67688 OL ymad 1ge93 18 OL yma Lg 7 726 OL ymad 1938368 oil ymad 1g 36048 61yman1937098 plynai 1937768 5lyna1937768 2 5lym1939268 1 lyn1939858 1 lym lgl L jlymas1g4 1856 2 Glyma 1g44918 1 OL ymad1g45 748 1 iL ymad 1g45 748 2 iL ymad2ge2 74a 1 OL ymad2ge 3460 1 Polym agados 7 gt il ym 2953578 L par p p p p gnus pl l 15 User Manual LEFT PRIMER TGG6TGAGGEGGGAC TGAAAG AMGAGTGTGAAGCCTGCGAT CAAGAGAACOGCCAANDANG COAG TACAATCOCCAGACAA AATOASOGCATGCCAATE TC CTTCGCCACTCCTATCAAGC GTGAATGTCCTAA
18. er Manual PRIMEGENSws3 2010 PRIMEGENS w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home Design Results Algorithm Query ei Fragment Probe Sequence start end Bindin 11529022 0000 0099 GCACGAGAACCGCTCCTCTGGTCCGTGGCCATGGCGTCGTCCATGCTCTCCGCTACCACCGTGCCACTCCAGCAGGGGGGCGG CGGCATCGACCTCGTCATCGAGGGCACCGGCGTCTICGTCGACCGCGAGGGCGCGGGGAAGCACATCCAGGCGGGGGCCAAG S11529022 1000 1099 AGGTGAACCAGGCGTTCCGCGACGCCGCGGCCAACGAGCTCACGGGCATCCTCGAGGTCTGCGACGTGCCGCTCGTGTCCGTC l 522334604 0000 0099 GACGACGTTGCGCAGAGAATCCCCAAGCAAACAAACAGGGTAGAGCGTgaggagaggagaggagaggagaggagaggI TGGGTCTG caus aa P 522334604 0500 0599 TGAGAAGCTGAAGAAGGTGCTAGAGGTGTACGAGGCACGCCTGACCAAGTGCAAGTACCTIGCTGGAGACTICCTCAGCCTCG References 811417968 0000 0099 TITTTTACCAAATCCGGACAGTCTTGAGCTCTCTCAGTCTCAGCATTTCAGTTCCCAGATTCACAGAGCCCCAGCCCCACAAGT Download User Manual 11529022 0500 0599 D m oDe Vesk p j 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 13 User Manual PRIMEGENSw3 2010 Input File Format PRIMEGENSw3 supports query file in
19. ess 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 PRIMEGENSws3 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 PRIMEGENSws Figure2 c shows the available genomes options on webserver a PRIMEGENS w3 Dig
20. ital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Input Files ome elect Available Algorithm ae Genomes option to e SSPD e PSPD Use Sample File Format 1 with sequence View Sample files SelactiVbiaaad Voti Owo e FSPD Upload your own file Browse Ty e Check S E fileSebettamtosaptvarted o sequence query format used database must contain corresponding sequences Binding Use gt chrX start end in no sequence query format for genomic database thedop isas or browsing Specificity Please check format from sample files Details Probe Design r file Only Upload Database file Implementation ile Viev Database Support Use Sample File View References Upload your own file Browse Download User Available Genomes None Manual D da j e Use S Cerevisae from available genomes if Format 3 Format 4 query sample is used d c pier PRISE iin ZIP corresponding files b click next to move on to ge P RI M E G E N S A w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Input Files Home Arabidopsis TAIR9 cDNA Algorithm i J Arabidopsis TAIR9 CDS Upload Query file pot Maize CDS e SSPD m l Medicago CDS e PSPD Use Sample File Rice all cDNA Upload your own file Rice all CDS FSPD pe Sorghum all CDS Check p I E base must contain corresponding sequences
21. l yaad TOS 2 10 LMJ Gali TT 1157 GL ya 0902 0 1152 Glyaadi3o6 Tm 186 Glati 00500210 184 Elymai TN LETLI Gl ymae sii 1171 Glysali396 7730 1072 GUgna i 6506218 11721 Glynda 7778 Li 130 GL yai 6 200860218 D38 Gl yaad 1901754 1 34 Glyaad 198 1758 1 3 blynad 1981758 1 86 Glad 1961758 1 85 Glysa 1561758 1 153 Ulysa 1581758 LUTE Glynd 1901753 1 18 Glas 1901758 1155 Glyaad 190 1758 1 82 Glynad 1981758 1122 Giyas 1964300 2 196 Goya 1984988 11451 Glyt 1564300 2 861 Gl yaad 1964300 LUNG Glysad 190300 2 118 Goya Lge 1188 Gisnad 1900 2 76 Goya 98400 LiTE Glast 194300 2 280 Goya 190 990 216 plyaj 1964360 2117 GLysad gl 14107 Glysa 1964300 2 121 Gl ynad 198436 1121 Glyt 1564300 2 125 GL yaad 1984288 111361 Giyas 1964300 2 133 Goya 198400 1133 Glad 1g 300 2095 Goya 190400 1085 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 parameters 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
22. ls 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 PRIMEG
23. nk You for using PRIMEGENS Algorithm All your input and outfiles will be available her http primegens org Garima cgi bin 8488 After the completion of primer design PRIMEGENS e SSPD is running It might take few minutes to hours to pdate the e PSPD e FSPD e Check n Binding View Status Specificity Click View Status button to check PRIMEGENS run status If PRIMEGENS fun is complete clicking this button will automatically rediredct user to result page ei Probe Design Only Te Return to our home page Implementation n Database Support 7 Click on this link to access References Download User Manual i your final results This link will show all the input and output files for the current Copyright Acknowledgement Contact Us Designed by Garima Kushwaha process Digital Biology Laboratory MU Informatics Institute University of Missouri Columbia Figure13 Page after running PRIMEGENS 10 User Manual PRIMEGENSw3 2010 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 E
24. on 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 sites 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 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Input Files PRIMEGENS w3 EH Upload Query file Use Sample File Upload your own file o sequence query format used database must contain correspond ng sequences se ch d sequ y format for genomic database ease chec ormat from sample files Details Upload Database file Use Sample File View Upload your own file Available Genomes None F WFo mple is used Cut Site Information Number of Cut sites Cut Sites seosrsted by comms eg CCTT CGCG GCCC Length of Cut site Region Next Reset Figure 5 Input file page for Around max cut site utility 5 User Manual PRIMEGENSw3 2010 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
25. rmed earlier MEGENS run for future use Only V E mail ade ees Implementation Please refresh this page to see the updated results 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 11 User Manual PRIMEGENSws3 2010 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 FSPD on its corresponding sequence
26. rs have Deen se D a deraul We Algorithm e SSPD e PSPD Parameters for Probe Design ONLY e FSPD e Check Probe BLAST hit e Binding Max cc Specificity ig f ps Min GC e e Probe Design Only Probe Length e Probe Period e Implementation Database Support Probe Region References Unique Download User Manual Parameters required by PRIMEGENS algorithm Email for notification optional print Result Display e detailed v Number of Primers e Allowed Hybridization in results e Maximum possible Amplicon Size e 96 Press RU N P RI M EG E NS Minimum Oligo size e i e eeufte uote button for final execution print Sequence in Bisulfite Mode Reet print Number of CpG sites in primer e efault 2 ote If uploaded sequences are sulfite aga of PRIMEGENS Figure 12 Page for setting parameters specific to PRI chosen EGENS 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 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 E G E N S w3 Digital Biology Laboratory High throughput Primer and Probe Design University of Missouri Columbia Home Tha
27. 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 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 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 Figure 6 12 below show the input parameter pages of PRIMEGENS tool Here user can provide PRIMEGENS their own values or just run PRIMEGENS using all def
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