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RTPCR User Guide - Gene

1. ExpornAs SVG Y Y Y sample4 1 1207 0 1064 FFFF55 vw S samples 0 809 0 026 E FFSSFF S sampleb 0 8212 0 0135 455FFFF vw 8 4 3 Quality Control Quality Control 1s only useful when multiple reference genes have been selected Page 41 63 Stephan Pabinger User Guide PCR The CV value represents the coefficient of variation of the normalized reference gene expression levels A value lower than 50 for heterogeneous panels lower than 25 for homogeneous is typically observed for stably expressed reference genes The M value geNorm represents the mean stability measure of the used reference genes A value lower than 1 for heterogeneous panels lower than 0 5 for homogeneous is typically observed for stably expressed reference genes The lower these quality values are the more stable the reference genes are expressed in the tested samples The next section checks if a NTC has been used for each selected target In the next table the differences between the technical replicates 1s calculated and the user can choose to color the ones that are above a certain user defined threshold Multiple Targets Single Target IHK Quality Control CV M geNorm detector1 8 04 0 2565 detector2 8 72 0 2565 Mean 8 88 0 2565 target has NTC
2. true Sample ddct SE ddCt SD dact sample 1 0 0 023332 0 032996 samples 1 0 0 028335 0 040074 samples 1 0 0 05177 0 073213 Avg replicates 1 1 0 0 017272 0 028815 replicates 2 1 0 1 0 0 0306 0 093 false sample ddCt SE ddCt SD dadct sampled 1 0 0 067074 0 094656 samples 1 0 0 06134 0 026818 sampleb 1 0 0 019126 0 027049 Avg replicates 2 1 0 0 025289 0 043801 10 Error propagation Throughout the whole analysis pipeline error propagation is performed to ensure statistical relevant results It is based on truncated Taylor series expansion and uses general valid error propagation laws Included in the pipeline is e Technical replicate handling e Efficiency correction e Normalization using reference genes e Inter run calibration e Referencing the normalized Cq values to sample s e Averaging of biological replicates Page 47 63 Stephan Pabinger User Guide PCR 11 User settings The user settings interface can be reached by clicking on the corresponding link described in chapter 2 2 1 Preferred Cq Analyzer s and Preferred Efficiency Analyzer s defines the analyzer s that is are used when the user starts a multiple parse described in 3 3 Use NTC s in Cq analysis Normalization defines whether the non template controls are used in the corresponding analysis Chart Background defines the background color that is used in every chart default 1s white User Settings memaw AnalvzerMiner Preferre
3. b o E E e e e e e E E oe Eo En ez F D emitted Bllempy Buses HEN available for calculation 13 6 2 Target Efficiencies By pressing on Target Efficiencies alist of the currently stored efficiencies is shown For each target the following information is displayed e Efficiency e Standard error of this efficiency e Slope e R2 e If this efficiency is used for this target e The run used to calculate the efficiency run used to perform the primer validation e If available a button to set this efficiency as the current on activate e Alink to delete this efficiency It is possible to perform several primer validations for one target and store each calculated efficiency Therefore the system provides the opportunity to set one efficiency as the active one which is loaded into the analysis settings when an experiment is analyzed see 8 1 4 To switch the currently active efficiency the button in the Activate column has to be pressed Page 57 63 Stephan Pabinger User Guide PCR Target Efficiencies Query gt Edit Display Settings There are currently 670 targets with na efficiency in the database a DetectorEfficiencys per page 15 25 50 100 5 DetectorEfficienceys found Page lofi go to page E go Secrecy supe p2 used Rm Acte 1858 rRIA cM 1 78402 0 05033 3 93969 0 98371 Jes xX E GAPDH hl a 81782 0 04924 3 85282 0 98583 om testi X E HAMS MM 2 07412 0 152
4. Clipped Files Component Files DeltaRn Files XML Files New File Upload Fe PO pDuehsuchen File uploads are visible for all users of the same institute When deleting files the user can choose to delete associated runs experiments and additional files by ticking Delete associated Runs and Experiment LE o You are going to delete Delete associated Runs and Experiments File s test Component By pressing the Delete button a list of entries is shown that test deltaRn are going to be deleted Run s test Experiment s Page 13 63 Stephan Pabinger User Guide PCR File Upload 9 Query fa Edit Display Settings FileUpload per page 15 25 50 100 19 FileUpload found Page loti go to page go Nr 1D Uploadame Category Added Date 20070129 di f 081 5200 20071108 2 2007 11 14 di 588 AA il rr sone ALEA A e O ok a e O AE LOLA 6 soso 2007 00 26 8 S 50 Files es pl EIA S EU ALA mim g 4500 test plate 2007 01 30 Page 14 63 Stephan Pabinger User Guide PCR 3 2 Multiple Upload The multiple file upload interface allows the user to upload several files at once The type of the file File Export File is determined automatically 1f option 1s selected Files are added using the Add Files button and after uploading them a status message 1s shown If this page does not show up please check if Java Version 1 5 1s installed
5. Multiple File Upload ey Add Files HE Remove Filename rectory Size kb Filel sds Pocuments and Settings 4dminist i T 4 Filed Component csv Ci Documents and Settings Administ File DeltaRn csv Ci Documents and Settings Administ E File sds Cu Documents and Settings Administ 16925 4 File s 18340 Kb to upload left Compress Files automatic Upload Cancel You need a Java Plugin Version 1 5 x Using compression is only recommended for remate uploading data that can be well compressed ed plain text files xml files and not images ar already compressed files Cancel Page 15 63 Stephan Pabinger User Guide PCR 3 3 Multiple Parse The multiple parse section is put into the Upload amp Parse menu in order to increase the usability because typically files are parsed and analyzed immediately after uploading This interface shows by default only files SDS IXO CSV which are not linked to a run and therefore have not been parsed yet Moreover the user can specify whether all files of the institute or only the files uploaded by the user are displayed In addition all uploaded files can be shown in this interface Each file can be accompanied by a maximum number of three additional export files Export file which are automatically linked to the according file Remark Export files need to contain the file name e g SDS myFile sds rn myHile clipped txt in order to be corr
6. example sampled detector 28 0017 1 2338 Page 29 63 Stephan Pabinger User Guide PCR 6 Experiment An experiment in the application QPCR maps a real world qPCR experiment It consists of a name a creation date an optional description and numerous runs New Experiment Mare Experiment Date 14 11 2007 ea Description Runs The experiment list is somewhat different to normal table views 2 4 4 because it contains an additional symbol for each entry By clicking on it A the user 1s guided to the analyze setting page 8 1 Experiments that cannot be analyzed have a grayed out symbol experiments that can be analyzed have a colored symbol Experiment amp Query f Edit Display Settings Experiments per page 15 25 56 100 2 Experiments found Page lofi go to pace go Nr Name Date T FEE exerment 20071108 2000 01 21 d S X 2 germen 20070104 150212 df S8 X Experiments per page 15 25 50 100 2 Experiments found Page lofi go to page go Page 30 63 Stephan Pabinger User Guide PCR The detailed view of an experiment contains its name creation date description and added runs The Go button next to analyze sends the user to the analyze setup page Each added Run can be viewed by pressing the Show button next to the run an by clicking on Show Cq and Efficiency Results the user is sent to the Cq Analyze Results page 5 3 Show Experiment
7. 85769 031478 0 971 371783 v Displayed below is the message shown when Cq values don t exist Cq Analyzer AnalyzerMiner Coq values do MOT exist Calculate Values The lower part of the page shows a graphical representation of the fitted line for the selected target Targets can be selected by choosing the corresponding entry in the nearby combo box Below the chart a representation of the plate layout 1s displayed that marks the wells currently used to calculate the efficiency The system allows the user to de select wells used for calculating the curve either by pressing on the point in the graph or by pressing on the colored well in the grid Results are updated on the fly after the particular well has been in excluded from the calculation The color code used in the plate layout representation 1s explained in the legend After the user is satisfied with the result of the calculated efficiency using the slope of the curve it can be stored in the database The choice which wells are used for the calculation is stored in the database and loaded when the primer validation is performed again Page 56 63 Stephan Pabinger User Guide PCR Standard Curve 185 rRNA MM E jm reuse 00 10 20 30 Log Quantity 185 rRNA MM C1 C2 C4 C5 C6 C7 C98 pug A md A e i ca B B 8 8 B8 o a a m m bo 5H B2 HEN p D os ps os DW o te bo
8. Normalize Hellemans q Calculation Method AnafyzerMiner oample Target Use replicate handling Reference Genes Depending on plate layout select Reference Gene s need to be on all plates Efficiency Use Efficiency of Analyzer otatistical testing Permutation test Student s t test log2 This flowchart displays the suggested methods printed in bold for each analysis step Please keep in mind that these suggestions do not consider special instrumental setups or practices of your laboratory The uniqueness of this application is the support of several different methods and a variety of setup parameters which are helpful to find the best setup for your data Page 5 63 Stephan Pabinger User Guide PCR 2 General information This chapter contains information about the basic layout the color and icon convention used throughout the whole application 2 1 Welcome screen QPCR Database Graz Mozilla Firefox Datei Bearbeiten Ansicht Chronik Lesezeichen Extras Hilfe Le M tes ay BABS https rtpcr genome tugraz at rtperfindex jsp 18 Meistbesuchte Seiten f Erste Schritte Aktuelle Nachrichten Oy Bioinformatics Graz QPCR real time PCR data management and analysi we e CUN i bv Lo vam x 4 Home User Guide Tutorial Information K login as other user Project ER QPCR Upload amp Parse real time PCR data management and analysis PCR Management Developed by Stephan Pabinger QPCR
9. Select the displayed error standard error standard deviation confidence interval e Select the samples used for referencing e Seta title for the chart e Specify the displayed minimum and maximum value e Set the tick size e Set the base to an arbitrary number or the lowest number displayed in the chart e Include the number of replicates of each sample in the chart e Customize and view the samples displayed in the chart O O Include Exclude the sample from the chart Set an alternative name Set a specific color for each sample Rearrange the list using drag and drop The selected color alternative name and position of each sample is stored in the database and loaded when this experiment is analyzed again Multiple Targets Single Target HK Quality Control Target fdetector2 Ty Show in title O Bar Chant SE 1 0 Type NRCq T M 1 31 Error SE 1 X References sample sample2 Customize Chart Title min y max y tick size hase use lowest Show of Replicates Copy sortto other targets Sample Alternative Name Expr SE Color Ie sample1 1 0287 0 0793 B FF5555 v Eam O NRCt i EN Ss E p B 5555FF v sample2 1 201 0 0552 sample3 1 1827 0 138 B 55FF55
10. detector false detector2 false detector3 false detector4 false detector5 false detectorB false detector false detector8 false ExperimentReplicates threshold 0 3 target cDNA difference detectors sample6 0 1799 2 detector8 samples RES 2 detector8 sample4 0 1712 2 detector8 sample3 0 2569 2 detector8 sample2 0 0497 2 detector8 sample1 0 0477 2 detector sampleB 0 2121 2 detector samples 0 1808 2 detector sample4 0 2016 2 detector sample3 0 1536 2 detector sample2 0 2071 2 detector sample1 0 0564 2 detectorb sampleB 0 0 1 2 2 detector6 samples 03483 detectorb sample4 EE a ata atarit vi de Page 42 63 Stephan Pabinger User Guide PCR 9 Statistical Test Statistical tests are used to test several groups in the software named as class of samples for significant difference between them It can be used for example to test whether several biological replicates of certain samples are differentially expressed to several biological replicates acting as a control 9 1 Test Setup The header section of the setup page provides links back to the experiment to the Analyze Setup see 8 1 and to the Normalization Result page The following parameters can be set for the statistical test e Define which samples are included in the test e Select the reference sample s used for referencing the calculated values This has nothing to do w
11. 34 3 15623 0 95177 w test X 4 TBP MM 1 61007 0 03878 4 83452 0 98241 w test X TF I B chiii 1 857589 0 11479 3 71783 0 971 om jes X 5 DetectorEfficiencys found Page lofi go to page EN go DetectorEfficiencys per page 15 25 50 100 Page 58 63 Stephan Pabinger User Guide PCR 13 7 Realtime Chemistry A realtime chemistry e g TaqMan Scorpions SYBR entry consists of name catalog number concentration provider and description Realtime chemistries are linked to wells New Realtime Chemistry Page 59 63 Stephan Pabinger User Guide PCR 14 Resource Management The management section groups properties which are of general information in the QPCR application 14 1 Hardware A hardware entry in the application QPCR describes a physical device It stores information about the name type creation date version shown attribute and description of a specific hardware Hardware entries are linked to runs Edit Hardware 2007 01 30 EDIT PLEASE Description Page 60 63 Stephan Pabinger User Guide PCR 14 2 Protocol A protocol entry is used to store protocols of analyzes runs and cDNAs It consists of a name a type its files and a description The specified files are uploaded to the system when a new protocol is created New Protocol Analyze Protocol CAE Analyze 44_57sk Page 61 63 Stephan Pabinger User Guide PCR 14 3 Provider A provider st
12. 351 oga 10793 0 0552 0 138 MARA MOFT 10328 01073 0 0497 0 027 ETE 0 0606 uag n 1 E a 1 0 ET EE lao 1 0 ammer ET 1 12507 UNECq 1n 11827 SECHRCA SD CHR q Log Nica Log Upper SE NRCq Log Lower SE NRCq Lon Uppe 0044 jor oo 0 1168 011272 0 1625 aons nory nn CE nna nossa uoza 00328 00 0 0332 0 034 0 0405 uoma 01073 ow 0 1056 0 1139 031471 00351 00497 00 00498 0 0516 0 0699 mom oor 00 0 0273 0 0278 0 0305 nomaa ann nns 04072 E n1492 U 552 ouem 02642 0 0649 0 0679 uan U138 01948 024231 01582 0179 02201 ninia Free f 1 ds 1308 l 1418 MIMA Stephan Pabinger User Guide PCR 8 4 Normalization Result Bars The normalization result bars page is divided into two sections The upper part contains information about the normalized experiment and provides links to the normalization result page statistical test setup page and normalization setup page The lower part consists of a tabbed navigation interface with three tabs Multiple Targets Single Target and Quality Control 8 4 1 Multiple Targets The multiple targets tab allows the user to choose one or many targets The chosen targets are shown in the legend and colored in different colors The x axis contains the selected samples and the y axis shows the normalized Cq value whereas each bar displays the calculated error T
13. CR 4 Run 4 1 Create a Run mum p absolute Sme Hardware ares T E o8 9 instrument Setting H B 13 Bes 15 16 48 2 27 28 29 30 a no experiment Experiments o A Run in the application QPCR represents a performed qPCR run To create a run three properties need to be specified a name the date of creation and a category relative or absolute run In order to attach existing plate information to a run a file for parsing needs to be specified SDS file gt the sds file of the thermocycler Rn Files exported Rn files Category Hardware Software Instrument Setting and Plate are created by the parser and are set during the parsing process Nevertheless they can be set manually by the user Page 17 63 Stephan Pabinger User Guide PCR 4 20 Run List Nr Name la X Date 1 20071108 EE jv 2007 11 14 ap MM X 2 Run a ER hv 2007 11 14 MX 3 20071108 2 Hb 2007 11 14 Mx 4 Run HH bv 2007 11 14 a 198 X 5 Run2 HAH bv 2007 11 14 ep 98 X The general layout of the run list is equal to the layout described in 2 4 4 In addition to the standard view the run list contains three more columns which indicate the state of a certain run The first column describes whether the run is currently parsed blue dot The second column green dot indicates that the run is currently analyzed The third column red dot informs the user that the run is currently ge
14. Display Normalization Results Experiment Back To Analyze Setup Perform Saatistleal Test Reference Samples Save Horne Keruma coy Legend cDNA sampiel simple Samples ampled samples sample sampler tamplez zamnind example Display Dars amp Quality Corb Shier samplel amplio Eme s Eger S5howHide laa Tan delectari telischard delector detector delectari delectari serpin i dolectar deleciora deler nelgrtar Task Samale arme Sample sample sample Zarngle Sample Samale zaral amp fiamme Page 39 63 av Ca 27 5851 26 4990 21 3751 36 7381 27 9565 26 9764 286 8743 23 5158 db 75 fea f show Pe SEmaCq Dra Cy 01086 01536 0 5936 00228 noma Anas 0 0298 00423 0 1097 01041 01472 0 3892 0 0451 00633 01614 0 02533 0028 00938 aome oian n3527 0 0303 owm 0 1188 onn 03038 08148 niara nonah nens rel cq 0 793 1 4323 0 8453 1 2306 0 6404 11097 lams 1 7201 1 118 1 anik SErelCq SDret bq NACA 0 0473 0 0747 0 0156 0 0677 0 0161 0 015 003598 0 0747 0128 1087 0 0660 00275 nons 0 0949 0 0220 0 0212 0883 0 0803 0 1825 nias 1 0 10 110 41 0 1 0 10287 1301 11821 13247 1 SE NRCq SD NACO 00844 04192 nons 1 0233 0 0759 0 0
15. QPCR User Guide Version 1 7 Create Date Sep 25 2007 Last modified May 25 2009 by Stephan Pabinger User Guide PCR T able of content Tale ol Content ai 2 VntrOQOUC HOP mn aaa 4 1 1 PATE BOSC FSR ES eM MEE M MEE MEM MEI MED MM MED IE 4 1 2 Whntsotustts the SOL DW ALC aia dicas 4 1 3 Example ofa standard WOEDKEIQN sal 4 2 General norman scu od ouai sd aii 6 2 Welcome Sres 6 2 2 Header econo ia cab 7 2 9 N vi sation SECOND Eu bes ase iue Doe du aed eae 9 2 4 Irifortmattob SeCUOB ocior au eo ee deoa are SE dalla used te EET aud TECUM UE 9 2 9 D Umbols usce O EUM t tec 12 9 plogd and Pdtse constituatur ue E eel Maan n Moe uM P a aS 13 3 1 il Keg Co alee greenery PT EE O eres 13 2 2 AMI Iple Upload pisa ita 15 3 3 Multiple Parse cacti tua d ee edubteod E i ufa ditata tee 16 LEE ium 17 4 Create d RD ea tedio pb od be OMA ated as od ee MAE adt da tata die tas 17 4 2 R n TS rarere eden e E renerne 18 4 3 Dusplas TU osi c an ede erne ener ada 18 4 4 Pasce OUI concito toda ee ici PE ee cdd rt uta CURIE 20 4 5 lg Tiger el Bo ceara mn NANI de O ON 21 Z0 Proeress Th Format OD rss tet nd RD bt didit vetere ue tue cud uiae t Outer bod we 23 ADU c Er En Ea E 24 SIMI bod E 25 2 2 HEUS Dui d iran Giu eie edat Tant toli atio Dat Co Ge imei london etie dal auauantaled ea dl ente 26 5 3 CATE RESUS sisas 28 Oc A lec desc ec Stet MAC cte SE cana NP Aca 30 A A o etos dice NET badvop al ttita edi TENDER SETE SSP tenis FE EDER SEE
16. S SE 32 S ANa 2 X VA 33 8 1 ADA ZU AE A E eee ee 33 8 2 Eq Amazon neat de rn steer otra ise 37 8 3 Norma ALTO Resol aia 38 5 4 Nornnalizati n Result Bats ias vue enit eod eere Mag esi ies eran 40 O Statistical estuarios las 43 9 1 TeSDSeDUDuetetnen eie i iate tatio a a E 43 9 2 Graphical Relais est udo e hur eiae dec fane hedde aub tuv Pio deci baee adds 45 10 Error propa SALON oo ted eue SU UU dS on cies etic a vas eee 47 11 O O MESE 48 12 iun DELIO EOS O cain eam sb Da ora Ed if 49 13 PER TVs UM crs doen cen tau Ee dac oo uas deut E Een ied ce 51 I9 CDNA mare an ciclo 51 SS PPP Tc 51 o IrstPitbie t 9c iros 52 3 4 Passive RelebellG e odia tete Qe o ed mud Ot a Cd RIMIS edid Otto 54 S POME ETT UU Tem 55 30 Pomer Validator 55 Page 2 63 Stephan Pabinger User Guide PCR 13 7 Realtime Chistosas 59 14 Resource Mana coment id 60 AL Hard E Em EET 60 14 2 TOU OC Otis dee cue ue tae M M at at M M M E E 61 A A II A A 62 HA A T TE 63 15 BSO HI RTT RR m 63 Page 3 63 Stephan Pabinger User Guide PCR 1 Introduction QPCR is a web application designed for storing parsing managing and analyzing qPCR data Including several different algorithms it can facilitate the analysis of qPCR results The application is not intended to be installed on client machines We suggest installing one instance for your group on a Linux server where u
17. SOUS experiment Date 10 09 2006 tutorial SDS experiment Description _ Show Ct and Efficiency Results Return Page 31 63 Stephan Pabinger User Guide PCR 7 Project Projects are used to group several different experiments Each project consists of a name date and description Experiments can be added to the project by assigning them to the right selection box New Project 09 05 2006 peame IRC test Livere Livers Experiments B Test Referencing Problem testLighti cler testNewSort a o Existing projects are displayed in a tree where the associated experiments and their runs are shown The user can sort the list and its child nodes according to name or date Moreover links to the detailed pages of projects experiments and runs are given and an icon is shown to quickly jump to the analyze setup page of an experiment Projects Sort Project Name amp Date m O a a Sort Experiment Nam Sort Run Mame amp Date W Projects empty 2009 04 09 Y t test 2009 02 27 A sl Test Project 2006 05 09 a test 2009 02 25 09 E 5 C liver GLUT1 1 2008 95 20 t Page 32 63 Stephan Pabinger User Guide PCR 8 Analyze 8 1 Analyze Setup The Analyze Setup page is divided into two parts the upper section contains information about the experiment gives the user the possibility to save the current setting and allows the user to load previously
18. alyzer e Target dependent efficiency the user can specify the efficiency for each used target If the target has a stored efficiency see 13 6 2 it is automatically loaded into the corresponding field Moreover the efficiencies calculated by Primer Validation see 13 6 can be loaded for a particular primer validation run by selecting the corresponding entry in the combo box To normalize the Cq values one of the three methods needs to be picked In addition to these methods efficiency can be calculated using dilution series Such a series is determined by the task attribute of a well which needs to be set to standard The selection of normalization algorithm is similar to the selection of Cq calculation method described in 8 1 1 Page 35 63 Stephan Pabinger User Guide PCR Cq Calculation Methods Sample Target Reference Genes Normalization Define Efficiency 4 C Calculate Efficiency if possible from dilution series Global Efficiency Efficiency 2 SE Efficiency 0 05 Use Efficiency of Analyzer AnalyzerMiner x 3 AnalyzerMiner implements the model described by Zhao A and Fernald in Zhao and Fernald 2005 PMID 16241897 It operates on the raw fluorescence data v Specify Efficiency for each Detector Use Primer Validation Plate mc Hg Detector Efficiency SE Eff Plate detector 2 10 05 detector2 2 0 05 detector3 2 0 05 detec
19. button nextto Display Charts sends the user to the charts interface described in 5 2 As soon as there are analyze results available the user can display them by clicking on Show nextto Display Cq Analyze Results Edit Plate me ER E Show Display Ct Analyze Results Show Design The lower part of the plate view shows the list of attached wells Displayed are well number whether this well is set to omitted passive reference target s cDNA and task Each well can be shown in detail by clicking on the well number Moreover the wells can be edited by clicking on the edit symbol of the according well Page 24 63 Stephan Pabinger User Guide PCR Task Well Number cDNA RI hs Sampl m sampl aampl sample Sampl m Sampl sampl aampl m sample AIO Sampl m sample sample sample DH y detector Sample By By By Bs By Be By By By Bs By Bs Bs By detector sample sample detector Sample Sampl at sampl nu Sample Samples Samples Sampled sampled samples Samples detector camnlak Carnie m sampl sample sampl Ha Sampl sample 22 B10 sample Pi ra ms a X IY I L I rm rm Oo m m rm T a LE E Pp FF P LE gt I im I Qu Pi oo eA T eu CT T ER Pi ls Eo Es By By Bs By Ep 73 5 1 Well A well represents the reaction room on a plate Each well consists of the following attributes well numb
20. colored in dark blue To change the chart to a linear logarithmic scale the user can click on the appropriate field By clicking on the button Clear All the current cell selection is cleared Page 26 63 Stephan Pabinger User Guide PCR Rn vs Cycle a m E re 10 0 12 5 15 0 17 5 20 0 22 5 25 0 27 5 30 0 32 5 35 0 Cycle 3412 3s 4 L5 Ls 7 8 8 T 2 a amp a a as as ar as ss m0 AM A3 B e amp B B B o s s Bo eM B2 c c c c a o e Te c c e em D Dr D o DI DM or amp amp E Eo EM ED F n m m mM re m m r ne m a e o e o c c c o 9 e oo oM e uH m m m m rm m W He m mo Hi Mo _ omitted E Clear All linear log Export s SVG Page 27 63 Stephan Pabinger User Guide PCR 5 3 Cq Analyze Results Analyzer Results Plate 20071108 Show Successful analyzed Analyzer Date SoFARAnalyzer 2007 11 14 at 15 14 54 csv Export View X Not successful analyzed Analyzer Date Error Message Return The Analyzer Results page displays a short overview over completed analyzes of the selected plate It is divided into two sections successful analyzing jobs and not successful analyzing jobs Each entry in the successful analyzed list contains the name of the
21. d Cq Analyzeris Analyzer caFARAnalyzer Analvzerkliner AnalyzerHutledGene LinRegAnalyzer Preferred Efficiency Analyzer s Use HTCs in Eq analysis Page 48 63 Stephan Pabinger User Guide PCR 12 Run Deletion Log Deleting a run is a very time consuming operation and is therefore performed in the background Whenever such an operation is completed the user gets informed that a new Run Deletion Log 1s available Results that have not been viewed are colored in blue and are put at the top of the list After a Run Deletion Log run deletion result has been viewed by a user it 1s automatically deleted Run Deletion Log 9 Query fe Edit Display Settings Legend Run Deletion Logs per page 15 25 50 100 1 Run Deletion Logs found Page 1 of1 go to page E go Nr Run Date T Viewed bi 1 2008 07 29 Color Legend Color Meaning Run Deletion Lag has nat been viewed Run Deletion Logs per page 15 25 50 100 1 Run Deletion Logs found Page loft goto page m go Each Run Deletion Result entry presents information about the deleted run whether the job was successful the submission date an error message 1f the deletion job was not successful and the view status Page 49 63 Stephan Pabinger User Guide PCR Show Run Deletion Log test Error Message Page 50 63 Stephan Pabinger User Guide PCR 13 PCR Management The menu PCR Management groups properties that are necessary to specify a qPCR r
22. e of Pathology University of Graz karin wagner gklinikum graz at aim Xe When selecting a user or an institute the checkboxes for editing f and deleting X are enabled and the user can additionally specify 1f the shared entry can be edited or deleted Page 11 63 Stephan Pabinger User Guide PCR 2 5 Symbols e D exa e e e a RE REY Page 12 63 Indicates that one can edit the data Indicates if there 1s some information downloadable Indicates that one can delete this entry Indicates that there 1s additional information available Indicates that the user can share his her data to other users of the system Indicates that the entry is currently parsed Indicates that the entry is currently analyzed Indicates that the entry is currently deleted Puts all entries from the left list into the right list Puts all entries from the right list into the left list Puts one or many selected holding ctrl entries from the left list into the right list Puts one or many selected holding ctrl entries from the right list into the left list Stephan Pabinger User Guide PCR 3 Upload and Parse 3 1 File Upload Files can be uploaded separately or in batches using the multiple file upload interface described in 3 2 When uploading a file the user has to specify the correct file type Currently there are 2 file types available e File SDS Applied Biosystems IXO Roche CSV generic e Export File
23. ectly identified by the system For each File Export file combination the user can specify whether the files should be 1 parsed and analyzed 2 only parsed or 3 skipped Pressing submit parses and analyzes the files in the background and creates a run for each selected file The methods one or multiple used for analyzing can be set using the user settings interface see 11 The legend explains which files need to be exported and parsed for each thermocycler in order to guarantee a successful analysis More information about parsing is provided at 4 4 Multiple Parse Display Files Owned By User i l Display Files All FEE Update Legend Nr File Export File 1 Export File 2 Export File 3 Parse Analyze 1 test test component zl test deltaPn v X Iv Iv 2 templateCSv Iv Iv 3 18samples 16samples 530Expc y x Iv Iv Submit Top Legend Thermocycler File Export File 1 Export File 2 Export File 3 ABI 7000 The saved SDS file Exported component file Exported deltaRn file ABI 7500 The saved EDS file Exported file including Sample Setup and Amplification Data ABI 7900 The saved SDS file Exported clipped file LightCycler 2 0 The saved IXO file Exported Fluorescence history over Cycles as XML file Generic CSY file The generated CSV file Page 16 63 Stephan Pabinger User Guide P
24. ements an algorithm similar to the one used by by the SDS 2 2 2 software from Applied Biosystems It uses a dynamic baseline created by a line fitted into the area prior to the exponential SDSAnalyzer Es me E me ls me lt MATOS Cq Values exist Efficiency Values exist 8 1 2 Sample Target selection The second tab allows the user to select samples and targets that will be used for normalization On the left side available samples and targets are displayed omitted wells are not considered for normalization in list form and on the right side the samples and targets used for normalization are shown Using the arrow buttons the user can add or remove one or many entries form each list When Use Replicate Handling 1s checked Cq values of replicates all sample target combinations on one plate are averaged When ticking Average technical replicates over plates then technical replicates are not only averaged within one plate but averaged over all plates that are in this experiment Cq Calculation Methods Sample Target Reference Genes Normalization Use Replicate Handling Average technical replicates over plates O Samples Used Samples samplel sample2 gt sample3 sampled sampled sampleb Targets Used Targets detectorl detector2 detector3 detector4 detectorb detectorb Cq Values exist Efficiency Values exist 8 1 3 Housekeeping Genes se
25. er omitted x and y position on the plate reaction volume task e g standard target sample quantity sample CDNA sample concentration passive reference realtime chemistries targets and description Wells are a subunit of plates and can not be created without a plate reference Page 25 63 Stephan Pabinger User Guide PCR Edit Well meea C PE ER ewe eo 5 2 Charts The chart interface can display four different datasets raw dissociation data derivative dissociation data Rn vs cycle and deltaRn vs cycle The Show button next to the plate name redirects the user back to the plate interface Tabs are used to navigate between the different datasets Charts includes a title axis annotation and a legend and can be saved as a picture by right clicking on the image or by using the Export As SVG button Below the chart a grid or list 1s representing the plate layout where each cell stands for a well of the plate By clicking on a cell the well is added to the chart and the image is updated Multiple cells can be selected by holding the ctrl key clicking on the start well holding the left mouse button and dragging the mouse to the desired end point of the rectangle When the mouse button is released the selected wells are added to the chart Moreover entire rows and columns can be added to the chart by clicking on the respective header Omitted wells are colored in light blue whereas empty wells are
26. er Guide link opens this document The Tutorial link opens the tutorial The Information link displays a page with useful information about the software At the right side there are 3 icons where the user can change the spatial usage of the browser window resizes the window to the default size Stretches the window to the full width of the screen EJ uses the full width of the window and the images at the header section disappear only the display bar and the authentication bar will stay If the user is not logged in the following bar is shown G Login please login Page 7 63 Stephan Pabinger User Guide PCR By clicking on the Log in link the user is directed to the login window a a Username Password Submit Javascript and cookies must be enabled in your browser Screen resolution of at least 1024x758 is strongly recommended Mozilla Firefox All Releases recommended Internet Explorer Windows Internet Explorer B Mac OS x Internet Explorer 5 2 Netscape Release 7 0 If the user is logged in the following bar is shown logout User Settings Progress Information New Parser Log New Analyzer Log Run Deletion Log Stephan Pabinger It provides the possibility to Log out Change user settings Display new Analyzer Logs only shown when new results are present Display new Parser Logs only shown when new results are present Display new Run Deletion Logs only
27. he database and loaded whenever the test 1s repeated Page 43 63 Stephan Pabinger User Guide OPCR Perform delta delta Cq calculation Back To Analyze Setup Display Hormalization Result Reference Calculation Reference Samples sample sample x Im class 1 Statistical Test nose Pamuaten eaten HE n Add Class Remove Last Class Set As Statistical Reference Green e sample samples sample4 sampleb sampleb O Set As Statistical Reference sample sampled Add Class Remove Last Class j Page 44 63 Stephan Pabinger User Guide PCR 9 2 Graphical Result The upper section of the statistical result page displays links back to the various analysis pages and provides the functionality to export the generated results Display Statistical Test Results Bars Back To Analyze Setup Display Hormalization Result Perform Statistical Test The Display Test Result combined target view displays the averaged results of each class 1n this case the classes replicates and replicates2 for the selected targets It is possible to select multiple targets and to set a title for the generated chart Combined Targets a ch E cm La 3S o LL Fold Change Combined SE 1 0 1 053 0 955 0 903 detectorl detectora detectarz E Class 1 Class 2 Export As SV Page 45 63 Stephan Pabinger User Guide PCR Next the resu
28. he user can customize the chart in the following ways e Select the displayed error standard error standard deviation confidence interval e Select the samples used for referencing e Seta title for the chart e Group the bars by sample or target e Select which sample should be displayed in the chart The order of samples is equal to the order set in the single target chart see 8 4 2 The chart can be saved either by right clicking on it and selecting the corresponding option orby using the Export AS SVG button Experiment example Back To Analyze Setup Display Hormalization Result Perform Statistical Test Multiple Targets Single Target HK Quality Control Bar Chart SE 1 0 detector i i detector3 detector4 detectorb detectorb v Error SE v Referentes sample sample Title O Group By sample Display Sample Elm E CNRCt s sample1 sample2 sample3 sample4 samples leege sampleb m detectorl M detector2 m detector3 Export As SVG Page 40 63 Stephan Pabinger User Guide PCR 8 4 2 Single Target The single target view shows samples of one specific target The user can customize the chart in the following ways e Switch between Cq value normalized Cq value and calibrated normalized Cq value and their log2 values e
29. in Temperature step Duration min Temperature Show Instrument Setting 2007 01 30 EDIT PLEASE 200 min Temperature 50 0 C Step n 10 00 min Temperature 95 0 C E 40 Duration 0 15 min Temperature 95 0 C NN 1 00 min Temperature 60 0 C mo 1 0 15 min Temperature 95 0 C Step 2 Duration 0 20 min Temperature 50 0 C Step 3 Duration 18 25 min Temperature 85 0 C Page 53 63 Stephan Pabinger User Guide PCR 13 4 Passive Reference A passive reference entry consists of name and description and can be linked to a well Edit Passive Reference Page 54 63 Stephan Pabinger User Guide PCR 13 5 Primer To create a primer six mandatory properties need to be set name sequence the actual DNA sequence sequence position primer length primer concentration and type forward reverse or probe Optionally temperature logNr provider author and description can be specified New Primer C IE seme Oooo CITO IS ee O OO OOE C ww LEN NNNM me mms j mm SS mn Create 13 6 Primer Validation Primer Validation is used to determine the efficiency of targets by using serial dilution series After calculating the Cq values linear regression is used to calculate the efficiency of the target The efficiency 1s then stored in the database and can be loaded into the analysis of an experiment see 8 1 4 In orde
30. ion please consult the paper qBase relative quantification framework and software for management and automated analysis of real time quantitative PCR data by Hellemans et al 2007 8 1 1 Cq Calculation Methods The first tab displays the available Cq calculation methods Shown is the name of the method and a short description Whenever a calculation method is chosen the application checks if Cq values are existent and displays the corresponding message at the bottom of the page Page 33 63 Stephan Pabinger User Guide Analyze Se PCR tup Experiment example Save Setting Setting Cq Calculation Methods Use Name Sample Target Reference Genes Normalization Description AnalyzerMiner AnalyzerMiner implements the model described by Zhao and Fernald in Zhao and Fernald 2005 PMID 16241897 It operates on the raw fluorescence data and calculates Cq value efficiency and starting AnalyzerCy0 Efficiency Use the efficiency calculated by another method or determined by primer validation AnalyzerCyO implements the model described by Michele Guescini and Davide Sisti et al in A new O SoFARAnalyze nalyzerSoFar implements the algorithm described by Wilhelm in Wilhelm 2003 and Wilhelm et al in Wilhelm et al 2003 PMID 12613255 SoFar stands for Software For the Analysis of Real time AnalyzerSDS impl
31. is a versatile web based Java application that allows to store manage im quantitative real time polymerase chain using the quest account in which youve e S gly recommended to use a priv rate account which guarantees C confidentiality and security of your data To request an account please contact qpcrgenome tugraz at To get started Read the tutorial which leads you through all important steps of the application For more information download the user guide which covers all aspects ofthe application Release Information aru E Institute for Genomics and Bioinformatics Graz University of Technology cs and Bioinformatics Graz University of Technalody Imprint rtpcr genome tugraz at a The picture above displays the welcome screen of QPCR The main view 1s divided into 3 sections 1 The header section consists of some images on the top of one bar managing the display settings and one bar displaying information about the AAS Authentication and Authorization System 2 The left bar contains the menu used for navigation The center frame displays the selected information UJ Page 6 63 Stephan Pabinger User Guide PCR 2 2 Header section 2 2 1 Display bar Oy Bioinformatics Graz QPCR quantitative real time PCR management system be ao AM c ith 4a ve DE LAM We D gt Home User Guide Tutorial Information o 3 login please login The Home link sends the user back to the start page The Us
32. ith the actual statistical test It is used to calculate the fold change ratios By selecting a class all samples in this class will be used as reference samples e Select which test should be used e Select the p Value type e Select multiple testing correction Choose between four established methods to correct the calculated p value Additionally calculate your p values without multiple testing correction e Select the data type choose between CNRCq and log2 CNRCq e Select if samples should be averaged in each class This causes that the output chart displays only the averaged value only on bar for each class The next part of the setup page is used to define the classes groups and their attributes The user can define as many classes as needed which are then used in the statistical test One class acts as the statistical reference reference class and all other classes are tested for their statistical significant difference to this reference class Do not confuse this with the sample references which are used to reference the samples to a given set of samples no statistical test Each class has a color or pattern associated is given a specific name and needs to consist of at least one sample In one class the property Set As Statistical Reference is set which specifies to which class all other classes are compared previously described Classes can be added or removed from the list and the complete statistical setup is stored in t
33. lection The selection mechanism for reference genes is the same as described in 8 1 2 If a reference gene is put into the used list the corresponding target is put into its corresponding used list because technically a reference gene 1s a target When analyzing multiple runs in one experiment the reference gene s can be Page 34 63 Stephan Pabinger User Guide PCR e on only one plate and Cq values of the other plates are normalized to the values of the reference genes present on only one plate e onevery plate and Cq values are normalized to the values of the reference gene on the current plate If the checkbox next to Reference Gene s need to be on all plates is checked reference gene s need to be present on every plate Cq Calculation Methods Sample Target Reference Genes Normalization Reference Gene s need to be on all plates Reference Gene list Used Reference Genes detector2 S detector detector3 rm detector4 ind detectorb detector 1 detector v Cq Values exist Efficiency Values exist 8 1 4 Normalization The last tab is used for setting the normalization parameters It is divided into two parts definition of efficiency specification of normalization algorithm The efficiency of each well can be specified in three ways e Global efficiency each well has the same user defined efficiency e Efficiency determined by an analyzer each well uses the calculated efficiency of a certain an
34. lts for each target are shown Both the calculated values and the created chart are displayed Displayed are the following values for each class e The calculated p Value e The average ddCq value of the class divided by the average value of the statistical reference class NOT reference samples e The average SD of the class divided by the average SD of the statistical reference class propagated standard deviation e Whether this class was set as statistical reference Target detector1 Class p Value ddCq reference SE ddCq reference SD ddCq reference Is Statistical Reference Class 1 true Class 2 0 1975 1 1214 0 0655 0 0655 0 1134 0 1134 false Fold Change detector1 SE 1 0 Fold Change 0 945 Class 2 Class 1 E sample B sample3 Msamplel sample4 m sample5 sample6 Export As SVG In addition to the graphical view results of the statistical test can be displayed in text format This view is accessed by clicking on show nexttoDisplay Test Result For each class the p Value average ddCq reference and the corresponding error are shown In addition the fold change values and the corresponding errors of the samples used for the statistical test are displayed in a list Page 46 63 Stephan Pabinger User Guide PCR detector1 Class p Value amp ddCt reference SE ddCt reference amp SD ddCt reference Is Statistical Reference replicates 1
35. ogress information list 1s automatically updated every five seconds For each process the corresponding method is shown and by clicking on the run name the user is linked to the specified run Progress Information The progress information page is reloaded every 5 seconds Progressinformations per page 15 25 50 100 10 Progressinformations found Page 1 of 1 go to page go Nr Type Method s Progress Run File 1 analyzing SoFARAnalyzer po 85 20071108 2 200711082 X 2 pina AbiSDS Parser ABIV1d1 MMM 100 20071108 2 20071108 2 x 3 analyzing SoFARAnalyzer A 15 20071108 20071108 X 4 parsing AbiSDS Parser ABI V1d1 rn gt 100 20071108 20071108 Tx 5 parsing ANSDS Panar von prre 10096 Run File5 d 6 analyzing SoFARAnalyzer 25 Run 2 File2 Ix 7 parsing AbISDS Parser V2d1 EE 100 Run2 File2 x 8 analyzing AnalyzerMiner 90 Run1 Files 9 analyzing _ AnalyzerMiner E 100 Run1 File4 x 10 parsing AbiSDS Parser V2d1 A 100 Run 1 File4 x Progressinformations per page 15 25 50 100 10 Progressinformations found Page 1 of 1 go to page go Page 23 63 Stephan Pabinger User Guide PCR 5 Plate The upper part of the plate view contains detailed information about a plate entry It shows the plate name which is equal to run name barcode description SDS amp Rn files and plate size The Show
36. ores information about name abbreviation street city province country phone fax email web address and description Providers can be linked to realtime chemistries and primes New Provider met gotham com Page 62 63 Stephan Pabinger User Guide PCR 14 4 Software Software entries describe the software of a particular hardware see 14 1 It stores information about the name type creation date version shown attribute and description A software entry can be linked to a run New Software 15 CSV file format If no parser for the used thermocycler is available the generic CSV file format can be used The information section provides a sample file and explains the specification Information about the CSV file format Page 63 63 Stephan Pabinger
37. played At the right side one can choose how many entries are shown per page Moreover the user can directly jump to a page by entering its number Runs per page 15 25 50 100 4 Runs found Page 1 of 1 go to page go Page 10 63 Stephan Pabinger User Guide PCR 2 4 4 Table view OS Upload Name Category Added Date EET Filet piate 2007 01 29 a 5 S8 X 2007 11 14 3 5201 20071108 2 Component additionalplatefiles 2007 11 14 dic sex 5202 20071108 2 DeltaRn additionalplatefiles lt _ 2007 01 20 at se X Files clipped additionalplatefiles LIL A roo di Sax The table view consists by default of the following parts e The header if one hovers the mouse over a column name the colour changes to blue and one can sort the list by this column e The number in the first column indicates the hit number of the entry corresponding to the order e Detailed information about an entry is loaded by clicking on links of the corresponding entry E Indicates that the data can be edited Indicates that there is information downloadable 3 Indicates that the entry can be shared X Indicates that the entry can be removed By clicking on the share icon the user is redirected to the sharing page Sharing You are about to share item 384 5208 27062005 E Mail mj Institute for Genomics and Bioinformatics Zlatko trajanoskiq tugraz at ej El E mj Inserm U255 jerome girgendwas fr cij p x 5 T dr XM MJ institu
38. r User Guide PCR can be added removed and saved When the operators LIKE and NOT LIKE are used a preceding or trailing asterisk needs to be entered The button Submit Query submits the entered query and updates the result table Reset Query removes all entered queries and submits a query without any user defined filters Restore Default restores the default set of queries and submits them Save Queries saves the current set of queries as default for this page Unless the user changes the queries the data on that page will always be filtered with this default set of queries i X y x sos vox submit Query Reset Query Restore Default save Queries Quel 2 4 2 Customizable display The information that will be displayed on the screen is customizable to the needs of the user One can select the desired columns by clicking on the checkboxes and update the view on the data by pressing the button Update Save Settings allows the user to store his her own display settings and whenever the user enters the same page his her settings will be displayed by default Available fields X Required Information M Name User Descriptian Submitter M Date Update Display all Display default Save Settings 2 4 3 Scrolling bar The left side of the scrolling bar displays the number of found elements depending on the query the user submitted In the middle of the scrolling bar the actual page and the total number of pages are dis
39. r to calculate the efficiency wells on the plate need to be marked by setting the task as standard 13 6 1 Perform Validation First a Run that has been used for primer validation standard curve needs to be parsed and analyzed using the standard procedure Dependent on the selected Cq analyzer the systems checks if Cq values exist for this analyzer If they are not present the Cq value calculation can be started directly from this interface If the system has found Cq values for this analyzer the calculation of efficiency values can be started by pressing the Analyze button If this plate has been analyzed before the system displays a warning that the results will be overridden When the calculation 1s finished the results for each target on this plate having a serial dilution series are shown in a list Displayed are the efficiency the standard error R2 and the slope of the calculated line The efficiency of each target can be stored by ticking the checkbox and pressing the save button Page 55 63 Stephan Pabinger User Guide PCR Primer Validation Ema le a CT AnalyzerMiner v po exist Validation Method Linear Regression Target E SE R2 Slope Save 185 rRNA MM EN 1 64409 0 07152 0 97647 376248 al GAPDH MM 1 81782 0 04924 0 98583 3 85282 i HBMS MM 2 07412 0 15234 0 95177 315623 E TBP MM 1 61007 0 03676 0 38241 4 83452 TFIIB MM 1
40. rnings occurred Farser result should be checked Blue Parsing was successful but result file has not been viewed Black Parsing was successful and result file has been viewed The coloring of the parser logs is explained in the attached legend Each Parser log shows detailed information about the completed parsing job It displays whether the job was successful when it took place and if a plate 1s attached to the run Warnings produced during the parsing job are displayed in a box Furthermore the result page shows the parsed run name and information about hardware software and instrument settings Information about the created plate contain its id name size input file and plate status Moreover the user can enter a description and update the plate using the Update goto plate button The three text fields for target cDNA and passive reference may contain entries that need to be added to the database Page 21 63 Stephan Pabinger User Guide OPCR Parser Result Hardware found fi Plate Hame Plate Description Plate Size Input File Plate Status Please add the following Detectors ta the System Please add the following cOMAs to the System Please add the following Passive References to the System Page 22 63 Stephan Pabinger User Guide PCR 4 6 Progress Information Progress Information is positioned in the run menu and shows the progress of parsing and analyzing jobs The pr
41. saved settings The lower part contains a tabbed navigation which is used to define the different settings for analyzing an experiment In general one can differ between two classes of analyzes e Cq value efficiency value calculation e Normalization Cq value efficiency value calculation needs to be done before normalization can be started because normalization is dependent on Cq values and if chosen also on efficiency values Therefore whenever a Cq calculation method is selected the application checks if theses values are existent If they are not present a message is shown at the bottom and the button changes from Analyze toCalculate values By pressing this button the calculation of Cq Efficiency values is started in the background Because of the fact that this process 1s very time consuming the calculation is performed in the background The normalization of Cq values is a very quick task and is therefore not performed in the background It is only started if Cq values and efficiency values if selected exist Normalization of the Cq values contains the following steps e Technical replicate handling samples having the exact same name cDNA are averaged e Efficiency correction e Normalization using the selected reference genes multiple reference genes are geometrically averaged e nter run calibration on a gene specific base samples that are present on all used runs are used as calibrators For more informat
42. se a thermocycler run at least the main file eds sds ixo csv has to be specified The following files need to be uploaded into the application Abi SDS 7000 SDS file Export and specify component file e g runl_Component csv Export and specify deltaRn file e g run DeltaRn csv Abi SDS 7500 SDS file Export file including Sample Setup and Amplification Data Abi SDS 7900 SDS file Export and specify clipped file e g runl_Clipped txt Roche Lightcycler 2 0 480 IXO file Export the fluorescence history in the Run gt Online Data Display window Select all samples then select Fluorescence history as the Chart and Fluorescence over Cycles as the Axis Then right click on the chart select export pick the data tab and select XML Generate a CSV file described in 15 GeneralMessage Parsing job started in the background Continue After a parsing job is complete a notification pops up in the display bar 2 2 1 which links to the parser log page Page 20 63 Stephan Pabinger User Guide PCR 4 5 Parser Log Displayed below is the parser log page Parser Result L Query Legend ParserResults per page 15 25 50 100 5 ParserResults found Page 1 of 1 qo to page go 5 ParserResults found Page 1 of 1 go to page go ParserResults per page 15 25 50 100 Top Color Legend Color Meaning Red Parsing was not successful Orange Wa
43. selected analyzer the time of submission and an export possibility By clicking on the Vi ew button the user is guided to a detailed list of the selected analyzing result described in 5 3 1 The same list is exported when the user presses the Export button whereas the file format can be chosen using the list next to the Export button Clicking on the delete symbol brings up a popup message which asks for confirmation to delete the analyzer result including its Cq efficiency starting amount and correlation value 5 3 1 Detailed Analyzer Results The detailed analyzer results page displays detailed information about a particular analyzer result The header section contains the name of the plate including a link back to it the name of the analyzer and the submission date The export feature 1s the same as described in 5 3 Each entry of the list contains information about a particular well of a plate Displayed are sample name target s Cq value and efficiency Page 28 63 Stephan Pabinger User Guide PCR Detailed Analyzer Results sample detector 26 5125 1 6769 AJ Ad detector 6 4674 J example samplez example samples detector rede 1 6978 example samples detector 27 305 1 7513 example sample4 detector 26 634 1 5808 example sample4 detector 6 0422 1 66 76 oo a amp in AS samples detector 27 9115 1 7334 ovammie H1 cammnlal rlatactnr E GR 1 BRA
44. sers connect to by a standard Web browser Additionally you can request an account on the server hosted by the TU Graz 1 1 Purpose This document is not written to be read from the first to the last page It is more a compendium that tries to tell what to do if one 1s puzzled However for an introduction it makes sense to read chapters 3 to 8 to get general information about the application These chapters are sorted according to a typical procedure of uploading parsing analyzing and normalizing a qPCR experiment 1 2 Hints for using the software e Always use meaningful names to be able to distinguish the different entries in the future 1 3 Example of a standard workflow e Upload files using the multiple file upload interface or upload them separately e Parse and analyze the newly uploaded files using the multiple parse interface e After parsing and analyzing is finished check the created runs Check hardware software instrument setting amp SDS Rn files Check plate including wells Display charts and check values save them as picture Check Cq analyze result values e Create experiment using the desired runs e Analyze experiment save analyze settings e Check normalized Cq values e Export charts picture and Cq values file e Perform statistical test e Export fold change charts and test results Page 4 63 Stephan Pabinger User Guide PCR Cq calculation efficiency calculation AnafyzerMiner Normalization
45. shown when new results are present Page 8 63 Stephan Pabinger User Guide PCR 2 3 Navigation section The navigation section 1s positioned at the right side of the screen Project Grey fields with bold text are headers that reveal a submenu when EE the user clicks on them A submenu can contain another submenu Run or links to a certain interface Upload amp Parse Multiple File Upload Hew File Upload Find File Upoad Parse PCR Management Management 2 4 Information section QPCR uses a list form to present overviews of data shown in the figure below The header section contains 2 links e Customizable queries e Customizable display The table with the data 1s always enclosed by bars used for scrolling and most columns in the table are sortable An arrow indicates the current sort direction of the corresponding column Sort settings are saved in the database for each user and are loaded when the user logs in the next time Run 9 Query 5 Edit Display Settings Runs per page 15 25 50 100 5 Runs found Page 1 of 1 go to page za go Nr Name Date 1 20071108 2007 11 14 2007 11 14 2 Run 3 20071108 2 Run 1 2007 11 14 XIX XIX 4 s e EZ Runs per page 15 25 50 100 5 Runs found Page 1 of 1 go to page go x 2 4 1 Customizable queries Queries are used to narrow the list of displayed entries according to the users needs They Page 9 63 Stephan Pabinge
46. ssing the button called Display Bars amp Quality Control sends the user to the Bars section of normalize results 8 4 The button Back to Analyze Setup sends the user back to the setup page Perform Statistical Test sends the users to the Statistical test setup page By selecting on or many samples in the Reference Samples box the calculated values of each sample are referenced to divided by the values of the selected samples If more than one reference sample is picked the average of the selected samples is used Save Normalize Results saves the current results to the database These entries are used if external applications request for normalized QPCR results MARS database Using the export feature the user can export the result list in various file formats Page 38 63 Stephan Pabinger User Guide PCR By pressing on the Show Hide log2 button the user can choose to display the calculated log2 results Each entry in the list contains information about cDNA target task average Cq value averaged value of replicates CV value relative Cq value rel Cq relative quantity of Cq value using gene specific efficiency E A normalized relative Cq value NRCq normalized using geometric mean of reference genes calibrated normalized Cq value CNRCq normalized NRCq values using calibrators their respective standard errors The legend at the bottom of the page explains each calculated result
47. tor4 2 0 05 Cq Values exist Efficiency Values exist v Page 36 63 Stephan Pabinger User Guide PCR 8 2 Cq Analyze Log When a Cq calculation 1s finished a message is displayed in the header section 2 2 By clicking on this link the user gets redirected to the Cq Analyzer Log page which lists the completed analyzing jobs Entries that have not been viewed are colored blue and are put at the beginning of the list CalEfficiency Analyzer Log gt Query f Edit Display Settings Legend Analyzer Logs per page 15 25 50 100 2 Analyzer Logs found Page 1 of 1 go to page msi ge musa vener pasenme Suecessta ate 2 Analyzer Logs found Page 1 of 1 qo to page m ge Analyzer Logs per page 15 25 50 100 Top Color Legend Analyzer Log has nat been viewed The detailed Cq Analyzer Log contains information about the plate name plate 1d whether the analyzing job was successful submission date and in case of an error the error message Page 37 63 Stephan Pabinger User Guide PCR Show Cq Efficiency Analyzer Log Viewed sd Analyzers Successful Error Message no errar AnalyzerMiner In case it was not successful plate has no values to analyze attached megna m 8 3 Normalization Result The normalization result view contains information about the normalization process of Cq values The header section displays the experiment and provides a link back to it Pre
48. tting removed Moreover the plate icon sends the user directly to the plate of the run and the chart icon directs the user to the chart view 4 3 Display Run After a run has been parsed the combo boxes for run category hardware software instrument settings and plate are automatically set to the corresponding entry by the parser The information section at the bottom of the page displays the current parse status and the latest successful parse job Using the checkbox next to Options the user can specify whether the names of wells specified in the thermocycler software are equal to sample names Pressing the Parse button starts a parsing job using the files specified above Whenever a parsing job is started the old plate including all plate information is deleted and a new plate is created Each run possesses a plate which can be displayed by clicking on the show button next to Plate If a run has not been added to an experiment the Create button next to Create Experiment including plate produces an experiment and adds the selected run to this experiment Page 18 63 Stephan Pabinger User Guide OPCR z 20 Names specified for wells are equal to sample names Parser Parse Page 19 63 Stephan Pabinger User Guide PCR 4 4 Parse Run Whenever a parsing job is started a notification message 1s displayed The progress of the Job can be monitored using the Progress Information list described in 4 6 To par
49. un 13 1 CDNA A cDNA entry represents a physical cDNA sometimes called sample in qPCR experiments Each entry consists of a name amount concentration an optional protocol an option to specify whether DNAse treatment was performed and a description RNA Extract defines the extract where the cDNA was created from and 1s linked to the MARS database New cDNA me CI IS CET IS no protocol Protocol DNAse Treatment E Description 13 2 Target A target contains information about the target s name type barcode description and used primers Page 51 63 Stephan Pabinger User Guide PCR New Target no primer Primers testPrimer eqre i LO 13 3 Instrument Setting An Instrument Setting represents a thermocycler setup for a qPCR run The general information contains a name and a description Each setting consists of stages which are divided into steps Each stage has a stage number and a number of repetitions Steps are also numbered and each step consists of a duration and a temperature During the parsing process these values are extracted form the SDS file and stored in the database Users can manually insert an instrument setting with at least one stage and one step in each stage The number of stages and steps is not limited Page 52 63 Stephan Pabinger User Guide PCR New Instrument Setting EN Duratian min Temperature Step Duration m

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