Coffalyser.Net Manual

Coffalyser.Net Manual

For the manual for Coffalyser digitalMLPA for digitalMLPA data analysis, see this download. However, for certain applications, the instructions for use may differ. In the event of conflicting information, the relevant instructions for use take precedence. A similar video about the analysis of regular MLPA data to determine copy numbers can be found here. In case you are unable to view the video on YouTube you can also download it here. The following articles are referenced in the video: Adding a capillary electrophoresis device to Coffalyser.Net Adding sheets to the sheet library Quickly defining the sample types in the fragment analysis tab Adjusting a bin set (creating a manual bin set) MLPA troubleshooting wizard However, for certain applications, the instructions for use may differ. Coffalyser.NET analysis manual beta version. Status. Release candidate. Classification. Confidential. VersieVersion. Date. Classification:Confidential. Version history. Version. Auteur. Owner. CommentsJordy Coffa. Jordy Coffa. Initial versionCoffalyser.NET analysis manual beta version. VersieThis document contains the analysis manual for Coffalyser.NET. ThisAmsterdam, 22 July 2013. Jordy CoffaCoffalyser.NET analysis manual beta version. VersieIntroduction to MLPA normalization.Organizations and user accounts. User Access. Connecting to a local database. Connecting to a server database.What is the Coffalyser.NET sheet manager. Downloading new sheets. About products, lots and version. Viewing the available products, lots and versions. Adjusting a sheet lot. Adding a new “custom” product to the sheet manager.What are the CE devices. Adding a new machine. Choosing the correct machine. Choosing the correct filter set. Changing the CE devices settings.What does a project contain. Creating a new project. Project settings.What does an experiment contain. Creating a new experiment.Coffalyser.NET analysis manual beta version. VersieSetting the experiment type. Setting the channel contents.http://cortemadera.com/wysiwygfiles/casio-ws-300-manual.xml

• manual, reference manual, manual.

Setting the channel settings.Importing the data files. Starting the fragment analysis. About the fragment analysis quality scores. Using the fragment results explorer.Setting up the comparative analysis. About the comparative analysis quality scores. About the comparative experiment results explorer. About the comparative sample results explorer.Introduction to MS-MLPA analysis. Setting up the MS-MLPA analysis. Comparative analysis experiment explorer with MS-Data.Coffalyser.NET analysis manual beta version. VersieMLPA kits generally contain about 40-50 oligo-nucleotide probes targeted toThe number ofFor each probe oligo-nucleotide in a MLPA kit thereThe ligase enzyme used, L igase-65, is heatinactivated after the ligation reaction. Afterwards the non-ligated probeAll ligated probes have identicalThe different length of every probe in the MLPA kit then allows theseTo make the data intelligible, data of aIf only oneCoffalyser.NET analysis manual beta version. VersieThis can beFigure 1 MLPA fragment profile of a patient sample with Canavan disease (top) andCanavan disease is the result of a defect in the. ASPA gene on chromosome 17p13. The fragment profile shows that the probeIt may however not be feasible to obtain reliable results out of such a visualTo make (complex) MLPA data easier understandable unknown andThis can beIn MLPA kits,Coffalyser.NET analysis manual beta version. VersieReference probe are usuallyThe results of data normalization areIn most MLPA studies, comparing theProbe ratios of below 0.7 orA delta value of 0.3 is a commonly acceptedAberrations can be recognized moreThis criterion alone may often not provide the conclusive results required forOther reasons forThe significance of each ratio can beIn this document we show the features and integrated analysis methods ofCoffalyser.NET analysis manual beta version. VersieIn the next chapters we explain how we can useCoffalyser.NET analysis manual beta version.http://lumieretvie.com/userfiles/casio-ws--100h-manual.xml

VersieThe client-server model has one mainEven though thisHaving both client and server on theWhen a new client is installed on aThe server application will then answerIn addition to serving as a common data archive, the database provides userOur database modelEach user receives a certain role within eachThese rights may for instanceThese same levels may also be applied onThe initial project creators will also be the project administrators who areCoffalyser.NET analysis manual beta version. VersieAfter installing Coffalyser.NET on a computer in a standalone configuration,To login to your localFigure 2 Coffalyser.NET login form for single PC, standalone configurations. If your login fails, please check the configuration settings of your database.Make sure that the configuration atCoffalyser.NET analysis manual beta version. VersieThe server nameIf you don’t know the IPaddress of your server computer, start command prompt on the serverYour IP address should turn up in the list.Coffalyser.NET analysis manual beta version. VersieCoffalyser.NET is equipped with MLPA sheet manager software, allowingNext to this, the sheet managerThe sheet managerIt can display scheduled update checks and canWith just one click, youEach Coffalyser.NET version starts with an empty database, containing onlyTo obtain the necessary information for data analysis, right click on “SheetCoffalyser.NET analysis manual beta version. VersieNext you will see the download form, click on “Start Update” to download theDirectly after you will see an update window, showing the progress of theWhen the update is finished you will receive aCoffalyser.NET analysis manual beta version. VersieFigure 6 Coffalyser.NET start screen and location to download sheet updates.To circumvent problems with designated products and lots, and to makeEach productR-number indicating products that can be used for quantification of RNABy selecting “Add” from the right click context menu you can create a newCoffalyser.

NET analysis manual beta version. VersieFigure 7 Coffalyser.NET sheet library product overview window.To view a product, first add it to the Coffalyser Work Sheet library by usingAfter you add a product the Coffalyser Work. Sheet Editor will automatically open allowing you to make the necessaryAt the moment when youThis copy can then beCoffalyser.NET analysis manual beta version. VersieYou can find who created this MLPA mix, who modified it, to whichFigure 8 Coffalyser.NET sheet library product lot version details window. Figure 9 Coffalyser.NET sheet library product lot version probes overview window.Coffalyser.NET analysis manual beta version. VersieYou can now adjust to the organization to which this version belongs, theThe second tab; “probes”, can be used to adjust the separate details of eachUse the rightPlease note, thatRight click on any probe and select “Validate Sheet Data” from the right clickYou may also change the control mix that is related to the probe mix byCoffalyser.NET analysis manual beta version. VersieYou may also add new “custom” product to the database. These areTo add a new product, start at theThis will openCoffalyser.NET analysis manual beta version. VersieCE devices within Coffalyser.NET are considered to be the capillaryCoffalyser.NET fragment analysis settings are specific for that machine andWhen such a device isThese settings will suffice in most cases, but inOur software is compatible with binary data files produced by all majorBefore any data import can be performed weDevices” and select “Add CE Device from the right click menu.Coffalyser.NET analysis manual beta version. VersieAfter selecting “Add device” a new window will open allowing you to defineNext to “CEFigure 11 Choosing your machine type.Coffalyser.NET analysis manual beta version. VersieAfter the correct machine was selected you should select the correct filterThe filter setsTable 1 contains the most common filter sets used by ABI.

If for instance youFigure 12 Choosing your filter set.Coffalyser.NET analysis manual beta version. VersieDye Set Filter Set Blue. Green Yellow. RedChanging the CE devices settings. By going through the different tabs of the CE Device properties window, youWhen you areIn that case you need to be aWhen performing detection of fluorescence in capillary electrophoresisCoffalyser.NET analysis manual beta version. VersieThe baseline wander of theOur software corrects forFirst, the signals of the first 200 (default is 80 for the size marker channel;Then for every 200 subsequentFor probe channels, this corrected baseline 1 is then fed as input for a filterAlternatively anRFU (see figure 19 “Baseline maximum signal for correction fine”) onBy applying this secondIn general it is not recommended to adjust the baseline settings, however ifCoffalyser.NET analysis manual beta version. VersieIn capillary-based MLPA data analysis, peak detection is an essential stepEven though various peak detection algorithms forWhile peak detection and peak size callingDue to the relatively nature of the MLPAOur peak detection algorithm exists of two separateCoffalyser.NET analysis manual beta version. VersieProgram administrators can modulate the peak detection algorithmThis threshold is used to filter out small peaks in flat regions. The minimalThese value are called thePeak area is computed as the area under the curve within the distance of aThese values are called theThe application of this criterion can consists of 3-4 steps:Coffalyser.NET analysis manual beta version.

VersieThis value is called: “detect fake peaks reset peakIn some cases the signal does not return to zero,This value is called: “MinimalThe median peak signal is calculated by the percentage of intensity of eachSince the minimum and maximum thresholds are dependent on detectedAfter peak end points have been identified, the peak width is computed asThe peak width should beThis filter is also applied after an initial peak detectionThis method is only applied for the size marker channel, and involves theIn case the correlation is less than 0.999, theThe minimal correlation quality is a default valueAfter each peak is detected it needs to obtain a size, which in general will beCoffalyser.NET analysis manual beta version. VersieLengths of unknown (probe)Once all peaks have been size called, the profiles must be aligned toPeaks corresponding to similar lengths of nucleotides may still be reportedOur software uses anThis procedureOur software algorithm follows four steps: reference profile analysis,The crucial task in data binning is to createWhile this procedureCoffalyser.NET analysis manual beta version. VersieSpecific settings can be applied for control fragments and probe fragmentsThis threshold is used to filter out peaks which may be detected in the firstThe minimal and maximal peak amplitudes are arbitrary units and defaultThese values arePeak area is computed as the area under the curve within the distance of aThe peak area ratio percentage of a peakThese values are called theThe search range determines the size in which the binning procedure willThe smaller thisCoffalyser.NET analysis manual beta version. VersieData filtering is the actual process where the detected fragments of eachThe binning procedure may forThe amount of fluorescence of each probe productAn algorithm can then be usedFiltering can be optimized by adjusting the settings shown in figure 16, theseFigure 16 Filtering settings tab.Coffalyser.NET analysis manual beta version.

VersieOur database setup contains a large number of subtraction levels, not onlyWhile all data normalization occurs perMultiple MLPA mix results mayBy creating projects, usersAfter creating an empty solution, users can add new or existing items to theCoffalyser.NET analysis manual beta version. VersieAfter a new project is created you can define the default capillaryFigure 18 Project settings window.Coffalyser.NET analysis manual beta version. VersieAfter creating an initial project, we can create experiment within this project. In each experiment data files can then be imported to the database andUsers then need to define the experiment type andSamples may further be typed as: MLPA test sample. MLPA reference sample, MLPA positive control, or MLPA digested sample.To create a new experiment within a project, right click on the project youCoffalyser.NET analysis manual beta version. VersieDirectly after you create an experiment you will be able to adjust theYou may howeverAfter you clickCoffalyser.NET analysis manual beta version. VersieAfter adding a name and description to your experiment, you may define theFirst we need to determine what type of experiment we are analyzing. ThereThese are experiments that are performed using standard MLPA probes orDNA target sequences present in each sample. These experimentsThis reference sample is usuallyThese experiment are combined experiment where both the copy numberFor MLPA probes thatCoffalyser.NET analysis manual beta version. VersieIn case only one ofRNA experiments are quite similar to copy number experiment, except thatSample DNA isIn the analysis you mayAlternatively users may only evaluateFigure 21 Experiment fragment analysis settings window.After settings the correct analysis method, you need to set for each dyeThe channels are usually setCoffalyser.NET analysis manual beta version.

VersieIf your channels are not set correct,The name of each dye should appear inNow you will be able to set the contentChannels are either set to “probes” indicatingFAQ). If you have set the content of a channel to be a probe mix, then youAfter settings the channel contents you will find some other settings behindNormalization in this caseReference probe areIn case a MLPA kit does not containIn population analysis mode, allTo change the analysis method click on the little arrow row define as aThe last two columns “DNA type” and “marker” will automatically be set forIf you chance to work with more than 2Coffalyser.NET analysis manual beta version. VersieAfter you have set the settings on the details page, you can go to the nextRight click anywhere on theAdd (from file)” from the right click menu (figure 22). Figure 22 Fragment analysis window for importing samplesFor ABI-devices,AgilentAt this point the files are not stored in theCoffalyser.NET analysis manual beta version. VersieIf all samples were importedThis form allows you to adjust the sample types that you have used in yourWe distinguish the following types:In case no reference samples areThe data forCoffalyser.NET analysis manual beta version. VersieIn case no reference samples areNext to this reference samples are used to estimateThey are used to make sure notCoffalyser.NET analysis manual beta version. VersieWhen you are finished adjusting all the sample types,The screen will automaticallyAfter you click on the fragment analysis button the fragment analysis settingsFirst tab contains all settings relatedThe exact differences and effectEnabling the advanced baseline methodFirst baselineCoffalyser.NET analysis manual beta version. VersieThe advanced baseline detectionInstead such peaks will be assumed to be split peaks and the fluorescenceThe peak recognition tab also allows you to change the way peaks are beingLinear regression refers to aThe order can beThis results in a regression line withCoffalyser.

NET analysis manual beta version. VersieAs discussed earlier (but also in the coming chapter about manual binning). Coffalyser a window or panel based approach to link peaks with comparableIn order to define these panels or bins we needThe more the lengths areCoffalyser allows two types of probe length to beCoffalyser lengths areHow to create a manual bin set is explainedCoffalyser.NET analysis manual beta version. VersieOn the second tab of the fragment analysis settings form you can find theThese settings are in generalThis page describes theCoffalyser uses several matrix correlations calculations. From the top downThis is the minimal correlation between the dataIn case a correlation wasCoffalyser.NET analysis manual beta version. VersieAfter this similarity matrixFrom this point a path will be tried to find follow aAfter a completeFinally the path that has the same lengthFigure 27 Fragment analysis probe alignment (auto bin) settingsCoffalyser.NET analysis manual beta version. VersieThese settings are in general optimized forThis page describes the properties thatThe method is essentially equal to theNote that the path retrieval correlation isThis is done becauseThis will be optimized in theBecause of problems arising from poor sample preparations, presence of. PCR artifacts, irregular stutter bands, and incomplete fragment separations,By creating a series of quality scores toDNA, MLPA reaction, capillary separation and normalization steps (figureThe quality of each step can fall roughly intoThe results of these analysis steps can be acceptedThese steps represent samples with contaminationThe results of these steps fall betweenThe related data and additional recommendations canCoffalyser.NET analysis manual beta version. VersieBased on the quality scores you may use the right click menu to open theTo get to a final score several different criteria areEach score that is dependent on the measurementCoffalyser.NET analysis manual beta version.

VersieBackground: theIs a change in one of theseFor MLPA the size callWe can use the technique ofWe deal separately withThe Correlation can be calculated by. Problems are indicated when: withoutWe thereforeFor correlations lower than 0.999, a subtractionRecommendation with problems: check the detected size marker peaksIf peaks are notProblems withCoffalyser.NET analysis manual beta version. VersieOptimal performance of the capillary system isProblems are indicated when: in case the measured average baseline, orFor an ABI-3130xl forRecommendation with problems: Remove the capillary array at the manifoldUse sterile water. DO NOT USE. METHANOL. Clean in one direction only. Use “Direct Control” from theBackground: while peak detection and peak size calling are very importantMost capillary electrophoresis devices use electro kinetic injectionBoth of theseBecause the entireLarger volumes will have moreSince we are not interested in quantification of the size marker peaks, butSignal strength isOptimal size marker signalsCoffalyser.NET analysis manual beta version. VersieProblems are indicated when: in case the measured median peak signalNotifications willNext to the median peak signal intensity we also check the maximum peakIn case the measured peakNotifications will be given in case the maximumFr an ABI3130xl the maximum peak intensity is set at 5600 for a notification and atRecommendation with problems: adjust the concentration of the size markerBackground: size markers are usually developed having fragmentsIn addition, the presence of the same multiple bandsA drop in signal of the fragments is thusProblems are indicated when: To make sure that the signal drop is causedCoffalyser.NET analysis manual beta version. VersieThen we measure theIn case signalIn case this sloping is accompanied by peakFRSS total. Recommendation with problems: rerun with alternative injection settings.

Run also a lane with only marker, since contaminant introduced from theBackground: we usually expect that all fragments of the used size markerIn some cases however the marker may onlyProblems are indicated when: not all marker peaks that were expected wereRuns with an incomplete marker therefore get aRecommendation with problems: rerun with alternative injection settings. Run also a lane with only marker, since contaminant introduced from theMLPA reaction. To get to a final score seven different criteria are evaluatedStart score of the FMRS is 100 points.Coffalyser.NET analysis manual beta version. VersieExcess DNA mayOverloading, and more specifically truncated peaks will result in a completeThe optimal range for peak quantification isProblems are indicated when: for most devices we give warning in case theFMRS. In case the signals are between the 4-5% or 50-60% of the absoluteIn case of an ABI-3130XL theRecommendation with problems: low raw data signal can be caused by aIt is vital to the success of fragment analysisIt is recommendedPCR products less than 3KB in length). In many cases the amount ofSpectrophotometer estimation of DNA samples isCrude preparation of DNA templates which haveMLPA reaction (as in the case of crude alkaline lysis minipreps). CorrectiveCoffalyser.NET analysis manual beta version. VersieAlternatively,Use the preheat treatment for highlyLow raw data signal due to “bad formamide”; formamide is used toThe formamide solution must be prepared and storedIf the formamide is notRaw Data signal Corrective actions: Use the special Sample Loading. Solution (SLS). Do not freeze-thaw the SLS or formamide solutions. StoreSome dyes are not stable inpure water solutionsLow Raw Data Signal Due to Insufficient Sample Injection; poor injection of. DNA fragments onto the CEQ capillaries will lead to low Raw Data signal.

Since the CEQ uses electrokinetic injection it is highly sensitive to excessThe excess salts compete with the DNAThe sources of the excess salts areCorrective actions: Follow a desalting procedure such as: ethanolDNA Polymerase inhibitor; if the correct amount of template was added andIn this situation furtherIn some cases a simpleCoffalyser.NET analysis manual beta version. VersieLow Raw Data Signal Caused by Poor Quality Mineral Oil; the mineral oilThe red and black dyesRecent experiments at MRC-Holland have shown that the use of patient orThese experiments indicate that a minimum of 5DNA samples eluted from a purification column withThis depurination of sample DNABackground: high probe signal intensity; in a few cases, the signal strengthThis can lead to an erroneousIf the peaks are “squared-off” at the top, then the detector isProblems are indicated when: next to the median probe signal intensity weA probe signalIn case the highest probeCoffalyser.NET analysis manual beta version. VersieRecommendation with problems: If the peaks are too high, the simplestUse less template DNA orBackground: high baselines decrease the dynamic range of detection of thatProblems are indicated when: in case the measured average baseline, orIn case theFor an ABI-3130xl forRecommendation with problems: Remove the capillary array at the manifoldUse sterile water. DO NOT USE. Use “Direct Control” from theBackground: An effect that is commonly seen with MLPA data is a drop ofCoffalyser.NET analysis manual beta version.

VersieUV irradiation which may result in differences in amplification rate orSignal to size drop may further be influenced byEven though some signal to size drop is expected,Problems are indicated when: to check if there are problems with probeIn case there is a problem with the capillariesThen we measure theIn case of a signal drop of more than 70% a severe warning will be givenIn case signal to size sloping isIn case of a severe warning another 30 penaltyRecommendation with problems: rerun with alternative injection settings. IfBackground: in a successful MLPA reaction more than 70% of the addedOften a lot of the available primer isDNA fragments that have complimentary sequences to one or both usedCoffalyser.NET analysis manual beta version. VersieA large primer-complex peakProblems are indicated when: to measure if there is too much primer left weSmaller mixes are allowed to haveRecommendation with problems: either use different primers, or make sureMRC-Holland decided to use specialBackground: the percentage of peaks that were detected, that were notLarge amounts of shoulder peaks mayProblems are indicated when: if more than 70% peak signals are detectedRecommendation with problems: increase the minimal peak detectionBackground: next to normal baseline heightening, baseline curving may alsoMost of this signal originates from theCoffalyser.NET analysis manual beta version. VersieBy measuring the fluorescence underneath the probesThis thus also indicated howProblems are indicated when: if more than 50% of baseline curvature existsRecommendation with problems: in most cases baseline curvature is likelyBackground: If the DNA concentration during the MLPA hybridization wasMLPA reactions can be performed in a concentration range between the 20500ng. We assume the DNA concentration is about 10ng if the medianProblems are indicated when: in case the ratio of 92 ligation fragment asFMRS, warnings will minimize the FMRS with 60 points.

A warning will beRecommendation with problems: in case there is a clear problem with the. DNA concentration, reaction should be repeated using higher DNABackground: incomplete DNA denaturation will not provide reliable results. We assume the DNA denaturation was incomplete if: the ratio of the signalCoffalyser.NET analysis manual beta version. VersieProblems are indicated when: in case the ratio of 88 control fragment andFMRS. In case the ratio of only the 96 or 88 as opposed to the 92 fragmentRecommendation with problems: in case there is a clear problem with the. DNA denaturation, the reaction should be repeated and DNA should beIn case the sample mayIf the ratio of the signal of control fragment X asIn case the ratio is betweenThe Y-control fragment isBy selecting “Open” from the right click menu on the fragments analysisThis can also be done by double clickingThis fragments analysisCoffalyser.NET analysis manual beta version. VersieThe fragment results explorer consists of 8 different tabs. The first tabFRSS and the FMRS (figure 29). Figure 29 Fragment results explorer sample overview screen. Other available screens are:In case there is a problem withThese factors are also separatelyThis screen can be used toCoffalyser.NET analysis manual beta version. VersieFragment profile: displays the baseline corrected signals of each. Displayed signals of channels set as probe mix content will also showAbove each peak topBy hovering over theGenomic profile: displays the baseline corrected signals of each.

Displayed patterns will also show which peak signals were identified asThe coordinates of the peak top of the peakFragment analysis steps: on this page you view each of the fragmentYou can alsoBinning: displays the peak heights on the y-axis and the estimated peakNext to this, using green andOn the X-axis right underneath each bin the probeIn case a signalBy hovering overNext to this the median, average and standardOur algorithm is also able to link more than oneThe amount of fluorescence of eachCoffalyser.NET analysis manual beta version. VersieHere you may view encoded data from each fileRPRN-1,, used size standard by STDF-1, run temperature by TMPR-1,Figure 30 Fragment results explorer genomic profile tab. Each tab of the fragment results explorer has several options that can beYou can for instance: view eachCoffalyser.NET analysis manual beta version. VersieYou may furthermore use manual zoomingOnce all peaks have been size called, the profiles must be aligned toPeaks corresponding to similar lengths of nucleotides may still be reportedOur software uses anOur software algorithm follows four steps: reference profile analysis,The crucial task in data binning is to createIn the first step ourNext, the largest peak inTo create a stable bin, we calculate the averageIf no referenceSince some probes may have a large differenceWe therefore check if the length that we have relatedWe do this by calculating howThe expected probe peak lengths may be predictedEven though a fullThe set of bins for each probe inCoffalyser.NET analysis manual beta version.