PeakStudio User Manual
Contents
1. PeakStudio screen shot Installation To install Peak Studio consult the requirements listed below then download the jar file To use the program you can double click on the icon or run from a command line details below Peak Studio PeakStudia jar Windows icon Mac icon System Requirements Max OS X e Mac OS X 10 5 Leopard or higher e Intel processor e 1GBRam e 4 MB available disk space e Java version 6 Peak Studio was built and tested on Mac OS X 10 6 4 Snow Leopard with 2 8GHz Intel Core 2 Duo processor and 2 GB of RAM Windows e Windows XP e Intel processor e 1GBRAM e 4 MB available disk space e Java version 6 Peak Studio was tested on Windows XP with a 3 0GHz Intel Pentium processor and 2GB of RAM Running from command Line Mac OS X Open a terminal window found in the Applications Utilities folder From here change to the directory where PeakStudio jar is located using cd yourpath without the quotation marks and run using this command userS java Xms128m Xmx1024m jar PeakStudio jar without the quotation marks Windows The Command Prompt application can be found in All Programs Accessories or accessed by clicking Start then Run and typing cmd and clicking OK From here change to the directory where PeakStudio jar is located using cd yourpath without the quotation marks and run using this command C path gt jav2. FE Peak Studio Peak Studio File Edit View Axis Analysis Help File Edit View Axis Analysis Help 1400 14 15 6 7 47 tano 6 7 18 4 11 16 8 9 4 17 L200 12 12 1 00 13 5 10 15 3 4 5 10 16 13 a 14 00 Boo 700 Boo 2 600 je pa RAQ Loo Q 3000 4000 5000 6000 7000 00 3000 4000 5000 6000 7000 a 2 Standard S LT ioo pem L o e e o T T ON F isn f eai sania L R 8 Standard A 8 SS B Standard Oo m Sind f Fa Before toggling the algorithm has missed this peak After toggling the peak is now correctly assigned You can also choose to edit peak calling parameters by going to Edit then Parameter Set and then allow the software to automatically call peaks again The modified peak calling parameters can be applied to a particular spectra or applied to all selected spectra Spectra Parameter Set Max InterSlope Distance Num to Trim from Beginning Num to Trim from End Regression Window Size Required Negative Slope Required Positive Slope Peak Thresholds Threshold Height Threshold MA Apply Abrubt Jump When Calling Peaks Apply to this spectra Apply To All Selected Analyze your data Once the standards spectra are acceptable you can choose to remove them from Table View by right clicking in Table View and selecting Spectra then uncheck the box for your standards color The sizing will remain in effect but you will only see sample data now As was the case for standards spec
3. ALRITE 48 wll no 22 260 3000 fa 30 23 728 4go 314 3602440012014029 PIT i Mouse Over Peaks Ctrl V Displays information box when the mouse cursor is placed on a peak 1 PEAK 14139 59 294 47 Shade Peaks Ctrl D Shades the area under a peak when the mouse cursor is placed on a peak Peak X Coordinates Ctrl J Inserts peak numbers based on base pair or scan number depending on the current view 6743 9993934470 7209 2 9128 5363 8162 10952 586137 Re d 97 9616 406 ai joe Tl my 1210o Line Thickness Ctrl U Adjusts the thickness of the displayed spectra Line Thickness 4913 2402 2 hes das Thin Line Thinkness Set X zoom Ctrl X Sets the x axis zoom level Input 9 Set the X zoom factor Anything other than a number between 1 and 500 0 will be ignored 1 9 Set Y zoom Ctrl Y Sets the y axis zoom level 2 Set the Y zoom factor Anything other than a number between 1 and 25 0 will be ignored 1 0 Zoom Out Ctrl Z Zooms spectra out to the original image size Raw Ctrl R View data in raw form Smooth Alt R View data in smoothed baseline corrected form Axis Dropdown Menu Display Displays the y axis with relative 2 Peak Studio fluorescence units RFUs and the x axis with File Edit View Axis Analysis Help either scan numbers raw
4. Relative Fluorescence Unit required for a peak to be considered Peak Height Threshold 25 0 Once you click Run a new window will pop up with a graph of your PCA This allows you to visually examine the grouping of samples You can mouse over data points to reveal which sample is represented or go to View then click the Name radio button and all the names will be displayed on the graph Hint you may find it useful to color code your samples to make them easier to visually inspect on the PCA graph To do this go back to Table View and click on the color of the sample you want to change You can assign any color to any sample It is possible to export the data used to generate the PCA for further analysis by going to Export then Matrix then select Data Chapter 2 Dropdown menu options File Dropdown Menu Open Spectra Ctrl O Use this function to open one or many fsa files Open Project Ctrl P Use this function to open a svaz project generated by PeakStudio Save Ctrl S Saves the current project as a svaz file Save As Save the current project to another location or with another name Import Allows the user to import metadata to associate with samples Peak Studio File Edit View Axis Analysis Help Open Spectra Ctrl 0 Open Project Ctrl P Save Ctrl S b Remove Sample s Ctrl C Close Project Ctrl A Exit Ctrl Enter When importing metadata into PeakStudio the file m
5. 2000 3000 4000 5000 6000 7000 s000 3000 10000 11000 Comp 1 Explains 57 30 Menus in PCA Window Export PNG Export a png screenshot picture of the PCA currently PCA displayed Expon View Edit Matrix Data Export the matrix of peaks and bins for any stats Components Export the matrix of the transformed data ene fo Components an View Shape Size This enables the size of data points to be PCA adjusted between 3 levels Zoom Out Ctrl Z Zooms PCA window out to the Original image size Name Displays the name of the spectra associated with Export View Edit Shape Size al zoomow cuz Medio Omme Lara each data point Edit Axis Displays dialog that allows components to be changed PCA along the x and y axes Change Axis X Axis Comp_1 57 30 m Y Axis Comp _2 13 32 7 Close Appendix A Peak Calling Heuristics Below is a diagram of a peak as it is determined by the peak calling algorithm Nonpeak Phase Nonpeak Phase Nonpeak Phase Down slope Phase Upslope Phase Initial Peak Calling Accurate identification of peaks is a critical step in ensuring that data is prepared for further analysis Our peak calling algorithm applies linear interpolation to separate signals of peaks from that of baseline in raw data from fragment analysis files The algorithm works by using a configurable parameter set that contains thresholds for
6. Icha nnel Number Type Peak Number Location Index Location Base Pair Height 2 1 105 Standard 1 2083 20 667 3 1 105 Standard 2 2216 30 571 4 1 105 Standard 3 2402 40 1061 3 1 105 Standard 4 2704 60 1097 6 1 105 Standard 3 3165 80 1004 i 1 105 Standard 6 3589 100 1378 8 1 105 Standard 7 3893 114 1330 9 1 105 Standard 8 4025 120 1334 10 1 105 Standard 9 4470 140 1329 11 1 105 Standard 10 4913 160 1074 QC Files Export a list of scan numbers and associated peak algorithm assignments B C D E basepair data feature 2 0 80 0806 F 3 1 80 0319 gt NON_PEAK 4 2 79 9833 1 NON_PEAK 3 3 79 9347 0 NON PEAK 5 4 79 880 2 NON_PEAK Fi 2 79 8374 2 NON_PEAK amp 6 79 7888 2 NON PEAK 9 J 79 7401 4 NON_ PEAK 10 6 79 6915 4 NON PEAK 11 9 79 0429 1 NON_PEAK 12 10 79 5943 6 NON PEAK 13 11 79 5456 2 NON PEAK Sizing Table Export a table that conforms to the format output of GeneMapper software 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa 2012 01 17 fsa Ma od wD a 13131 83 7322 92 B715 2 9987 64 9194 59 11409 38 10406 45 10430 55 10530 54 6571 19 10403 78 5826 93 9465 72 6417 16 dinne n Remove Sample s Ctrl C Highlight one or multiple files in the Table view and remove Clos
7. data or base pair I Display numbers based on peak calling algorithm BasePair Ctrl K Converts the spectral display I BasePair Ctl K from scan numbers raw data to base pair numbers based on peak calling algorithm Analysis Dropdown Menu PCA Ctrl 4 Opens a new window displaying Peak Studio File Edit View Axis principle component analysis PCA on the Analysis Help PCA Ctrl 4 spectra open in the Table View Select the range of basepair values the size of the bin to use and a value for the peak height Start BasePair 400 0 threshold then select Run End BasePair 400 0 Bin Size 10 0 Peak Height Threshold 300 0 The PCA output opens in a new window Hovering over data points reveals the identity of each point Colors are associated with the Peak Color from the PeakStudio viewer window See Chapter 3 for description of PCA in PeakStudio Help Dropdown Menu About Ctrl I Contains version information Peak Studio File Edit View Axis Analysis Help About Ctr Table View Table view is the bottom panel of the PeakStudio window Columns can be sorted by clicking on the column headers File Name Dye Color Show Graph Peak Color Rejected Peak Color NumPeaks Spectra Type e Blu H L nnm a 89 Data Orange ROICtay 68 Standard Default columns in Table view are File Name Displays the file name associ
8. 1 Blue FALSE 0 0 255 255 0 255 89 Data 4 10 Orange FALSE 255 153 0 255 0 255 68 Standard bi 10 Blue FALSE 0 0 255 255 0 255 167 Data 6 11 Orange FALSE 255 153 0 255 0 255 68 Standard T 11 Blue FALSE 0 0 255 255 0 255 148 Data B 2 Orange FALSE 255 153 0 255 0 255 68 Standard 9 2 Blue FALSE 0 0 255 255 0 255 66 Data 10 3 Orange FALSE 255 153 0 255 0 255 68 Standard 11 3 Blue FALSE 0 0 255 255 0 255 75 Data 12 4 Orange FALSE 255 153 0 255 0 255 68 Standard 13 4 Blue FALSE 0 0 255 255 0 255 131 Data 14 5 Orange FALSE 255 153 0 255 0 255 68 Standard 15 5 Blue FALSE 0 0 255 255 0 255 172 Data 16 6 Orange FALSE 255 153 0 255 0 255 68 Standard 17 6 Blue FALSE 0 0 255 255 0 255 115 Data 18 7 Orange FALSE 255 153 0 255 0 255 68 Standard 19 7 Blue FALSE 0 0 255 255 0 255 132 Data 20 8 Orange FALSE 255 153 0 255 0 255 68 Standard 71 8 Blue FALSE 0 0 255 255 0 255 159 Data 22 9 Orange FALSE 255 153 0 255 0 255 68 Standard 23 9 Blue FALSE 0 0 255 255 0 255 179 Data Binned Peak Matrix Export the matrix of peaks and bins for any stats A W a g in ua Mi m Pa j 0 S1S5 4 6 6 5 o o c D BP _ 25 28 BP 28 31 BP_31 34 BP _ 34 37 BP 58 61 1 0 0 33 10 0 54 0 11 0 44 0 2 0 0 0 3 0 0 0 4 0 32 0 5 51 45 62 6 35 50 0 7 0 0 8 35 AT 0 9 42 58 0 All Peaks Export peak information for a selected file D B C E F G
9. If you are using LIZ 1200 from ABI you should have 68 size standard peaks Notice that sample 4 only contains 67 identified size standard peaks We will correct this shortly File Name Spectra Type MumPeaks Peak Color j Standard GSi mE 10 Standard 6o ME Wo Standard 63 E 2 Standard 3 Standard __ 40 Standard _ 8 7 DE So Standard 60 E 6 Standard 63 mE To o Standard 63 E go Standard 6o mE 9 Standard 63 E Right click anywhere in the Table View and select Show columns then click on QC number Generally the lower the number the more confidence that peaks have been called correctly As a general guideline a QC between 0 18 and 0 30 is good see Appendix B Any column in Table View can be used to sort the data by clicking on the header Click the check box for Show Graph to display the spectra If you prefer you can go to Axis and check Basepair to display the spectra in basepair rather than raw data format Also in the Axis dropdown you can choose to Display the X and Y axes To see which peaks have been assigned with each size go to View Show Peak Numbers If you find that you are missing peaks or that they have been incorrectly identified you can manually correct this problem Double click on the misidentified peak and then right click this will bring up the option to Toggle Peak which allows a peak to be reassigned from accepted to rejected or vice versa
10. Peak Studio Contents Chapter 1 Chapter 2 Chapter 3 Appendix A Appendix B Appendix C Using PeakStudio Introduction Installation System Requirements Running from Command Line Terms used with PeakStudio An overview of the workflow Dropdown menus in PeakStudio File Edit View Axis Analysis Help PCA in PeakStudio Peak Diagram and Peak Calling QC Number Automated Peak Adjusting Acknowledgements Chapter 1 Introduction Welcome to Peak Studio a program for viewing and analyzing fragment analysis files generated by ABI capillary electrophoresis instruments PeakStudio is an open source program developed at UNCC in the department of Bioinformatics and Genomics by Jon McCafferty PeakStudio was developed in order to help make more objective decisions about fragment analysis files It allows users to view any file with a fsa file extension assign sizing to peaks manually edit sizing calls and generate PCA plots to analyze the grouping of data Peak Studio Jog File Edit View Axis Analysis Help ARAP WW AL Ls Ja Daad i AoA Ad S PERENE bas ia D S AA aAA A E Aado pipl LSH Ul ow AL ih ere ere 12000 13000 14000 15000 16000 17000 18000 _ 19000 20000 21000 22000 23000 24000 25000 260017 File Name Dye Color Orange Be 10 ue 1 Hy Bue
11. a Xms128m Xmx1024m jar PeakStudio Jar without the quotation marks Before using PeakStudio we recommend reading through the user manual and or watching our tutorial videos at http www fodorlab uncc edu PeakStudioPage html In the next section we will discuss some terms used with PeakStudio that may be unfamiliar Terms used with PeakStudio This manual contains some terms that are specific to PeakStudio and some which have a particular meaning for our purposes in regard to PeakStudio A list follows with several of these terms which will help as you read through the rest of the manual Bin Bins are used to group peaks for analysis Smaller bin sizes are more exclusive while larger bin sizes cause peaks of similar size to be grouped with each other Spectra In the context of PeakStudio the word spectra is used to refer to one of the 5 possible colors captured by the CCD camera in the genetic analyzer Every fsa file contains the data from the different colors channels but a spectra is one color separated from the others Table View This is the bottom portion of the PeakStudio window Table View is essentially a customizable table that lists details regarding samples that have been imported for analysis Peak Studio JEE File Edit View Axis Analysis Help 0 Two spectra from the same fsa file 0 which have been overlayed for viewing 0 in the Viewing Window adhd x Fes Oe Hee n
12. ated with each sample Data channel Displays the data channel associated with the dye used Show Graph Check the box to display the electropherogram in PeakStudio One or many graphs can be displayed at a time and peak colors can be adjusted to tell them apart Peak Color Peak colors are associated with dye colors but can be modified by clicking on the color swatch in Table view gt Pick a Color Swatches _ a Pe LI E m an E I ERATAN O E a a a U 0 S a a a a EAA EAA L 0 a a E a go Sample Test Sample Text Rejected Peak Color Peaks which are identified by the peak calling algorithm but are rejected as non real display as blue on the spectra Spectra Type This indicates whether the file represents data from a sample or the size standard associated with that sample NumPeaks The number of real peaks identified by the peak calling algorithm Additional columns available under Show Columns right click the mouse when cursor is within Table View include ber v File Name Graph Color v Dye Color Show Graph v Peak Color wv Rejected Peak Color Selected Peak Color v NumPeaks Smoothed v Spectra Type we Standards File NG Num Size Standard Peaks Spectra Y QC Number Base Pairs Called Show b Data Channel eene v Smoothed Add A Column Min atin LIE IELE RENAME Color by Graph Color The color of the spectra which is not identified by th
13. called peak Table 1 Automated Peak Adjusting Filtering Algorithm SD standard deviation Acknowledgements Dr Michael Thomas Flanagan for making his code publicly available www ee ucl ac uk mflanaga which we used for smoothing and area calculations in Peak Studio
14. cation of the left and right peak to predict the location of the middle peak The QC Score is then the sum of the absolute value of the difference between the predicted location and the observed location of the middle peak divided by the number of total peaks called Equation 1 When the user toggles a peak on or off the QC Score is updated in real time with lower scores being better The score represents the overall accuracy of the peak calling algorithm therefore a smaller score especially less than 0 5 indicates that the predicted peak was very close to the actual peak that was called Higher scores are a signal that something is wrong and the user may have to manually adjust peaks 2 predicted observed l QC Number __ Equation 1 number of peaks Appendix C Automated Peak Adjusting To increase the user friendliness and the speed at which data can be processed we implemented a feature that allows the user to automatically adjust peaks in the standard spectra correcting any potentially misclassified data regions We try to separate background noise from what should be actual peaks by applying filters to the spectral data Our first filter assumes that because peaks will have larger areas and higher heights than non peaks they will contribute more to the variation in the distribution of areas under the curve for the spectra Setting a default threshold of 3 standard deviations user preference in the parameter set we can filte
15. e Project Ctrl A Closes the current project but keeps PeakStudio open Exit Ctrl Enter Closes PeakStudio Edit Dropdown Menu View Axis Analysis Parameter Set AQ Standards AW Data Smoothing Help Parameter Set Ctrl Q Allows the user to adjust peak calling parameters Spectra Parameter Set 0 B aa Max InterSlope Distance Num to Trim from Beginning Num to Trim from End Regression Window Size Required Negative Slope Required Positive Slope 100000 Peak Thresholds Threshold 25 0 Height Threshold NA Apply Abrubt Jump When Calling Peaks Apply to this spectra Apply To All Selected Standards Ctrl W Use this to import a standards file eer ae TEIH Standards File StdSizeValues6 amp txt Import Standards Standard Weights W Automated Standard Peak Adjusting Se Smoothing Parameters a e man Baseline Window Baseline Window increment Savitzky Golay Smoothing Window Savitzky Golay Polynomial Degree Smooth All Smooth Selected Only View Dropdown Menu ANO File Edit Axis Analysis Help Show Peak Numbers B v Mouse Over Peaks AN v Shade Peaks D Peak X Coordinates J Line Thickness LU Set X zoom AN Set Y zoom way foom Out SE v Raw R Smoothed XR Show Peak Numbers Ctrl B Inserts numbers above peaks which have been called by the peak calling algorithm 15 J p 2 41801213 10 2004
16. e peak calling algorithm as a peak or rejected peak Selected Peak Color The color a peak displays when it has been selected by double clicking on it QC Status Displays Good Data or Bad Data depending on whether the peak identification algorithm was able to correlate the size standard peaks to their correct sizes Standards File The file name which was used to assign sizes to spectra is displayed Num Size Standard Peaks Displays the number of peaks in the assigned standards file QC Number A numerical representation of the quality of peaks based on the interpretation between expected peaks and observed peaks See appendix B for further detail on QC number Base Pairs Called Displays whether basepairs have been called for each sample Smoothed Lets user know if smoothing algorithm has been applied to the spectra Additional right click options Spectra b Show Show Columns Add A Column Spectra 1 blue dye FAM HEX 2 green dye VIC 3 yellow dye NED 4 red dye PET ROX 105 orange dye LIZ Show Select all marks all the checkboxes to show all spectra on the screen at one time Unselect all unmarks all the checkboxes to clear the screen of all spectra Add A Column Adds a blank column in which you can add your own text Delete Deletes a column that was added using the Add A Column function Rename Allows you to rename the column that was added Co
17. fy that now Click the Open button to open them into the Peak Studio window E LIZ1200 68 txt Notepad File Edit Format View Help Data Channels Standards lv Bewe Coreen Cvolow CRed Grange Look In Sample data final D 3 fsa 4 fsa 5 fsa Ea 1 fsa B 6 fsa 10 fsa 7 fsa 11 fsa 8 fsa D 2 fsa C 9 fsa File Name Files of Type Arisa T RFLP fsa Cancel Files are displayed in the Table View bottom portion of the PeakStudio window Each fsa file that you import will generate a separate spectra for each dye previously specified With ARISA for example there should be two spectra one for the blue FAM labeled products and one for the orange LIZ labeled size standard fragments File Name Oye Color snows Graph Color ey Add a standards file Go to Edit Standards click the Import Standards button then find your standards file and apply You can opt to apply the standards to all the files or you can select certain files in TableView Standards File 1LIZ1200 68 txt M Import Standards Remove tandard Weights 860 Apply To All Spectra Close Once you have applied a size standard file to the spectra you will want to determine whether the peak calling algorithm has identified the size standard peaks correctly Begin by checking the NumPeaks column
18. ines I be ee ee ei ee eA Mla A A Kaaka Wh IMa 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 x4 NumPeaks Spectra Type 68 Standard pata O S Orange A aA 68 Standard Bue a Data Orange TTTTTTTTTTTTTTTCTC wCcaLaS EE Be ata Standard Data 31 Data gt 115 C Orange TTL a 6eStandard Ie a ee 2Data Bo range OQ Ld TTT A 6estandard po Data 149 Data C I ee 17o Data o TableView An overview of the workflow PeakStudio has been designed to provide a straightforward method of data viewing and analysis Below is a walkthrough of a typical project To begin You should have the PeakStudio jar file downloaded on your computer or the source code You will need fsa files to analyze and a size standard file The size standard file indicates which size standard fragments were used and should be in a txt file format that contains a list of each size separated by a return each size on its own line Import samples At the upper left of the window go to File Open Spectra and select the file s to be analyzed You must select the color of the dye labeled products in ARISA this is typically Blue for FAM and the color of the size standard dye by default this is Orange for LIZ If you are using other dyes for your products or size standards you can speci
19. lor by Changes the color of the spectra associated with the label in the column created Chapter 3 PCA in PeakStudio PCA or Principal Component Analysis is an algorithm that identifies patterns by revealing similarities in a dataset This is accomplished by transforming a high dimensional multivariate dataset into a set of principal components allowing you to project the data onto a new coordinate system such that the greatest variance by any projection of the data comes to lie on the first principal component the second greatest variance on the second component and so on Data can now be visualized in a lower dimensional space creating a more informative view of the dataset The Data Matrix used as input to the PCA algorithm is generated with user defined parameters Using a start and stop location bin width and peak height threshold the spectra is divided up into bins and the peaks which meet the height threshold are grouped into the bins A binned matrix with columns representing bins and rows representing spectra is created with the cells containing the sum of peak heights for each peak over the defined threshold The PCA algorithm uses this binned data matrix as input i Jog Export View Edit Oo oOo 3 5 7 00 4 3 5 6 e D 00 5 si oo 10 11 9 e eS N NGEN E E ee pp EE E E ee ee ee es ee E 000 000 9 kd i 000 ag 000 000 7 000 000 000 7000 6000 5000 4000 3000 2000 1000 j 1000
20. r out larger peaks from background noise The second filter is applied if the number of called peaks differs from the number of actual peaks according to the user provided weights We walk through the current set of peaks and make adjustments by turning peaks on or off trying to minimize the QC score Our third and final filter is applied if the previous two filters were not successful in correctly adjusting the peaks This filter uses the current set of features peaks amp non peaks and gathers potential peaks by incorporating features that are a default 2 standard deviations away and also requiring that a peak meets a default height threshold of 33 user preference in the parameter set the height of the nearest called peak Post automated adjustment of the standard spectra the weights should be applied correctly to the called peaks Understanding that this is a heuristic and will provide the optimal solution but not always the correct solution the user has the ability to manually adjust any standard spectra that passed the filter steps yet has miscalled peaks This adjustment procedure is intended to reduce the amount of time a user needs to process data from raw fsa files to usable data for downstream analysis Identify features with SD greater than 3 label them as peaks Toggle features on and off trying to minimize the QC score 3 Gather features with SD greater than 2 and whose height meets a threshold of 33 the height of the nearest
21. tra the Show Graph check box brings up each data spectra in the window You can show one or many spectra at atime Sometimes when viewing multiple spectra it can be helpful to change the display color This is easily achieved by clicking on the Peak Color box for the spectra then selecting a new color in the pop up window l Pick a Color Swatches HSB RGB Carrer OG GO EE EO Recent Cancel Reset Preview 700 Base pair Peak Color A Lo S y Od m E E Sample Text Kl a C nn Sample Text Sample Text ox Now you can determine how closely related your samples are to each other using PCA To do this go to Analysis and click on PCA This will bring up a window where you can specify which range of bases you would like to compare how large your bin size should be and what threshold to use for cutting off peaks In capillary electrophoresis very small and very large fragments are often not as reliably sized as those in the middle so we often limit our analysis to what we consider to be the best fragments for those samples Note the Start BasePair of 25 and End BasePair of 900 Depending on the range of fragments of interest you may Start BasePair 25 0 want to make that range narrower or wider Using the current End BasePair q00 0 setting bins would be 25 28 28 31 etc Peak Height Bin Size 30 Threshold indicates the minimum y axis value
22. ust be formatted as a tab delimited text file and it must be formatted using the following template sample name anyheaderl anyheader2 anyheader3 The first column must be titled sample name 1 2A u and the remaining metadata columns can 2 45 J contain any title you choose The sample i A names included in the metadata file must 5 mm K match the sample names loaded in PeakStudio 6 12D J 7 12A U 8 25 U 9 2A U 10 AS U 11 7X J Once metadata have been assigned to the spectra right ea nn clicking in a header column in Table View allows the spectra to REGIE b be colored based on the data in that column Show Columns P Add A Column Delete header 1 Rename header 1 Color by header 1 Spectra which have been colored by header2 then sorted by header2 Export aO Edit View Axis Analysis Help Open Spectra o Open Project p Save 5 Save_AS Import fe PNG XG Encapsulated PostScript CN Remove Sample s AL Table View xX Close Project A Binned Peak Matrix WM Exit Aal All Peaks Aa a OC Files K Sizing Table LO PNG Export a png screenshot picture of the spectra currently displayed Encapsulated PostScript Export a eps screenshot picture of the spectra currently displayed Table View Export a spreadsheet of the samples details in Table View B C D E F G File Nam aDye Color Show Graj Peak Colo Rejected NumPeak Spectra Type 2 1 Orange FALSE 255 153 0 255 0 255 68 Standard 3
23. values such as slope and peak heights assigning each data point to one of five possible phases non peak peak up slope down slope or inter peak After assigning all the data points to one of the four phases the peaks can be identified A peak is recognized as a collection of points that meet the requirement of beginning at an up slope phase and ending at a down slope phase Taking the difference between the highest and lowest data points in the region containing the peak determines peak heights If the peak height does not meet the threshold from the parameter set the region is relabeled as a nonpeak region Adjusting the parameters allows the user to redefine what constitutes a peak with the resulting peak calls seen in real time Since any peak calling algorithm has the potential of missing peaks or miscalling peaks Peak Studio combines automated peak detection with the ability for the user to visually inspect and manually select peaks that need to be adjusted Through the use of the program samples that have misidentified peaks can be salvaged by manual user selection of peaks Appendix B QC Number A quality control method was developed to allow the user to rapidly identify any spectra where the peak calling algorithm mislabeled peaks Quality control scores are calculated through a linear interpolation process We start with a standard spectra and walk through all of the peaks that have been called Taking a set of 3 peaks at time we use the lo
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