Department of Pathology School of Medicine. Author Zach Dwight Location University of Utah Wittwer DNA Lab

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1 Author Zach Dwight Location University of Utah Wittwer DNA Lab

2 Introduction to uanalyze Functionality Users can compare experimentally obtained high-resolution melting data and theory based melting prediction for a single amplicon Melting curves can be identified spatially on an interactive plate map Assess melting curve data with curve metrics Curve Spread (Precision) 2-D Offset (Accuracy)

3 Input Required Examples files are located on home page of uanalyze Generic Instrument Data (generic *.gen) HR-1 Data Files (format) Instrument Data (HR1 *.vs2) LS32 Data Files (format) Instrument Raw Data (LS32 *.txt) LS Data Files (format) Temperature file (LightScanner *.tem) Fluorescence file (LightScanner *.mlt)

4 Walk Through Paste amplicon sequence into interface Must import first to set preliminary analysis region Upload Data Files Adjust Parameters Download Chart Image (.png)

5 1a ) Paste sequence into sequence box located under chart

6 1b ) Launch import file browser window after selecting instrument by clicking Folder Search icon to right of instruments Department of Pathology

7 2 ) Select all files required for instrument Department of Pathology

8 3 ) Move cursors to adjust normalization calculations Horizontal sliders adjust normalization cursor regions Vertical slider adjusts fluorescence threshold (curves will skip normalization if they fall under threshold) Note : Green lines indicate proper adjustment

9 4) Adjust chart axis for better viewing of data

10 5a) For LightScanner : 96-well geometric display links sample locations to displayed melting curves

11 5b) For LS32 : 32 carousel geometric display links sample locations to displayed melting curves

12 5c) For HR-1 & Generic : Data files are listed in a DataGrid. Click on a melt file to switch visibility.

13 6) Curves, or wells, may be turned on or off by single well, row, column, or entire plate selection ( All or None );

14 7) Predicted data can be optimized for monovalent salt concentration, DMSO content and free magnesium concentration

15 8a) Chart toolbox includes a chart download button (.PNG), data download (.TXT) and current normalization indicator ( E for exponential, B for baseline)

16 9a) Shift experimental or predicted curves to the experimental average using the overlay option drop down box options include : Predicted : Overlay predicted on experimental average Experimental : Overlay instrument data on experimental average *Note : Lab parameters are hidden for overlay

17 9) Curve color definitions Color Green Grey Normalization Normalized Not optimal normalization or does not meet fluorescence intensity threshold Curves normalized with exponential & baseline background removal methods as discussed by Palais and Wittwer (2009). Chapter found at S

18 Curve Metrics Curve Spread : Average max helicity difference between all experimental curve pairs 2-D Offset : Area between predicted curve and average experimental curve (sum of helicity % multiplied by temperature ) Scores found in lower right hand corner of GUI

19 Formatting Other Data Files Data output from other instruments must first be converted to our generic data format A generic file format is currently available Tab delimited Temperatures in first column, fluorescence in the second One melt curve per generic file Example generic data can be found on uanalyze splash page

20 Predictions A predicted melting curve is hardcoded to use the following parameters: Thermodynamics Unified-SantaLucia (Pubmed ID : ) Melting Algorithm umelt (Pubmed ID : ) Cooperativity Blossey and Carlon (Pubmed ID : )

21 Questions & Comments We welcome feedback, questions, and comments about all of our software. Lab Contact : Zach Dwight zach.dwight@path.utah.edu

22 Icons Icons used in uanalyze are freely available from FatCow us/

23 Disclaimer The following disclaimers apply to any use of uanalyze SM and related materials provided on dna.utah.edu. Access to uanalyze and related materials are provided as a courtesy, free of charge, and are intended for informational and educational purposes only. Any use of uanalyze and such related materials shall be at the sole and exclusive risk of the user(s). The University of Utah and its affiliates make no representations or warranties of any kind, express or implied, about the completeness, accuracy, reliability, suitability or availability of uanalyze or such related materials, or any related products and services. Any reliance you place on uanalyze or such related materials is therefore strictly at your own risk. In no event will the University of Utah or its affiliates be liable for any loss or damage arising in connection with your use of uanalyze or such related information including, without limitation, any direct, indirect or consequential loss or damages.neither uanalyze, nor results generated through the use of uanalyze, may be incorporated into any publication or formal or official report or document without appropriate reference and use of the service mark ( SM ). Users of uanalyze may not use the name or marks of the University of Utah, without the prior, written consent of the University of Utah.

24 Thanks and enjoy Department of Pathology