Molecular Biotechnology and Genomics Lecture 20: Data Analysis of RAPD and STR Files - Pro, Study notes of Bioinformatics

Download Molecular Biotechnology and Genomics Lecture 20: Data Analysis of RAPD and STR Files - Pro and more Study notes Bioinformatics in PDF only on Docsity! BINF 6010/8010 Fall 2008 Molecular Biotechnology and Genomics Lecture 20: Nov 11th, 2008 Lecturer: Dr. Weller C W bourse e pages: http://webpages.uncc.edu/~jweller2 Fall 2008 Dr. Weller BINF 6010 ITSC 8010 UNCC 1 Agenda • Data Analysis of RAPD and STR files • Practice files for both RAPD and STR have been posted on the class Web page . MetaData Start GeneMapper 4.0 Open Panel manager Create the Kit Define the panel of markers D fi h k ( i d d )e ne eac mar er s ze an ye Define a Project Define size standards Add Sample files to Project Proceed to analysis Note on Practice • When you get ‘real’ data you will have to , set up a custom Panel, and define a new size standard using ROX , – we did not use any commercial kit we used a different dye on the standards– – For the practice part you will use the provided files (GeneScan500-LIZ) GeneMapper Manager =i Projects Anabelé Methods | Table Seting=| Pioieting=| Matices| dire Stendarcts | Marne Last Saved Gamer Instant Analysis Tyne | Cascretion 1 0700 DO-J2 late 2002-00-19 THO S2.0 | gin or Mic rose belie | Paehon: Mr ovicked é Microsatellite Darah 2002-08-18 1616220 | gm Microsatellite | Factor Provide Neve... | Open. | Bare As... Import... | Export... Delela one Setting size reference Project window Samples tab Analysis Method For practice use Default For new set choose None Editing an Analysis Method Analysis Method New Microsatellite OK Type sets the algorithm and presents the fields Create the Method Start with General (metadata) Give it a name (yours and T t i l b f )u or a , y pre erence . If this is to be exported to a LIMS system then Instrument information would be important. Now select Allele tab For the tutorial use the Microsatellite Binset For new data you can also use these values If you had a new locus you might have to customize. QC and cutoffs This provides a set of weighting values for a number of peak height/ratio/shape criteria here – all except out of bin allele have been given the same importance. Once the final QV has been determined the final step is to give a cut-off for accepting a call. Save the settings At this point you are done with the editor. Analyzing Data Back to the Project screen Select the Samples tab Select an Analysis Method: Microsatellite Default for practice Microsatellite type and None for bin set for your own data. Then: Select a Panel – click on the top cell in the column A Select window opens Expand the Microsatellite Tutorial folder Double click on Tutorial Panel 9 – is entered in the first cell Begin the actual analysis • Finally for the Size Standard column, assignment Click on top cell in column– • Select GS500-LIZ from the list for the tutorial – The top cells are now assigned • Click and drag to highlight all three – From EDIT Fill down (=Ctrl+D) – Under Analysis Analyze » You now have a project that results must be saved to, so when the dialog box comes up provide a name (MyName_MicrosatelliteTutorial, for example) Select Binning target • Expand the Tutorial sample folder (Panel 9) The markers in the folder are displayed– – The Add Reference Data icon is enabled Marker Name, Dye Color, Min Size, Max Size, Control Allele, number alleles, etc) Create new Bin set • Select Bins Add Reference Data – In Dialog Box a folder (the current project) is seen in the lower pane with other projects , that used this panel Select project, click Add to list Project moves to the right Click Add, then OK Now Bin • In Panel Manager, Select Bins – For options: Allele Naming:– Rounded basepair – Auto Bin options: Auto Bin – OK Ch k th bi b Vi i l t– ec e ns y ew ng: se ec a marker • Reference alleles where bars are shown • GQ scores on Y-axis • To see the electropherogram, l t th l thse ec e samp e – en you can edit, create or delete the bin (options under Bins menu or right-click in the pane for a N bi th bi tew n, or on e n o edit/delete it) – To change the Range you can drag the handles on the boundary lines Editing Allele Calls Start in Plot Window, drop down menu under Alleles Editing Mode Peak Selection Access labels (in single- dye mode) under View labels (either horizontal or vertical) Click on the peak to be edited, right click for editing options Labeled peaks: edit or delete Unlabeled peak: add an allele call A Change History is recorded GQ scores are. grayed out. Editing Marker/Bin Calls Start in Plot Window, drop down menu under Alleles Editing Mode Binning To change a marker size range click a red – triangle marker and enter new values OR drag the handles To change a bin, select it, then right click and enter new information or delete it or drag the range, handles. To move a bin, select, then click and drag