myPEG User Manual
Contents
1. SIFT and consensus predictions for the functional impact of nSNVs as well as infer ancestral alleles using the 46 species UCSC reference peptide alignments hg19 assembly for a given coordinate using Maximum Likelihood ML or Maximum Parsimony MP methods EvoD PolyPhen2 and SIFT predictions as well as related data are pre computed and stored on the EvoD server for large numbers of variants it is recommended to use the Evod server directly myPEG is built on top of the MEGA Molecular Evolutionary Genetics Analysis software package and is developed using the Delphi programming language Citing myPEG Kumar S Sanderford M Gray VE Ye J Liu Li Evolutionary diagnosis method for variants in personal exomes Nature Methods 2012 Sep 9 9 855 6 doi 10 1038 nmeth 2147 Disclaimer Although the utmost care has been taken to ensure the correctness of the software it is provided as is without any warranty of any kind In no event shall the authors or their employers be considered liable for any damages including but not limited to special consequential or other damages The authors specifically disclaim all other warranties expressed or implied including but not limited to the determination of the suitability of this product for a specific purpose use or application Note that brand and product names e g Windows and Delphi are trademarks or registered trademarks of their respective holders Copyright 22. X tropicalis LogL 42 72 34 Input Data Overview In order to retrieve predictions for a given nSNV myPEG requires three pieces of information 1 RefSeq proteinid e g NP 000082 2 amino acid position e g 43 3 mutant allele e g R There are three ways to provide this coordinate information to myPEG Upload a text file Manually enter the information Use the Gene Search and integrated Sequence Data Explorer Upload a text file with the coordinate information for all nSNVs of interest Create a text file with coordinate information for all nSNVs to be explored following the format below NP 000758 99 NP 000761 264 NP 000762 144 NP 000762 335 NP_000773 374 NP 000838 71 NP_000886 131 NP_000887 271 Each line contains coordinate information for one nSNV and each value is separated by white space i e spaces or tabs 4 IMA4ASO ZM In the Mutation Explorer window select File gt Import Query Data From File or click the upload data button and browse for the newly created text file myPEG will first validate the format of the coordinate information file and then request prediction information for all specified nSNVs from the EvoD web server As data is retrieved the Mutation Explorer window is updated The myPEG application has no limit on the number of entries that can be included in the coordinate information file However depending on your internet connection speed and the current load on the EvoD server re
3. Copy copy selected values to the system clip board e Select All select all values in the table e Clear Table clear all data from the table Format e Increase Precision increase the precision of all numeric values in the table and also in the Mutation Detail View window e Decrease Precision decrease the precision of all numeric values in the table and also in the Mutation Detail View window e Resize Columns to Best fit resizes all columns in the table to achieve the best fit and optimize the view Useful when hiding showing columns and column widths change sub optimally note if there are many records in the table more than several thousand this operation may take a few moments or more during which time the window will be unresponsive Search e Find text search for values in the table e Find Next find the next value matching the search query search goes to the right and then down to the next row Options e Keep detail view on top toggle this action on off to keep the Mutation Detail View window from staying in front of other myPEG windows on by default e Show Toolbar toggle on off the display of the toolbar on by default e Toggle Auto Column Width when off default a horizontal scroll bar is used to view columns that don t fit in the window When off the horizontal scroll bar is removed and all columns are squeezed into view Windows e Detail View Form show the Muta
4. Cysteine Q Glutamine E Glutamic Acid G Glycine H Histidine I Isoleucine L Leucine K Lysine M Methionine F Phenylalanine P Proline S Serine T Threonine W Tryptophan Y Tyrosine Li V Valine Input Data Manually enter the coordinate information using the integrated entry form In the Mutation Explorer window select the Coordinate Info tab Enter the RefSeq protein id and the amino acid position for the nSNV of interest Select a mutant allele from the drop down list Click the submit button and myPEG will send the request to the EvoD server and add the returned data to the Mutation Explorer prediction data view Emei BM ME SOM OP ee el File Edit Format Search Options Windows Help HAG Ca 2 3E gt AA AA O Gene Search Prediction Data Coordinate Info RefSeq Protein ID Amino Acid Position Mutant Allele INP 000509 124 IR Arginine v Y Submit 21 mutations Request completed References EvoD Evolutionary Diagnosis Kumar S Sanderford M Gray VE Ye J Liu Li Evolutionary diagnosis method for variants in personal exomes Nature Methods 2012 Sep 9 9 855 6 doi 10 1038 nmeth 2147 PolyPhen2 Adzhubei IA Schmidt S Peshkin L Ramensky V Gerasimova A Bork P Kondrashov A Sunyaev S A method and server for predicting damaging missense mutations Nature Methods 2010 7 248 249 SIFT Pauline C Ng and Steven Henikoff SIFT predicting amino acid chan
5. Site drop down list When an allele is selected from the list myPEG will query the EvoD server and append the returned prediction data to the Mutation Explorer prediction data view The Sequence Data Explorer window also provides much other functionality such as alignment export and composition based exploration AA myPEG Sequence Data Explorer SE X Data Display Search Groups Highlight Statistics Help aja 2 amp l eje eye del v m s o gt Spi amp 44 5 amose saaste es Name All E 1 Human ERMFLSFPTTKTYF PIH A d Alanine 2 Chimp La o a ea asa a R Arginine 3 Gorilla ERMFLSFPTTKTYFPH A partic Acid 4 Orangutan ERMFLSFPTTKTYF H Cysteine 5 Rhesus ERMFLSFPTTK TY F H Q Glutamine 6 Baboon ERMFLSFPTTK TY EF H E ado 7 Marmoset ERMFLSFPTTKTYFPH i H Histidine 8 Tarsier Boo ii TEE 9 Mouse Lemu E RMF HS FPTTKTYF H L Leucine v10 Bushbaby ERMFLSFPTTKTYF H K Lysine 11 Tree Shrew 2 E M Methionine F Phenylalani 12 Mouse GRL i pi anine 4 m S Serine Data T Threonine W Tryptophan Y Tyrosine Le V Valine n m 09 Tl 1 4 om A 4 lt 45 143 Highlighted None Data Analysis Preferences Dialog The Analysis Preferences Dialog is used for specifying the substitution model to use as well as the distribution of rates for ML based ancestral sequence inference Selection Analysis Ancestral Se
6. 013 This software is protected under copyright law No part of this manual or program design may be reproduced without written permission from the copyright holders Please e mail all inquires to s kumar Wasu edu Development Team Sudhir Kumar Project Director Glen Stecher myPEG application developer myPEG and EvoD websites developer Maxwell Sanderford EvoD database administrator EvoD calculation pipeline developer Koichiro Tamura myPEG application developer Dan Peterson myPEG application developer Center for Evolutionary Medicine and Informatics Biodesign Institute Arizona State University ASU Tempe AZ 85287 School of Life Sciences ASU Tempe AZ 85287 Department of Biology Tokyo Metropolitan University Hachioji shi Tokyo 192 0397 Japan myPEG Windows Mutation Explorer The Mutation Explorer window displays data associated with the nSNVs being explored and provides functionality for text searching sorting importing exporting for matting gene search and manual data entry This window displays three main views each located on a separate tab Gene Search Tab Prediction Data Tab Coordinate Info Tab m myPeg Mutation Explorer File Edit Format Bj E Gene Search MA MA MA 17040070 MA 17040070 MA MA MA MA MA MA MA 1204000 N NP 000 la 23 of 44 9 mm DD 0 Feptide ID MP DOOU MP DOOU MP DONO MP DOOU MP DOOU MP DOOU MP DOOU MP DOOU
7. 556 Q Ambiguous Neutral 2 1E 001 NA Deleterious 51 27 NA NP_000549 NM 000558 Deleterious Deleterious 1 8E 001 NA NA 62 95 O ot OC Nf Request completed 21 mutations Coordinate Info tab The Coordinate Info tab can be used to input the RefSeq protein id amino acid position and mutant allele instead of loading the data from a text file After required information is entered and the Submit button is clicked myPEG will send query the EvoD server for prediction data When the data is returned it is added to the Prediction Data view for further exploration Help daa 10 0 235E nB8 800 2 Gene Search Prediction Data Coordinate Info RefSeq Protein ID Amino Acid Position Mutant Allele NP_o00509 24 IR Arginine v Y Submit Mutation Detail View The Mutation Detail View window displays all available information for the currently active record selected in the Mutation Explorer window Additionally this window provides access to the 46 species reference alignment for the given gene as well as the ability to infer ancestral alleles using the Maximum Likelihood ML or Maximum Parsimony MP methods When the Get Alignment button is clicked myPEG will retrieve the 46 species reference alignment from the EvoD server and display it in the Sequence Data Explorer from which it can be exported or further exploration can be done When the Get Ancestors button is clicked the choice of ML and MP methods are presente
8. MP DOOU MP DOOU MP DOOU MP DOOU MP DOOU MP DOOU 007 Request completed Mutations mANA ID MhA_OOO016 MhA_OOO016 MhA_OOO016 Mh_OO0016 MhA_OOO016 MhA_OOO016 MhA_OOO016 Me 00007 6 Mei 00007 6 MhA_OOO016 MhA_OOO016 MhA_OOO016 MhA_OOO016 MhA_OOO016 Mh 000076 4 Search Options Windows oe hi H Prediction Data Coordinate Info Reference AA Mutant AA E R E E E E E E E E E E E E E E A Ea Ed m Be Consensus Deleterious Deleterious Deleterious Neutral Neutral Deleterious Neutral Neutral Likely Deleterious Neutral Neutral Neutral Deleterious Deleterious Neutral Predictior ad EvoD Dieleterious Dieleterious Dieleterious Neutral MA Deleterious Neutral MA Neutral Neutral NA Neutral Deleteriqus Deleteriqus Neutral Erob P bE 2 3E 2 aE E Ja 2 BE TEE Ja 2 2E 2 JE RE e DE ee ae 13E 7 2 records The actions provided by the Mutation Explorer are divided into several categories and are accessed using the main menu bar or the main tool bar File e Import Query Data From File load coordinate information form a text file e Search for a Gene access the gene search page e Export Table to Excel File save all prediction data to an MS Excel file e Export Table to CSV File save all prediction data to a Comma Separated Values text file e Exit Close the application myPEG Windows Edit e
9. d After a method is selected the Analysis Preferences Dialog is displayed from which the analysis can be launched with custom settings e g substitution model distribution of rates etc When the analysis is completed the reference topology will be displayed in the Tree Explorer along with inferred ancestral alleles for the amino acid site designated earlier m myPeg Detail View Get Alignment Property 33943097 Feptide ID NE 0005499 mRAMA Accession Mh 000556 Reference AA H Mutant AA L Predictions Consensus Dieleterious Evol Deleterious Evod Fwalue p a E 002 PolyPhen e MA SIFT Deleterious Impact Evol Gane FolyPhen MA SIFT 0 0 Grantham Distance 93 Blosurn be a Evolutionary Features Substitution Rate Position Time Span Mutation Time Span Coordinate Info Chromosome Chromosome Position Strang Nucleotide Position AA Position Wilg Nucleotide Mutant Nucleotide 2 44 4016 05 0 00 chr 16 cobrir si be e A myPEG Windows Sequence Data Explorer The Sequence Data Explorer is used to display the 46 species alignment for a given gene and provides a graphical interface for specifying amino acid position and mutant allele for nSNVs of interest With an alignment activated the amino acid position is specified by selecting the site of interest which will be highlighted With the site of interest selected the mutant allele or all alleles can be specified from the Diagnose Selected
10. ges that affect protein function Nucl Acids Res 2003 31 13 3812 3814 doi 10 1093 nar gkg509 Index A Analysis Preferences Dialog 9 C Citing myPEG 1 Coordinate Info tab 6 Copyright 1 D Development Team 1 Disclaimer 1 E EvoD Evolutionary Diagnosis 17 G Gene Search tab 4 I Input Data 13 M Manually enter the coordinate information using the integrated entry form 14 Mutation Detail View 6 Mutation Explorer P Poly Phen2 17 Prediction Data tab 5 S Sequence Data Explorer 8 SIFT 17 Specify the coordinate information using the integrated Sequence Data Explorer 13 T Tree Explorer 10 U Upload a text file with the coordinate information for all nSNVs of interest 13 W Welcome 1
11. ha hemoglobin subunit gamma 1 C C chemokine receptor type 5 hemopexin precursor C x C chemokine receptor type 1 C C motif chemokine 4 like precursor C C motif chemokine 3 like 1 precursor hemoglobin subunit mu C x C chemokine receptor type 4 isoform a leukocyte cell derived chemotaxin 1 isoform 2 precursor Get Alignment View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen View Alignmen de xCRI NP_001019815 chemokine XC receptor 1 View Alignmen HI t 21 mutations Displaying 30 results Prediction Data tab The Prediction Data tab displays all prediction data retrieved from the EvoD server ina list view Complete information for the currently active record is displayed in the Mutation Detail View Columns of data are banded together into categories e Mutations identifiers as well as mutant and reference alleles are given here e Predictions consensus EvodD PolyPhen2 and SIFT predictions are given here e Impact the impact scores for EvoD PolyPhen2 and SIFT predictions are provided along with the Grantham distance and Blosum62 value e Evolutionary Features hidden by default substitution rate position time span and mutation time span are displayed e Coordinate Info hidden by default additional coordinate information is s
12. hown here including chromosome strand nucleotide position amino acid position wild nucleotide and mutant nucleotide To toggle on off the display of a given band click on the indicator button which is located to the far left in the band headers row A popup menu will appear from which bands can be selected deselected Often times when changing the display of bands column widths will change in undesirable ways To remedy this you can execute the Best fit Columns action by clicking Format gt Resize columns to best fit or clicking the toolbar button Alternatively columns widths can be adjusted by dragging their header edges The toolbar and main menu provide access to several actions for importing exporting data formatting the view sorting text search and setting view options myPEG Windows im myPeg Mutation Explorer daa Ha 22 eo AB ov P Gene Search Prediction Data Coordinate Info Mutations Predictions a mRNA ID Reference AA Mutant AA Consensus EvoD EvoD P value PolyPhen 2 SIFT Evod PolyPhe NM 000558 Deleterious Deleterious 2 0E 001 NA NA 62 48 leterious 3 0E 001 NA NA pose NM 000556 Best fit columns tool 33943087 NPAP00549 NM 000558 1i r vemenuus veleterious 9 8E 002 NA Deleterious 69 48 NA NP D00 N9 NM 000558 H H Neutral NA NA NA NA NA NA i Q Ambiguous Neutral 2 1E 001 NA Deleterious 51 27 34708054 Bands indicator a As cra ee WW Deleterious Deleterious 6 7E 002 NA NA 72 03 NA NP 000549 NM 000
13. myPEG User Manual e i Ea A FEF Table of Contents A ee SS O DS DD a a 3 US e er e 3 A A a ee A o o O 3 nn a ander 3 COI sen else Di Dee 3 Development CM dsd 3 MY RE VV INIOWS ss aie ties tae asian alias oa eier ete Gah ante aed ante RT ee 4 MUta non EXPORTIERT 4 Gene Search tab as Sasa ace asd RT a NR 5 Prediction E o o PE EE OOO A 6 Coordinate e O E En een 7 M taton Detail VIEW asr eera a o a ee aorta tidad 8 Sequence Data Explorer sas ROSA VN DS RE nenne ER O OR OR RR AR ERER ENNE DES 9 Analysis Preferences Dialog ars o een delle 10 TOS EXPO anni ar al lada 11 A sta an RR RR a RD CU DDR DR REAR RR RR A 13 A aha o CRS DE os Caso aaa 13 Upload a text file with the coordinate information for all NSNVs of interest nenn nnnnnnennnenene nenn enenen 13 Specify the coordinate information using the integrated Sequence Data Explorer eee 14 Manually enter the coordinate information using the integrated entry form nen nnn nen nnn nen nnn nennen en 15 o OR NRO ONA CERA RR PRE PERDOE ERR ERR RREO RR 17 EVO Evolutionary Diagnosis anne ie 17 PON RACE rca E A aa 17 o E a DT DR AR AR i 17 ale gt AA o a a a a E a a a a a a 19 Introduction Welcome myPEG my Personal Evolutionary Genomics is a client server software application that is used for exploring the functional impact of non synonymous single nucleotide variants nSNVs Using myPEG one can obtain EvoD PolyPhen2
14. quence Inference Statistical Method Maximum Likelihood Substitution Model Substitutions Type Amino acid Model Method Jones Taylor Thornton ITT model Rates and Patterns Rates among Sites Gamma Distributed G No of Discrete Gamma Categories 5 7 Data Subset to Use Gaps Missing Data Treatment Use all sites Branch Swap Filter myPEG Windows Tree Explorer The Tree Explorer displays the results of the ancestral sequence inference analyses When an ancestral sequence inference analysis is complete the 46 species reference phylogeny is displayed in the Tree Explorer with the inferred ancestral alleles for the nSNV amino acid site shown The Tree Explorer provides many capabilities which are described in detail in the MEGA5 user manual which can be obtained from www megasoftware net manual paf E Mo TreeExplorer CAUsers kumarlab AppData Local Temp NM_000558 meg File Image Subtree View Compute Ancestors Caption Help Er Ni ff E rte 22 A Human Chimp Gorilla A Orangutan A Rhesus A Baboon A Marmoset Tarsier A Mouse Lemur A Bushbaby Tree Shrew Y Rabbit A Pika G Squirrel A Guinea Pi Kangaroo Rat 5 Mouse Helen a og Shrew o Dolphin A Cow Alpac ar A Horse Cat llo Sloth Y Tenrec A Elephant A Rock Hyrax gt Opossum Wallaby G Platypus Lizard A Chicken A Zebra Finch Lamprey A Zebrafish A Tetraodon A Fugu G Stickleback A Medaka A
15. tion Detail View window e Search for a Gene jump to the Gene Search tab in the Mutation Explorer window e Sequence Data Explorer show the Sequence Data Explorer window e Contents Display this help document e About show the About myPEG window Gene Search tab The Gene Search tab facilitates searching for genes by keyword based on gene product or alternatively by RefSeq identifiers mRNA ID or Protein ID Search results limited to 1000 are displayed in a list view with cursory information and a link for retrieving the 46 species reference protein sequence alignment from the EvoD server When a sequence alignment is retrieved it is displayed in the Sequence Data Explorer which can be used to specify the amino acid site and mutant allele for a nSNV of interest myPeg Mutation Explorer File Edit Format Search Options Windows Help Am E o gt A ai amp Search By Gene Product or RefSeq ID mRNA ID or Peptide ID hemo Search HBG2 DEE HBAZ2 HBB HBD HBAI HBG1 CCR5 HPX CXCR1 CCLAL1 CCL3L3 HEM CXCRA4 LECT1 Peptide ID NP 000175 NP 000401 NP 000506 NP 000509 NP 000510 NP 000549 NP 000550 NP 000570 NP 000604 NP 000625 NP 001001435 NP 001001437 NP 001003938 NP 001008540 NP 001011705 Gene Product hemoglobin subunit gamma 2 hereditary hemochromatosis protein isoform 1 precursor hemoglobin subunit alpha hemoglobin subunit beta hemoglobin subunit delta hemoglobin subunit alp
16. trieval of many predictions may take some time anything less than 5 000 should not be problematic For situations where myPEG does not perform optimally due to high numbers of nSNVs the EvoD serverEvoD Server can be used directly www barn asu edu EvoD The same text file can be uploaded to the EvoD server which will process the file and send you an email for retrieving prediction data once the processing is complete Specify the coordinate information using the integrated Sequence Data Explorer If a 46 species sequence alignment has been retrieved see Gene Search for a given gene the Sequence Data Explorer window can be used to first navigate to the amino acid site of interest and then specify a mutant allele x At myPEG Sequence Data Explorer Data Display Search Groups Highlight Statistics Help dm ROH Bl tejvelelv r s 0 2 Special 4 2 Name v1 Human ERMFLSFPTTKTYF H v2 Chimp ee ee ee ee gt 3 Gorilla ERMFLSFPTTKTYF H v Orangutan ERMFLSFPTTKTYF H v5 Rhesus ERMFLSFPTTKTYF H v6 Baboon ERMFLSFPTTKTYF H 7 Marmoset ERMFLSFPTTKTYF H 8 Tarsier 5 2 a gt E 9 Mouse Lemu E RMFHSFPTTKTYF H 710 Bushbaby ERMFLSFPTTKTYF H v11 Tree Shrew 12 Mouse EUA AAA H 4 IM Data 45 143 Highlighted None Data gt Diagnose Selected Site 45 v All DE A Alanine R Arginine N Asparagine D Aspartic Acid C
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