IronChip Evaluation Package (ICEP)
Contents
1. Text file C Ptogram Files EMBL ICEP arrays process tt Browse Browse Browse Browse This version of text to Excel convertion utility allwos to browse manually text files from different locations and save it as a excel file Another automated text converter is available by request see Contacts This version can save automatically all text files from specified folder to a multi sheet Excel file top I CEP Example data set Example data set contains 4 text files corresponding to 2 arrays hybridized with dye swaping Each file is a result of ChipSkipper normaliyation of raw array images top Analysis of a generic microarray ICEP recognizes any generic tab delimited text tables from any type of gene microarray containing the normalized signal intensities and background data The native format for ICEP is a text tab delimited file generated by ChipSkipper application ICEP could be conigured to work with other array types in 3 steps Prepare array definition file running numbers gene name txt Prepare gene description file running numbers feature descriptions txt Prepare column definition file Custom columns configuration txt Deinition files located in the program folder Array Definition File ADF contains unique feature IDs characteristic for every feature cDNA clone for example spotted on the array and corresponding gene ID like GeneBank accession number or EnsEMBL gene ID ADF fi2. D work WWW I CEP I CEP images I CEP WorkFlow png 11 02 2010 15 30 12 file D work WWW ICEP ICEP images lronChip WorkFlow png file D work WWW ICEP ICEP images lronChip WorkFlow png 11 02 2010 15 30 12 file D work WWW ICEP ICEP images ICEP S2 A jpg Transcripts Nr vs time A 54 VER DE 3l 50 runtime sec 49 4s dj 46 45 50 600 690 900 950 Transcript No file D work WWW ICEP I CEP images I CEP S2 A jpg 11 02 2010 15 30 13 file D work WWW ICEP ICEP images ICEP S2 B jpg Feature Nr vs time runtime sec d 2000 4000 6000 6000 10000 Feature No file D work WWW ICEP ICEP images ICEP S2 B jpg 11 02 2010 15 30 14 file D work WWW ICEP ICEP images EST2 rules png Relative error RE DSF EST2 DSF EST TRUE m Hone Regulated DSF EST2 Hone Regulated IDSF gene None Regulated DSF EST2 Absent RF EST1 IRF gene NONE RF EST2 Regulation gene Average Relative Error 25 yes DSF gene TRUE JRF gene RF EST1 Regulation gene verage file D work WWW ICEP ICEP images EST2 rules png 11 02 2010 15 30 14
3. files Columns definition file is simplified XML file oO Custom columns configuration txt SciTE File Edit Search View Tools Options Language Buffers Help 1 Custom_columns_configuration txt lt config columns Custom gt lt SpotNumber gt Spot number lt SpotNumber gt lt MetaColumn gt MetaColumn lt MetaColumn gt lt MetaRow gt MetaRow lt MetaRow gt lt Column gt Column lt Column gt lt Row gt Row lt Row gt lt Reporterld gt Reporterld lt Reporterld gt Comp ChannelizComp Channeli lt Comp Channeli gt Comp Channel2 gt Comp Channel lt Comp Channel gt lt CompRatio gt Comp Ratio C1 C2 lt CompRatio gt lt Comment gt Comment lt Comment gt lt config gt ICEP extracts from input files only specified columns Following columns reguired to run analysis correctly a name SpotNumber Number of each spot on the array if available used for internal purposes MetaColumn O Spotting pin X coordinate 40i Ub J ii MetaRow Spotting pin Y coordinate ii Column X coordinate of a feature inside a block spotted by single pin Row O Y coordinate of a feature inside a block spotted bysinglepin file D work WWW ICEP ICEP ICEP_manual html 13 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP Unique Clone ID Should contain flags for negative control positive Control empty spot gene name and running number trf1 352 probe gene tfr1 has a uni
4. ArrayLayout png file D work WWW ICEP ICEP images ArrayLayout png file D work WWW ICEP ICEP images ArrayLayout png 2 of 2 11 02 2010 15 30 12 it file D work WWW l CEP I CEP images I CEP WorkFlow png UM normalized data including spike in data EST P call EST Up Down flag average log ratio relative error St Dev assign P M A call Pete Cy5 Cy3 4 yo d background cut off channel signal Hs ratio cut off BG signal IronChip Version 7 d r ke p 1500 positive controls assign 4 N L i Up Down fla data transformation analyze dye swap analyze single array convert to the experiment experiment logarithmical scale compare expressio one P group similar 1800 negative controls 5900 probes 200 spots feature level assign EST Up Down flag IronChip Version 7 820 ESTs minimum 6 features per EST EST Level 7 between ESTs and assign dve swap flag log ratio di roup all repetitive N P M A call EEE py features together IronChip Version 7 430 transcripts 2 ESTs per transcript Up Down flag transcript level calculate StDe and Relative Error calculate average log transcript regulation flag transcript expression ratio log normal transcript P call transcript quality flag assign EST P M A call file
5. using rule 1 for 2 ESTs recurrently top Copyright European Molecular Biology Laboratory 2009 ICEP Disclaimer Support Yevhen Vainshtein file D work WWW ICEP ICEP ICEP_supl html 3 of 3 11 02 2010 15 30 00 IronChip Evaluation Package I CEP IronChip Evaluation Package ICEP About CEP Download application User Manual Windows installation packages Supplementary figures ICEP msi 11 8MB ICEP zip 11 5MB Downloads Windows version without installation Contact ICEP_noinstall zip 11 4MB Links Unzip to a separate folder and run ICEPstart exe to start analysis Download user manual icep chm 411KB Download test set Original data files only test data set zip 3 32MB Original data and results I CEP test arrays and results zip 5 84MB Note file results xls with final result of color swap experiment was post processed with Excel sorting filtering color code e t c top Copyright European Molecular Biology Laboratory 2009 I CEP Disclaimer Support Yevhen Vainshtein file D work WWW ICEP ICEP ICEP_download html 11 02 2010 15 30 01 IronChip Evaluation Package I CEP IronChip Evaluation Package ICEP About CEP With general question related to IronChip platform please contact Martina Muckenthaler Martina Muckenthaler med uni heidelberg de User Manual With questions related to I CEP please contact Yevhen Vainshtein vainshte embl de Supplementary figur
6. Analysis settings folder With standard sequrity settings Windows Vista do not allow I CEP to modify own configuration files batch settings and array analysis settings Batch analysis top Batch editor Package content Analyse Color Swap CEP consist of 5 major utilities Convert text to Excel CEP start pannel I CEPstart exe Example data set Single array analysis Analyse IronChip exe Color swap analysis Analyse ColorSwap exe G S Batch mode starter ICEPbatch exe SS oo Convert multiple text files to Excel workbook Text2xls exe Supplementary figures Each utility can be run separatly without ICEP start pannel Downloads top Contact Start using CEP Links Prepare array data Analyse feature background subtraction and normalization by the ChipSkipper images derived from Cy3 and Cy5 channels of an IronChip microarray file D work WWW ICEP ICEP ICEP_manual html 1 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP Create a separate folder for an experiment Figure below represents typical folders structure for color swap experiment with several tissues and treatments Favorite Links Folders v 4 Data Evaluation Folder 4mm14_all_ETSs txt a 3 Tissues FE vs Untr 4mm14_genes txt 4 k Duodenum E S 4mm14 genes xls gt FE treated 4mm24_635 txt gt LPS treated 4mm24 635spk txt a None treated Amm24_all_ETSs txt B2m HFE vs SV129 Tini Genesee B2mvsSV129 Si 4mm24_gen
7. IronChip Evaluation Package I CEP IronChip Evaluation Package ICEP About CEP introduction user interface User Manual Supplementary figures Downloads Contact Links Introduction The IronChip Evaluation Package ICEP is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip but can be easily adapted for other cDNA or oligonucleotide based microarray platforms of similar design CEP is using decision tree based algorithms to assign quality flags and performs robust analysis based on the chip design properties regarding multiple repetitions ratio cut off background and negative controls top User interface The I CEP package is designed for post processing of the IronChip microarray data It consists of several utilities to operate IronChip data including single chip analysis and batch processing 76 ICEPstart 74 Analyse IronChip Open Raw data table Open Spike data table i Settings Display settings Exit Spike data table substitute wrong IDs assign probe control spike flag Raw data table D work 3 Tissues FE vs Untr Duodenum LPS treated TFR LPS vs SV129 3cmm89 t 74 Compare 2 tables color swap Open table 1 Run M ds Save Unique Entries Compare all ESTs tables Table 1 D work 3 Tissues FE vs Untr Splee
8. ell to compare 2 text files and identify common entries and unique entries present either in first file or in second file This function is activated automatically when ACS can not recognise default header line format In this case ACS will prompt you to select colum containig unique ID file D work WWW ICEP ICEP ICEP_manual html 9 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP Overall P call Regulation n 30129654526366 FIP FIPIPIP F NONE 13185 875 640256 724300 0177938507557082 PIPPIFJPJP P WE 600235 6581 77 5727 00 0 20069600883158 FIFIFIFIFIF p NONE 71154 32971 68 3239900 0 0252781134605083 PIPIPIPIPIP P NONE 28278 2333333333 T Column 0 selected 0 Spot number Pressing Run button will start either color swap analysis or table comparison Results will be Saved automatically in the same folder as a text files To convert results to Excel press Save as xls button top Convert multiple text files to Excel workbook Output file read C Program Files EMBL ICEP running numbers martinas name bt This is a stand alown application designed to convert multiple text files to multi sheet Excel workbook Browse Text Files button activates a text file selection dialog file D work WWW l CEP ICEP ICEP manual html 10 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP 8 Browse text files
9. er of analyzed features On average CEP evaluates 208 features per second top file D work WWW ICEP ICEP ICEP_supl html 2 of 3 11 02 2010 15 30 00 IronChip Evaluation Package I CEP EST 1 Dye swap flag DSF EST Regulation fiag RF pP flag DSF Relative error RE jon flag RF Relative error RE Deg CMS no Berrend no iri ad DSF EST2 ine esta rem DSF EST1 N ui N e 2 e 2 gt gt d gt BD None gt Le pat Pom an Ze e b uus Absent 7 TRUE 2 N TRUE 7 pss pe ke he es PP Gene Pp T Gene y pm E 22 ves DSF gene Absent _ osF est2 gt Ye DSF gene None Regulated d 5 Absent VI IE NINE lt p gt ara NONE no 8 Uu e RE Gene NONE eae p p re NONE gt RE EST1 S Regulation gene 0 e Regulation gene Average RF EST2 z e no yes y Gene RF gene NONE Reliable att Reguiation gene 0 DSF gene HighRE _ Relative Yes PSF gene TRUE lt Error lt gt RF gene RF EST1 N 25 77 RF gene RF EST1 lt gt Regulation gene yes DSF gene None Reliable DSF EST2 Regulation gene 0 v g g TRUE Average RF gene NONE TRUE Regulation gene Average Figure 3 Schema of grouping two ESTs The flow diagram indicates different decision steps in the analysis tree The grouping procedure for three and more ESTs is done similarly
10. es Downloads Contact Links top Copyright European Molecular Biology Laboratory 2009 I CEP Disclaimer Support Yevhen Vainshtein file D work WWW ICEP ICEP contacts html 11 02 2010 15 30 01 IronChip Evaluation Package I CEP IronChip Evaluation Package ICEP About CEP IronChip related projects User Manual lronSystems Supplementary figures IronLI MS Downloads Contact Relative Expression Software Tool REST Links REST REST 384 REST MCS REST RG RefScout PDFManager Hentze Group Page Main Page of the EMBL Heidelberg Copyright European Molecular Biology Laboratory 2009 I CEP Disclaimer Support Yevhen Vainshtein file D work WWW l CEP I CEP links html 11 02 2010 15 30 01 top file D work WWW ICEP ICEP images ICEP_gui gif file D work WWW ICEP ICEP images ICEP_gui gif 11 02 2010 15 30 01 uunjo2 it epatta Dep PC TTT eg saca 45800 op ee heenae 260 se e e dq sessast ffehoage sosse eeonscee sa asas e ane 2008 a8 TTT TS ose cos sn sos e sag e 6660688 sesi O eb dete lana saa o epp ate a de 2L LZ E eo io es eniss sesa vi Ba Ze Sai KS TVT UE KEEN mma H E amaaan C 2 O O D O MOMEI9S N file D work WWW ICEP ICEP images ArrayLayout png 1 of 2 11 02 2010 15 30 12 file D work WWW l CEP I CEP images
11. es xls C282Y vs SV129 B2m HFE vs SV129 xls HFE vs SV129 A Rag vs sv129 TFR vs SV129 v al TT On the right pannel two files 4mm14_635 txt and 4mm14 635spk txt is a gloabl normalized data and spike normalized data derived from hybridisation of an murine IronChip batch 4mm Such two files normalized and spike normalized are required to start analysis with the I CEP Start ICEP with a shortcut on the desktop ee top RW sl CEP start pannel d file D work WWW ICEP ICEP ICEP_manual html 2 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP Start pannel contains buttons allowing to start correponding utilitis Table below contains a description Single array analysis Color swap analysis Convert multiple text files to Excel workbook Batch mode starter Batch mode editor shortcut Change I CEP analysis settings Single array analysis gt IronChip Open Raw data table Open spike deta table Execute Execute Settings mua TER spike data table C Program Files EMBLACEPACEP test data set arrayl spk txt Raw data table Cf Program Files EMBLACEPACEP test data set farrayl norm ii file D work WWW l CEP ICEP ICEP manual html 3 of 15 11 02 2010 15 29 58 top IronChip Evaluation Package I CEP Single array analysis application Analyse_lronChip exe AIC is a major application of
12. le Default IronChip columns configuration txt en t false use customized column definitions specified in the file See Kg Custom columns configuration Lt for detailes see section Analysis of a generic microarrays true AIC creates 3 output files Excel workbook containing all statistcs relevant to this analysis original data filtered data and results of single array analysis Text file containing intermediate results for color swap analysis Text file with results of single array analysis Save results as a text true false AIC creates 1 output file Excel workbook ff file D work WWW ICEP ICEP ICEP_manual html 6 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP true display current analysis configuration overview at each application start 7a Configuration I Relative Error Trashold Group Present Call cut off Fixed ratio cut off Always show ER false 2 configuration info Use fixed ratio cut off Use Median as a BG cut off Save EST evaluation result as a text file Always automatically open this window Use default column names Displays default columns definition for ICEP analysis for detailes see section Analysis of a generic microarrays Displays default gene names definition file used by ICEP for feature extraction for detailes see section Analysis of a generic microarrays Displays default gene descriptions file used by ICEP for fea
13. le links internal feature IDs with corresponding genes file D work WWW ICEP ICEP ICEP_manual html 11 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP oO running numbers gene Dame Di SciTE File Edit Search View Tools Options Language Buffers Help 1 running numbers gene name txt iunning Number Official name Contamination GFP FIH1 FIL TFRC SLCI1A2 IRE RELA form2 HSPB2 MI2A SO0D1 U1A JUN MYC PMP22 HMOX1 GAPD ACO B2M IREB 1 2 3 a J 6 F 8 9 To edit ADF file press button from ICEP analysis settings dialog IronChip ADF file from the example above contains Running Numbers cDNA Clone IDs IronChip internal unique clone identification numbers and Gene Names Genes description file as well link unique feature ID running number or IronChip with any arbitary text entrie for example gene description This table as well should be tab Edit features description file delimited To edit genes description file press button from CEP analysis settings dialog Columns definition file allows to teach I CEP recognizing new array file firmats To enter Edit columns definition file columns re definition mode press button This file links internal column file D work WWW ICEP ICEP ICEP_manual html 12 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP IDs with actual columns from input files By default ICEP recognize ChipSkipper output
14. n LPS treated TRF PLS vs SV129 3cmm90 all E Column number Column number Table 2 D work 3 Tissues FE vs Untr Spleen LPS treated TRF PLS vs SV129 3cmm88_all_ 74 Step 8 save multiple text files as Excel WorkBook BAX Browse Test Fies Save muiste tex fles as tee Excel fie Ent Every utility can be operated both via user friendly graphical interface generated with perl Tk file D work WWW ICEP ICEP index html 1 of 2 11 02 2010 15 29 52 IronChip Evaluation Package I CEP and using command line Copyright European Molecular Biology Laboratory 2009 ICEP Disclaimer Support Yevhen Vainshtein file D work WWW ICEP ICEP index html 2 of 2 11 02 2010 15 29 52 IronChip Evaluation Package I CEP IronChip Evaluation Package ICEP About I CEP System requirments User Manual Any version of MS Windows testet on Windows 2000 XP Vista Minimum 256 MB RAM recommended 512 MB System Requirements installing CEP Installing CEP Package content download I CEP from the Downloads page Start using I CEP After downloading I CEP msi start installation by duble clicking on I CEP msi and follow on screen instructions I CEP start pannel If required download as well example data set to the separate folder Start I CEP using shortcut on the desktop Single array mode Windows Vista users please install I CEP to alternative location not to the default c program files
15. o from 1 to 100 Smaller value of relative error treshold makes filtering conditions more stringent Each EST on the array represented by 6 to 24 features By teqnical resons some features could hybridise less efficiently than others or could be contaminated P call grouping creteria 60 P call grouping creteria determines how many dublicated faetures in from total number of replicates should share similar P call Bigger value of a P call treshold makes filtering more stringent Single feature is marked as regulated if the ratio of signal Fixed EE 13 intensity between channels is bigger than the pre defined ratio cut off For IronChip this value could be very low 1 3 because of special design and high quality true use fixed ratio cut off 1 3 by default Use fixed ratio cut off false false calculate ratio cut off using signals from spiked in controls file D work WWW l CEP ICEP ICEP manual html 5 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP true use median of signal intensities of empty buffer spotted features for background signal compensation Use median as BG treshold false false use average of signal intensities of empty buffer spotted features for background signal compensation true display preview of a original data files Display preview false false do not display preview true use default column names specified in the fi
16. out arrays data files at the following lines Below is a table from example data set e to the working directory p data file P ke data file file D work WWW l CEP ICEP ICEP manual html 8 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP C Program Files EMBL ICEP ICEP test data set arrayl norm txt arrayl spk txt C Program Files EMBL ICEP ICEP test data set array2 norm txt arrayl spk txt Warning in the batch analysis mode both original data files global normalized and spike normalized from one array have to be stored in the same folder Original data from different arrays could be stored and analysed from different folders top D e 5 7 m T j 7 z Ir m a GAL UE ese x Compare all ESTs tables Color swap analysis application Analyse ColorSwap exe ACS allows to compare intermediate results of single array analysis from different IronChip batches When Save Unique Entries option is activated ACS creates separate text files for ESTs present in one array batch and absent in another batch By default single array analysis intermediate resulting file has a following file extention all ETSs txt This file contains all flags and values for each ESTs on the array Header line formaof of such file recognised automatically To force ACS to recognise text file with different from the default file name activate option Compare all ESTs tables The ACS application allows as w
17. que running number 352 and this is a probe empty empty spot 2xssc negative negative control spot firluc Spike spike in positive control Comp Channel Channel 1 signal intensity local background compensated Comp Channel Channel 2 signal intensity local background compensated Comment Am text comment related to this spot Reporterld To illustrate how to define MetaColum MetaRow Column Row look at the following figure MetaColumn 0009050000000 4005100009995 10900 999 0 40070000000 000 010000000 058115 oo eee8e08 000000 lt see MetaRow file D work WWW ICEP ICEP ICEP_manual html 14 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP Copyright European Molecular Biology Laboratory 2009 ICEP Disclaimer Support Yevhen Vainshtein file D work WWW ICEP ICEP ICEP_manual html 15 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP IronChip Evaluation Package ICEP EE Supplementary figures User Manual A B Supplementary figures Figure 1 Figure 2 IronChip Version 7 Figure 3 1500 positive controls 1800 negative controls 5900 probes 9200 spots Downloads feature level Contact Li nks IronChip Version 7 430 transcripts 2 ESTs per transcript transcript level IronChip Version 7 820 ESTs minimum 6 features per EST EST Level Figure 1 IronChip analysis wo
18. rk flow A Flow chart of I CEP data processing and evaluation Data evaluation with I CEP is organized into three functional modules Single feature EST and transcript evaluation B In our application example hybridized microarrays were scanned on a GenePix 4000B Microarray Scanner Axon Instruments Union City CA USA and processed feature background subtraction and normalization by the ChipSkipper software 8 ICEP uses these output files generic tab delimited text tables containing the normalized signal intensity and background data for further analysis top file D work WWW ICEP ICEP ICEP_supl html 1 of 3 11 02 2010 15 30 00 IronChip Evaluation Package I CEP Transcripts No vs time Feature No vs time TPP TYP TTT TTT STS ATT TTT TTT TTT TTT TTT TTT ATT PTT TTT ATT TT ATT EEE EEE EEE H S i 2000 4000 6000 8000 10000 Transcript No Feature No Figure 2 ICEP run time chart A Run time analysis of di erent microarray versions containing an increasing number of features The plot shows the resulting increase in I CEP run time for di erent ronChip versions B A set of virtual arrays of 1000 to 9000 features was analyzed We used a general tab delimited format Robust statistical analysis included analysis of background noise ratio cut o evaluation of multiple repetitions detailed feature extraction and grouping results CEP Run time increases linearly with the increase of the total numb
19. the I CEP It allows to perform analysis of a single array by default IronChip View and change analysis setting both for single and batch mode Application has a very simple user interface One can load gloabl normalized Open Raw data table button and spike normalized Open Spike data table button data files generated by ChipSkipper start analysis Execute button change analysis settings Settings button and view current analysis settings Display settings button Start array analysis load gloabl normalized file load spike normalized file Press Execute button to start analysis top I CEP analysis settings Settings button activates the following window mones i Relative Error for repetitions P call grouping creteria Fixed ratio cut off 15 DU 1 5 Iv Use fixed ratio cut off 1 3 M Use Median as BG threshold Display Preview Edit columns definition file Edit gene name file Edit features description file M Use default column names I Save results as a text Always show configuration info Below is a description of all analysis options Field name Default Description file D work WWW ICEP ICEP ICEP_manual html 4 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP Relative error treshold determines stringency of a filtering according to a spread of a signal intensity values of repetetive Relative error 15 featureas from the average It can g
20. ture extraction for detailes see section Analysis of a generic microarrays Edit features description file file D work WWW l CEP ICEP ICEP manual html 7 of 15 11 02 2010 15 29 58 IronChip Evaluation Package I CEP Note settings could be modified directly by editing configuration file process ironchip ini located in the program directory top GO Batch analysis Batch analysis will activates automatical array analysis mode It does not assume any user Input TO cange analysis settings activate single array analysis mode and press Settings button or modify process_ironchip ini file Batch mode starter reads information about path to arrays data files from arrays2process txt file This file should be prepared in advance see Batch mode editor shortcut section All resulting files will be saved in the source data folders top Batch mode editor shortcut Pressing this button will pop up default system text editor with the batch command file arrays2process txt Edit Saath View Tools Options Language Butter Help D Hl im l arrays2process bt 1 Path to the working directory Raw datafile Spike data file 2 C Program Files EMBL ICEP ICEP test data set arrayi_norm txt arrayi_spk txt 3 C Program Files EMBL ICEP ICEP test data set array2 norm txt arrayi_spk txt Hli 1 co 1 INS CR LF This is a tab delimited text file with a header at the first line and information ab
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