ALEXA-Seq User Manual (v.1.5) - Canada's Michael Smith Genome
Contents
1. ccc cccccseccssecesecessceeseecseceseceeeeessecseceseseeseeessecsaeenss 8 COMTI ATION eaei enni n a aplbiiiada te dana aden eae hed E Modena aaa 8 1 Analysis configuration Si 2i iies cia ealkcr madenie a anden aes aclncttal anaes 8 2 Project onfig ration eisice cadet si ee asks ahs Sane a a ts Ae ls A 9 3 Installation of ALEXA Seq annotation database ec ccccsseesteceteeeeeee 9 A Download and install an existing database recommended 5 9 B Request the database o oo ccc ccesceessecsseceseeeeseecssecsecessceeseeessecsseceseeeeaes 10 C Create new ALEXA Seq database 0 i ccc ccc ccesceesceesseesseceeeesseeesseesseeens 10 PINAY SUS Nee shat e eave fac Mi e Fela gadced A a Aleta E E nade aeuateNtatey Some ciet 11 Command CREAT ON iseer oee aa E AO REO EEE oaa EATE EAEAN ASS 11 0 Install the ENSEMBL AP Is s555 cess scstsssactansvnntsrineaauimasiite ans Naa 11 1 Create target directories excl es seat ccact dues set behdae arisen eratnapad ba eadbsrciueddedavaedeactandy 11 2 Import raw read data is cei Mist he cateet ace biases eda Ria hestenees Aue adios teen 11 3 Basic data statistics isi cicnace Asia Ue oa cei Mta des ceacicetleabeiascadecds shades anece Naess Maedicrat 12 4 Generate read fasta files ooo eee cccecseeeseeseeeseesecsaecseeeseeeesseeseeseeseeeeeees 12 5 M ppInNg aina a yaa elas E E E OE E 12 6 Read assignment aka parsing 0 0 0eo0eoeeseesoesesseseesessos2. Unassigned and if possible are assigned to transcripts junctions introns etc analysis_dir read_records library_id Mapping results files Within the read_records directory mapping results files are stored in sub directories For convenience of downstream analysis individual mapping results are provided for each target sequence type repeats transcripts junctions boundaries introns and intergenic regions analysis_dir read_records library_id sequence_type Figures and statistics A large number of statistics summary files and figures are generated for each lane of data library and comparison Summary of annotations used for the analysis analysis_dir figures_and_stats Generic Copyright 2009 2010 Malachi Griffith and Marco A Marra 15 Basic lane by lane library quality statistics analysis_dir read_records library_id Summary Feature expression expression correlations read assignments average coverage values library statistics etc analysis_dir figures_and_stats library_id Expression_v Differential expression results by sequence feature type analysis_dir figures_and_stats DE project_name feature_type Alternative expression results by sequence feature type analysis_dir figures_and_stats SI project_name feature_type Log files Log files from the ALEXA Seq analysis analysis_dir logs library_id Temporary files Working directory for creation of temporary files during data
3. Expression calculations Using the coordinate information in the ALEXA Seq annotation database in combination with the mapping results generated above the expression of 13 types of sequence features will de determined These features consist of Genes transcripts exon regions exon junctions known and novel exon boundaries known and novel introns active intronic regions silent intronic regions intergenic regions active intergenic regions and silent intergenic regions Refer to the manuscript methods for further details on the annotation of these features The result of this analysis for human is an expression value for 4 million sequence features Transcript specific expression for all Known EnsEMBL transcripts is calculated by using only those exon regions and exon junctions that are unique to each transcript For each feature several expression metrics are calculated These include cumulative coverage average base coverage cumulative coverage divided by the length of the sequence feature the percentage of bases of a feature covered at 1x or greater etc See the schema description for a detailed explanation of all expression measures http Awww alexaplatform org alexa_seq data ALEXA Seq Schema _ Description htm The expression of exon junction and boundary features are systematically lower than those for exon regions This is due to an inherent mapping disadvantage for these sequences see manuscript for discussion For this r
4. Each script can be run without arguments to display a list of instructions Configuration Before starting your analysis you will need to track down basic information about your computer system as well as specific info relating to the project A project relates to a biological question Many examples of such projects that have already been analyzed by ALEXA Seq are provided at our website http www alexaplatform org alexa_seq results htm For example a project could consist of a tumour versus normal comparison or treated versus untreated cell lines etc Each project generally has two or more sequence libraries corresponding to these conditions Each sequence library may itself consist of multiple lanes of paired end sequence data that were generated as one or more runs of a high throughput sequencing device Each project generally also has at least one comparison defined e g tumour vs normal 1 Analysis configuration For tidiness you should have three main directories for ALEXA Seq processing Briefly these contain the code itself the sequence databases and the analysis files For example these directories could be nome user ALEXA alexa_seq nome user ALEXA sequence_databases nome user ALEXA alexa_seq_analysis Other folders you may wish to create nome user ALEXA perl_storables for BerkeleyDB perl storables nome user ALEXA www for web files in case you can t write directly to your web s
5. expression differential expression and alternative expression values for transcripts as well as their component exons junctions boundaries introns etc Copyright 2009 2010 Malachi Griffith and Marco A Marra 3 In order to complete an ALEXA Seq analysis you will need to download the source code and other resources from our website The website also contains example data and the results of many ALEXA Seq analyses performed by us as part of various collaborations As a department of the BC Cancer Agency our focus in these collaborations has primarily been the analysis of cancer samples However the method described below was designed to work for any comparison between paired or grouped conditions Although we have primarily analyzed human and mouse data the method should also work for any species currently annotated by EnsEMBL For further details please visit our website www AlexaPlatform org Before Starting Before you can run the ALEXA Seq analysis you will need to ensure that your system meets certain requirements We make use of numerous existing bioinformatics tools Many of these are commonly used but you should check the list of dependencies below to ensure that all necessary tools are installed If you have problems that are not covered by this manual please contact us Contact information is available on our website www AlexaPlatform org System requirements The ALEXA Seq pipeline is designed to run in a 32 or 64 bit Linu
6. gunzip ALEXA hs 53 36o0 tables tar gz tar xvf ALEXA hs_53_360 tables tar alexa_seq sql restoreAlexaDb pl B Request the database If your analysis involves a species we have not included as a download you can request that we add it by contacting us through our website www AlexaPlatform org We currently have ALEXA Seq annotation databases for Chicken Chimp Fly Human Mouse Rat Yeast and Zebrafish C Create new ALEXA Seq database If you wish to create a complete new annotation database you can use the following tool to create the necessary instructions alexa_seq createAnnotationCommands pl You will be asked to supply two configuration files One was created above For the other use the following file as an example alexa_seq config_files examples ALEXA_Seq_dr_57_8c conf This script will create a new commands file containing detailed instructions for building your own ALEXA Seq annotation database A detailed description of the database schema is provided here http www alexaplatform org alexa_seq data ALEXA_ Seq Schema_Description htm Copyright 2009 2010 Malachi Griffith and Marco A Marra 10 Analysis Command creation Once you have created or updated your system and project configuration files as described above execute the following to create the analysis commands cd alexa_seq createAnalysisCommands pl alexa_ seq config file config files examples ALEXA Seq PIPELINE conf
7. processing analysis_dir temp Temporary storage for ALEXA Seq data viewer files analysis_dir temp project_name Copyright 2009 2010 Malachi Griffith and Marco A Marra
8. project_config file config files examples ALEXA Seq lt ExampleProject gt conf commands file ALEXA Seq ExampleProject commands Open the commands file created by this script and follow the instructions The tasks that will be performed are briefly described below Each step described below corresponds to a step in the commands file with the same number 0 Install the EnsEMBL API and BioPerl The version to use is specified in the configuration file It will only be installed if not already present 1 Create target directories All directories need for the analysis and creation of results files statistics figures etc will be automatically created 2 Import raw read data This step will concatenate if necessary the _seq txt or _qseq txt files from a source directory to create a combined raw seq data file There may be many tile files corresponding to a single lane or they may already be joined together These files are expected to be named as follows s 1 1 0001 gseq txt bz2 i e s_ lt Lane gt lt Read 1 or 2 gt Tile lt seq or qseq gt txt lt gz or bz2 gt Note compressing seq or qseq files is not required but recommended to save storage space The format of sequence data in the raw seq or qseq files is as follows Lane Tile X coord Y Coord Sequence In this format the sequence for read1 and read2 is pasted together for paired reads Bases which could not be resolved are re
9. this step An example of this search functionality can be found here http www bcgsc ca xapian search omega Copyright 2009 2010 Malachi Griffith and Marco A Marra 14 Result file locations The following is a brief description of files created during the ALEXA Seq analysis and their relative storage location all files are created automatically during the analysis In the following descriptions analysis_dir refers to the base path where all the results are stored e g home user alexa_seq_analysis This parameter is set at the beginning of the analysis in the ALEXA Seq configuration file Similarly Sproject_name is the name of the project defined in the configuration file and library_id is a variable used to denote each of the libraries being processed for that project Batch files All command files to be executed for a particular project are stored as follows analysis_dir batch_jobs project_name Raw sequence data Raw sequence files imported from the high throughput sequencing device are stored as follows analysis_dir raw_seq_data library_id Fasta sequence files Fasta files containing all reads passing basic quality filters are stored for each lane of each library here analysis_dir fasta_seq_data library_id Read record files These files store the read sequences of each pair on a single line along with basic quality metrics for the read and its current assignment status All reads start as
10. you do not have access to such computer resources this analysis may not be practical for large datasets Copyright 2009 2010 Malachi Griffith and Marco A Marra 4 Example data Example datasets can be downloaded from our website as they become available http www alexaplatform org alexa_seq results htm Dependencies You will need the following components to be installed Some of these may be included with your Linux distribution by default Others will need to be installed For a detailed walkthrough of the installation procedure including all dependencies refer to the separate ALEXA Seq Linux installation manual included with the source code Cairo http cairographics org In order to dynamically produce graphics in R you will need to install both the cairo and pixman packages for Linux R The R Project for Statistical Computing http www r project org Once R is installed begin a session and install the following additional packages RColorBrewer Cairo Bioconductor biocLite http Awww bioconductor org Bioconductor is used for certain data processing and graphing functions The biocLite version contains all necessary packages Perl http www perl org Perl should already be installed with Linux Both Perl 5 6 x and 5 8 x should work BioPerl http Awww bioperl org Core Latest index shtml BioPerl is used by the EnsEMBL API BioPerl should be installed into the ensembl_ api
11. CANADA S MICHAEL SMITH CS en Tol wce s Centre www bcgsc ca ALEXA Seq www AlexaPlatform org User Manual v 1 5 23 April 2010 Can E Michael Smith Foundation for Health Research C 2 Genome BritishColumbia Pye Ey vaur NU Asingle dream A world of hope The Terry Fox Foundation mse National Institut national Cancer Institute du cancer of Canada du Canada Copyright 2009 2010 Malachi Griffith and Marco A Marra Table of Contents Table of Contents ieee ieee ee ee as ieee 2 A NOTS a onae r teuce e EE E N E O N auace ease ae 3 Citation 2s sz esac ie cohen E cues E AR A a E Pa E A T E TE 3 LICENS Oie A EEEE E aaa ts ATA AEA AR EL E i 3 Acknowledgements set Sep ce dal coast aan Sea eta R AEA e sb ged Madame deed ead aves 3 Affiliations analen a deeded ta wade and ada R ee eae Bernas 3 Introduction Tenne A Gate E a E e E a e TA 3 Betore Startihg acn anet aie eae Ui ea Baa A a Et 4 System requirements o0ooeoenseeeeeesseseesessesoseesosrtsestestssestestssesessestestssestosessoseesesseesessesee 4 Warming S eee etn ea a E sh aaa a tana A est aude Raa aE aA need EE AE AES 4 Example data mnanaa aaa a a a a a 5 Dependencies raea n a O O A RSL Bh MERE POLE POMPEO Rane CSC 5 Obtaining ALEXA Seq code i 22 ctctseeeniatedicedadnae natn email ies Beaten ceneectels 7 Installation of ALEXA Seq COC cece ccssceesceessecssecesecesseeessecssecesececseeessecsseceseesaes 7 Script and Module Locations
12. a general overview of the installation procedure and assumes all dependencies p 5 6 are already installed For a detailed walkthrough of the installation procedure including all dependencies refer to the ALEXA Seq Linux installation manual Before proceeding download and unpack the ALEXA code base For example mkdir home user ALEXA cp ALEXA Seq v 1 5 tar gz home user ALEXA cd home user ALEXA gunzip ALEXA Seq v 1 5 tar gz tar xvf ALEXA Seq v 1 5 tar Unpacking the code is not necessary if you obtained it from our svn repository Once the code is installed Open the INSTALL txt file and copy the indicated section of environment variables to your bashrc file or an equivalent shell parameters file that is automatically loaded on log in These variables should edited to reflect your own specific file paths and install directories Finally once this is done log out of your session and log in again to make sure the environment variables take effect Copyright 2009 2010 Malachi Griffith and Marco A Marra 7 Script and Module Locations The root reference directory for all scripts is alexa_seq where is wherever the code was unpacked Many of the scripts described below make use of methods that we have written and stored in utility modules The details of these functions are beyond the scope of this document These Perl modules follow POD plain old documentation format and are stored in alexa_seq utilities
13. ate the databases blast provides a utility for this or create new databases mdust http compbio dfci harvard edu tgi software The version we use in the pipeline is included with the ALEXA Seq source code package in the external_tools sub directory Unpack and compile this tool Note the location of the directory containing the mdust binary This information will be needed when setting up your ALEXA Seq configuration file Xapian Omega http xapian org Used to index results created by the ALEXA Seq pipeline This tool will need to be installed somewhere that is accessible by your web server It will also need permission to run as an executable process on the web server Copyright 2009 2010 Malachi Griffith and Marco A Marra Obtaining ALEXA Seq code There are several options for getting the ALEXA Seq code A From our website The source code package as well as ALEXA Seq annotation databases can be downloaded from here The code is available http www alexaplatform org alexa_seq downloads htm B From SourceForge net https sourceforge net projects alexa seq C From our public subversion repository If you have subversion svn installed you can check out the code directly from our subversion repository For example execute the following from a command prompt svn co https svn bcgsc ca public ALEXA Seq tags ALEXA Seq v1 5 ALEXA Seq v1 5 Installation of ALEXA Seq code The following is
14. eason these values are empirically adjusted to compensate for the disadvantage 9 Summarize expression statistics In this step various expression statistics and graphs are generated For each library gene specific expression cutoff values are determined by examination of intronic and intergenic noise levels see manuscript for details The number of features of each type that are expressed above background is then determined The overall sequence coverage of genes is also determined in this step 10 Regenerate expression values Using the expression values determined above the junction boundary correction factors and gene by gene cutoffs values are now used to re evaluate the expression of all features To accomplish this steps 8 9 are repeated 11 Create custom UCSC track files to visualize data Expression values will be used to create custom UCSC track files a mixture of GFF and wig tracks to display all features expressed above background and the base level sequence coverage of the genome Similarly a wig track displaying base level differential expression will also be created in this step for each comparison you define Copyright 2009 2010 Malachi Griffith and Marco A Marra 13 12 Calculate Differential Expression DE of all features Differential expression of all features Genes Exons Junctions Boundaries etc will be assessed as the log2 difference in expression level for each comparison you define Each differen
15. eosssseessssesesesseseesseseeses 12 7 Read assignment SUMMALPY cece cceesceeseeseceseeesecseeeseeeeessecsseeseeseeeseeeeeees 13 8 Expression calculations iciisc ta2 ciecerlicsiticmaeed dure d laisse nienidionde 13 9 Summarize expression StatiStiCs ccc ces ceecseeeseesseeeeeseesseeseenseeeeeees 13 10 Regenerate expression values 0 i ee ccc ecceccetecsseesceeseeeeeeseceaecseeeseeseeeeeeeaees 13 11 Create custom UCSC track files to visualize data 0 00 0 eee 13 12 Calculate Differential Expression DE of all features 00 0000 14 13 Calculate Alternative Expression AE of all features 0 00 ee 14 14 Populate ALEXA Seq data viewer oo cc cccccccecsceeseeeeceseceseessesseeeeeeeeeeaees 14 15 Index all gene records using the Xapain Omega utility 000000000 14 RESUIt Tile locations oe n iE E saan aio AR iota te dene apap ag eae 15 Copyright 2009 2010 Malachi Griffith and Marco A Marra 2 Authors ALEXA Seq is the work of Malachi Griffith and Marco A Marra Citation Malachi Griffith Obi L Griffith Ryan D Morin Michelle J Tang Ying Chen Hou Trevor J Pugh Rodrigo Goya Jill Mwenifumbo Suganthi Chittaranjan Adrian Ally Jennifer K Asano Susanna Y Chan Haiyan I Li Helen McDonald Kevin Teague Yongjun Zhao Thomas Zeng Allen Delaney Martin Hirst Gregg B Morin Steven J M Jones Isabella T Tai Marco A Marra Alternative expression analy
16. eral example project configurations are provided The validity of values entered in this configuration file will be tested in the first step of the analysis 3 Installation of ALEXA Seq annotation database You will need an ALEXA Seq annotation database to perform the analysis This database defines all the canonical alternative and hypothetical sequence features that will be considered in the analysis The database is specific to a particular species and genome build of that species You have three options for obtaining these databases Instructions for each of these options are as follows A Download and install an existing database recommended Pre computed databases are provided here ftp ftp03 bcgsc ca public ALEXA alexa_seq Each of these is described here http Awww alexaplatform org alexa_seq downloads htm Most of the analyses described on our website used the Human build hs_53_ 360 NCBI Build 36 UCSS hg18 but databases for several other species and builds are available Install as follows using hs_53_ 360 as an example Copyright 2009 2010 Malachi Griffith and Marco A Marra First log into your mysql server and create the target database mysql CREATE DATABASE ALEXA hs 53 360 exit Then download and install the database cd home user sequence databases wget ftp ftp03 bcgsc ca public ALEXA alexa_seq hs_ 53 360 tar gz gunzip hs_53_ 360 tar gz tar xvf hs 53 _ 360 tar cd hs_53_ 360 alexa_db
17. ere both reads of a pair are identical library artifacts 5 Mapping Reads will be mapped to a database of repeat elements transcripts known exon junctions and boundaries hypothetical exons junctions and boundaries and intronic and intergenic regions of the genome Where possible read pairing information is used to resolve ambiguously mapped reads but if only one read of a pair can be mapped this read will still be retained i e paired mapping is not required With default parameters reads are mapped with up to 3 mismatches and 1 gap Sub string alignments are also allowed A perfect alignment of 60 of the read length will be allowed again assuming default parameters 6 Read assignment aka parsing Based on the alignments reads will now be assigned to their most probable source If possible each read is unambiguously assigned to a repeat element known transcript novel exon junction novel exon boundary intron or intergenic region At this stage the apparent distance between reads of a pair is determined as well as other statistics pertaining to mapping efficiency Reads that can not be assigned to a repeat or human genome or transcriptome sequence are retained but are marked as Unassigned and excluded from downstream analysis Copyright 2009 2010 Malachi Griffith and Marco A Marra 12 7 Read assignment summary The number of reads assigned to each category read class for each library will be determined 8
18. erver nhome user ALEXA commands for commands files for each project nome user ALEXA config_files for config_files for each project Before performing your first analysis you will need to create an updated analysis configuration file This file stores basic system specific parameters mostly paths to directories or binaries Create this file by starting with the example file provided cp alexa_seq config_files examples ALEXA_Seq_PIPELINE conf Copyright 2009 2010 Malachi Griffith and Marco A Marra 8 config_files ALEXA_Seq_PIPELINE conf Edit all values in the config files to reflect your own file locations The validity of values entered in the configuration files will be tested in the first step of the analysis by createAnalysisCommands pl see below 2 Project configuration Before starting analysis for a new project and for the first analysis you run you will also need to create a project configuration file This file stores information about the data to be analyzed Specifically you must define each LANE LIBRARY and COMPARISON For example you will need to determine the paths to your input sequence data Also included in this file are configuration values that might be project specific e g the species being analyzed the genome version to use etc Create this file by starting with the example file provided here alexa_seq config_files examples ALEXA_Seq_ lt ExampleProject gt conf Sev
19. presented by a and will be converted to N s During this step the complexity of each read will be determined by mdust and various statistics pertaining to each lane will be summarized total read counts low complexity reads poor quality reads etc During this step it is also possible to trim reads if there was a problem with the quality of read ends Copyright 2009 2010 Malachi Griffith and Marco A Marra 11 3 Basic data statistics Gather basic info about each lane library and comparison defined in the Project configuration file Also get number of quality reads the average read length and the overall tag redundancy of each library 3 A Gather info about each lane of data Imported from the project configuration file 3 B Gather info about each library Imported from the project configuration file 3 C Gather info about each comparison Imported from the project configuration file 3 D Generate statistics for each library This step will determine the number of quality read counts in each lane the average read length of the library and the overall tag redundancy of each library If the library consists of a mixture of read lengths not advisable it may be useful to know the average read length 4 Generate read fasta files One fasta file will be created for each lane of data after filtering out reads that are poor quality too many ambiguous bases i e N s low complexity e g polyA reads or wh
20. sis by RNA sequencing Pending publication License ALEXA Seq is open source and available for public use under the terms of the GNU General Public License version 3 For details refer to http Awww gnu org licenses gpl txt Acknowledgements We are grateful for funding provided by the following organizations the University of British Columbia Faculty of Graduate Studies and Faculty of Medicine the Michael Smith Foundation for Health Research the Natural Sciences and Engineering Research Council Genome British Columbia the National Cancer Institute of Canada and the Terry Fox Foundation Affiliations British Columbia Cancer Agency Genome Sciences Centre University of British Columbia Faculty of Medicine Department of Medical Genetics Introduction This manual provides instructions to assist in the completion of an ALEXA Seq analysis Alternative Expression Analysis by massively parallel RNA sequencing The purpose of the ALEXA Seq method is to comprehensively profile the expression differential expression and alternative expression of a transcriptome and compare transcript specific expression events between two or more conditions of interest Novel transcript discovery is also facilitated These analyses require as input paired end RNA Seq aka WTSS data from a high throughput sequencing device such as an Illumina GAII For examples of the output of the analysis please refer to our website Briefly the output consists of
21. sub directory of alexa_seq This installation will handled automatically by the pipeline Berkeley DB Perl Module http search cpan org dist BerkeleyDB BerkeleyDB pod P Needs to be installed for some components of ALEXA Seq to work Ideally it would be installed globally in your system so that Perl knows where to find it If this is not possible instructions are provided in INSTALL txt with the source code for setting the appropriate environment variables EnsEMBL API http ensembl org info data api html Updates should be installed into the ensembl_api sub directory of alexa_seq This installation will handled automatically by the pipeline Copyright 2009 2010 Malachi Griffith and Marco A Marra 5 MySQL Community Server http dev mysql com downloads MySQL may be included with your Linux distribution ALEXA Seq pipeline uses mysql databases to access EnsEMBL data via the EnsEMBL API as well to store custom annotation data We have been using MySQL version 4 1 MySQL DBI DBD http search cpan org dist DBI http search cpan org dist DBD mysql DBI DBD may be included with your Linux distribution by default These are two Perl modules that allow Perl to interact directly with MySQL database BLAST ftp ftp ncbi nim nih gov blast executables LATEST Download the 32 or 64 bit version as needed To ensure compatibility of sequence databases use the same version as us 2 2 18 Otherwise you will have to upd
22. tial expression value will also be associated with a p value 13 Calculate Alternative Expression AE of all features Alternative expression of all features Genes Exons Junctions Boundaries etc will be assessed by calculating splicing index SI values reciprocity index RI and percent feature contribution PFC values for each comparison you define See manuscript for details 14 Populate ALEXA Seq data viewer To help summarize and visualize the expression of known and novel isoforms as well as differential and alternative gene expression between conditions of interest all expression differential expression and alternative expression results will be imported into the ALEXA Seq data viewer essentially a dynamically generate web interface This viewer also facilitates data sharing with collaborators design of validation experiments and interpretation of the results In addition to these results detailed summaries of the characteristics of each library are also produced For example http www alexaplatform org alexa_seq Morgen MM0472 htm Several examples of data sets processed and displayed in the ALEXA Seq data viewer are available at our website http www alexaplatform org alexa_seq results htm 15 Index all gene records using the Xapian Omega utility For every project a summary is available for every gene To allow searching for arbitrary genes the results will be also indexed with the tool Xapian Omega in
23. x system We use RHEL 4 and CentOS 5 but any distribution should work fine Due to the extremely large datasets generated by next generation sequencing devices you will require considerable CPU and storage resources to perform an ALEXA Seq analysis We used about 20 100 Gb of disk space to process each of the datasets listed in the results section of our website www AlexaPlatform org Most of this is needed only temporarily and the final result should take 5 10 Gb of storage space We also had access to a Beowulf style CPU cluster of 1000 nodes Although not technically needed to perform ALEXA Seq analyses it is highly recommended to increase the rate of data processing Detailed instructions will be provided for submission of jobs to a CPU cluster However since configuration of the CPU cluster will likely differ at your centre these steps may need to be modified slightly The jobs themselves should not change but the steps involved in submitting those jobs to your cluster may vary from ours Finally in order to create your own ALEXA Seq data viewer to help visualize and distribute data you will need access to a web server we use APACHE2 Warnings You will require at least a basic familiarity with Linux and Perl to perform the ALEXA Seq analysis Furthermore certain aspects of this pipeline are extremely computationally intensive have included scripts to assist in the creation of parallel jobs to be run on a cluster of computers If
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