SOLiD Software Quick Facts
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1 SOLiD Sftware Quick Facts 1. What s New in SOLiD Sftware Data Prcessing and Data Analysis SOLiD Data Prcessing Overview SOLiD 4 Result Size and On-Instrument Cluster SOLiD Offline Analysis Cluster Specificatin SOLiD BiScpe Sftware BiScpe v1.2 Sftware vs. Crna_Lite fr Offline Analysis Data Visualizatin
2 1. What s New in SOLiD Sftware Data Prcessing and Data Analysis SOLiD Accuracy Enhancer Tl The SOLiD Accuracy Enhancer Tl (SAET) is a spectral alignment errr crrectin tl, which, when applied t raw data generated by the SOLiD platfrm, reduces the clr calling errr rate by factr f three t five withut having the reference genme. Decrease in errr rate imprves mapping, SNP calling, and de nv assembly results. Use the SAET t pre-prcess raw reads befre alignment. SAET reduces the clr calling errr rate by a factr f three t five withut having the reference genme. SAET is nt recmmended fr use with whle genme resequencing f large genmes where large > 600 Mbases. The decrease in errr rate imprves mapping, SNP calling, and de nv assembly results. Mapping becmes mre accurate and the number f mapped reads increases by 40-50%. SAET is available thrugh the BiScpe Sftware cmmand line nly. Histry tab Use the histry tab in the Web brwser t dwnlad r view files generated during a selected plug-in sessin. The histry feature is available nly frm the BiScpe Sftware web brwser. Barcde script The barcde script runs a given BiScpe Sftware data analysis n a set f barcde library read files in batch mde. Use the script t run simultaneus secndary r tertiary tests n barcded libraries. BAM frmat utput BiScpe Sftware secndary analysis (mapping and pairing) nw prduces a BAM file as the main alignment frmat. Mate pair and paired end analysis prduces a BAM file while a single file cnversin is needed fr fragment libraries. Depending n the utput filter selected, unmapped and secndary alignments can be included. HD-300 perfrmance HD-300 increases thrughput. BiScpe Sftware v1.2 can prcess larger files at increased thrughput, which results in mre reads while maintaining current speed and increasing density. 2
3 SNP detectin pst-errr file changes BiScpe Sftware nw prvides the ptin f pre-generated Prbe Errr files. The feature f prviding pre-generated files has the main advantage f time saved in bypassing the regeneratin f Prbe Errr files every time SNP detectin is executed. Using SOLiDBiScpe.cm Users f clud cmputing can wrk with BiScpe Sftware using SOLiDBiScpe.cm. This feature prvides the fllwing benefits: N up frnt (capital) cst and n cst cmmitments Pay as yu g; predictable peratinal csts Scale up/dwn as demand needs Uplad data via Internet r physical shipments f hard drives Fr mre infrmatin, please visit here. Fusin/Splicing A fusin junctin is a sectin f transcribed RNA that maps t an exn frm ne gene fllwed by an exn frm anther gene. It can ccur as the result f a translcatin, deletin, r chrmsmal inversin. It excludes exn-exn bundaries that arise frm alternative splicing fr a gene. ChIP-SEQ BiScpe Sftware v1.2 gives yu the ptin t perfrm ChIP-Seq resequencing thrugh the BiScpe Sftware brwser. Paired-end mapping Biscpe Sftware v1.2 added supprt fr paired end experiments in additin t mate pair. In a nrmal paired end experiment, the F5 and F3 tags matched t the genme n different strands and facing tward each ther, and satisfies a distance cnstraint determined by insert size. Exprt Cnfiguratin Yu can nw use the web brwser as well as the cmmand line t create the cnfiguratin file that is required t perfrm experiments n barcded libraries. 3
4 2. SOLiD System Data Prcessing Overview SOLiD Instrument Cntrl Sftware (ICS) Prvides autmated instrument peratin and submits data prcessing jbs fr primary analysis: Imaging Bead finding Image registratin Filtering Clr call SOLiD Experiment Tracking Systems (SETS) Web-based applicatin that enables users t view n-instrument real-time data and cmpleted run analysis reprts frm the SOLiD Analysis Tls. Secndary analysis (mapping) is enabled n-instrument, althugh it is recmmended t run secndary analysis ff-instrument using BiScpe Sftware. SOLiD BiScpe Sftware SOLiD BiScpe Sftware prvides a cmmand line and simple web interface that builds cnfiguratin files fr running applicatin-specific sequence analysis tls. The BiScpe Sftware framewrk enables the user t perfrm ff-instrument secndary and tertiary analyses, and it allws cnfigurable 4
5 biinfrmatics wrkflws fr resequencing (mapping, SNP finding (dibayes), cpy number variatins, inversins, small indels, large indels) and whle transcriptme analysis (mapping, splicing/fusin detectin, cunting, UCSC WIG Files creatin). Results can be exprted as BAM frmats. The resulting industrystandard files frm BiScpe Sftware can be used with third-party visualizatin and analysis sftware tls. 3. SOLiD 4 System Result Size and On-Instrument Cluster The SOLiD Sftware analysis pipeline generates results in BAM frmat cntaining base space sequence. Mapping results in BAM frmat cntain base space sequences, clr space sequences and quality values. The data sizes frm the SOLiD 4 System, as indicated in the table belw, are fr fully laded experiments and directly crrelate with the thrughput (ttal bases generated frm a run). Table 1: Result size generated n SOLiD 4 System sequencing (Assume 2 slides and depsitin densities f 300K beads/panel, 2357 panels) 50 nt tag/300k/panel,2357 panels Image data size Primary analysis Primary analysis data results size in.spch size (flatfile:.csfasta, frmat _QV.qual,.stats ) 1 slide 1 tag 1.84 TB 646 GB 170 GB 1 slide 2 tags 3.6 TB 1.29 TB 340 GB 2 slides 1 tag 3.6 TB 1.29 TB 340 GB 2 slides 2 tags 7.2 TB 2.58 TB 680 GB Nte: Image data is nt needed after analysis is cmplete and intensity files are n lnger required fr submissin int NCBI. 5
6 w.pdf On-Instrument Cmpute Cluster Specificatin SOLiD 4 System Specificatin Cmputer Cmpnents 19-inch flat screen mnitr, muse and keybard Instrument cntrller Head nde Cmpute ndes (3) Shared strage Gigabit Switch Pwer distributin units (2) Pwer crds (2), attached Sftware Suite Instrument Cntrl Sftware v4.0 SOLiD Experimental Tracking System (SETS) v4.0 Instrument Cntrller Hardware: Intel Xen prcessrs Operating system: Micrsft Windws XP Prfessinal, Service Pack 2 Installed RAM:8 GB Hard disk strage: dual 250 GB SATA hard drives (RAID-1) Peripheral: CD-RW/DVD ROM, 19-inch flat screen mnitr, keybard, muse Head Nde Cmpute Ndes (each) Shared Strage Gigabit Switch Pwer Distributin Units Hardware: Intel Xen Quad Cre prcessrs (2) Operating system: 64-bit LINUX Installed RAM: 24 GB Hard disk strage: 6x 1 TB SATA hard drives (RAID-5) Hardware: Intel Xen Quad Cre prcessrs (2) Operating System: 64-bit LINUX Installed RAM: 24 GB Hard disk strage: 2 x1 TB SATA hard drives (RAID-0) Hard disk strage: 15x 1 TB SATA hard drives RAID-5 w/ ht spare 16 prt Gigabit Switch Rack PDU (2) with 16 utput cnnectr While full mapping is enabled n-instrument (cluster) fr small genmes (i.e. Bacteria), we d nt recmmend secndary analysis (mapping) n the instrument cluster. Instead we highly recmmend secndary analysis (mapping) using a separate ffline analysis cluster. Hwever, fr large genmes (i.e. 6
7 human), a sub-mapping can be perfrmed n-instrument fr quality assessment f the run nly. 4. Offline Analysis Cluster Specificatin The SOLiD 4 System allws cycle by cycle aut-exprt and manual exprt f primary analysis results t an ffline cluster where the secndary analysis can be perfrmed independently f the instrument. This feature enables the instrument t be utilized fr additinal experiments while secndary analyses are being perfrmed. Offline Analysis The ffline analysis cluster specificatin is shwn belw and represents the minimal and the recmmended (Penguin Cmputing) specificatin fr ffline data analysis. BiScpe Sftware Offline Cluster Specificatin Minimal Offline Cluster Specificatin Minimal Offline Cluster Specificatin Head Nde Cmpute Ndes (minimum 3 cmpute ndes) Gigabit Switch > 2 GHz prcessrs 16 GB RAM 100 GB strage lcal disk space fr OS+ sftware installatin > 2 GHz prcessrs > 2 GHz prcessrs,16+ GB RAM, 8+ cres per nde > 500 GB strage lcal disk space fr OS+ sftware installatin 1 GB Switch Operatin System Cents 4.x, 5.x RedHat 4.x, 5.x Strage >10TB 7
8 Penguin Entry Level Cluster fr SOLiD Offline Analysis 8
9 5. SOLiD BiScpe Sftware OVERVIEW SOLiD BiScpe Sftware is a framewrk fr biinfrmatics tls t perfrm ff-instrument secndary and tertiary analysis. BiScpe Sftware cnsists f a cllectin f biinfrmatics tls that are integrated int a single cmmand line shell. Additinally, BiScpe Sftware prvides a simple web interface t help build instructins (cnfiguratin files) t run these tls. BiScpe Sftware includes mapping, pairing, SNP finding, structural variatins and whle transcriptme analyses. FEATURES BiScpe Sftware 1.2, using a flexible pipeline architecture, enables maximum flexibility and ease f use fr perfrming high thrughput data analysis fr SOLiD instrument data. It cntains fllwing features: 9
10 Mapping BiScpe Sftware 1.2 features Applied Bisystems newly develped mapping algrithm MaxMapper, which drastically imprves the mapping rate and mapping speed ver the previus generatins f mapping techniques. It prduces mapped data that gives excellent sensitivity and specificity in SNP finding using Applied Bisystems dibayes SNP finding algrithm, a cmpnent in BiScpe Sftware. Pairing BiScpe Sftware 1.2 features Applied Bisystems updated pairing algrithm which handles mapped data frm MaxMapper. It prvides new pairing categries D and E and prvides reads file that d nt pair. Resequencing Applicatins BiScpe Sftware 1.2 features five resequencing applicatins: SNP Finding, Cpy Number Variatin fr Human, Small indel finding, Large indel finding and Inversin. SNP Finding BiScpe Sftware features Applied Bisystems latest SNP finding technlgy fr SOLiD System instrument data which allws sensitive and specific SNP detectin even at mderate t lw cverage. It allws varying levels f stringency and prvides full cntrl ver many filters fr custmizatin t fit particular experimental designs. CNV (Human) BiScpe Sftware features Applied Bisystems latest prgress n human cpy number variatin detectin technlgy which allws detecting variatins as small as 5KB and as large as the whle chrmsme in humans frm single sample sequences. It can detect such variatins at very lw cverage, even at 1x. Small Indel BiScpe Sftware features Applied Bisystems latest prgress n detecting small indel variatin. By using a nvel split read technique alngside a pwerful indel caller n pileups, is able t detect deletin up t 500bp and insertins up t 20bp. Large Indel BiScpe Sftware features Applied Bisystems latest prgress n identifying large insertins and deletins cmpared t a reference genme using SOLiD mate pair libraries. It is able t 10
11 identify large insertins and deletins (indels) frm 100bp t 100Kb with great cnfidence. It is able t accept multiple mate pair libraries t increase cverage. Inversin BiScpe Sftware features Applied Bisystems latest prgress n detecting genmic regins that are inverted with respect t reference. Taking advantage f lnger insert size and lwer inverted dimmer nise f SOLiD mate pair technlgy, it prduces a cnfident list f inversins Whle Transcriptme Analysis The Whle Transcriptme Analysis (WTA) in BiScpe Sftware aligns t a reference genme. With mapping results, it cunts the number f tags aligned with exns, and can cnvert the BAM file t WIG fr display f cverage n the UCSC genme brwser. WTA als supprts experiments in fusin detectin Create UCSC WIG file This plug-in takes the BAM file and cnverts it int WIG files cntaining cverage data. Cverage is the number f reads cvering a given genme stranded psitin. Cunt knwn exns Given a BAM file f mapped reads and predefined regins (such as exns) prvided by the user in a.gtf frmat file, this plugin generates tag cunts fr anntated regins. Fusin, splicing A fusin junctin is a sectin f transcribed RNA that maps t an exn frm ne gene fllwed by an exn frm anther gene. It can ccur as the result f a translcatin, deletin, r chrmsmal inversin. A fusin junctin excludes exn-exn bundaries that arise frm alternative splicing fr a gene. There are five mdels f alternative splicing: Exn skipping r cassette exn: in this case, an exn may be spliced ut f the primary transcript r retained. This is the mst cmmn mde in mammalian pre-mrnas Mutually exclusive exns: One f tw exns is retained in mrnas after splicing, but nt bth. Alternative dnr site: An alternative 5 splice junctin (dnr site) is used, changing the 3 bundary f the upstream exn. 11
12 Alternative acceptr site: An alternative 3 splice junctin (acceptr site) is used, changing the 5 bundary f the dwnstream exn. Intrn retentin: A sequence may be spliced ut as an intrn r simply retained. This is distinguished frm exn skipping because the retained sequence is nt flanked by intrns. If the retained intrn is in the cding regin, the intrn must encde amin acids in frame with the neighbring exns.. 6. B iscpe 1.2 Sftware vs. Crna_Lite fr Offline Analysis Offline Data Analysis BiScpe 1.2 Crna_Lite v4.2 Analysis executin Integrated cmmand line and Integrated cmmand. Simple web interface Can run batch mde. Prgramming Java Scripting languages language User Interface fr parameter setting and analysis GUI (Brwser interface f BiScpe); Cmmand line interface Expanded rich parameter setting thrugh cmmand-line interface Multiple run cmbinatin analysis Mapping Algrithm Max Mapper fr SOLiD 4 MapReads System Default Mapping Max Mapper: Anchr and Full length with fixed setting Extend number f mismatches Iterative Mapping User cnfigurable NO/Manual Multi-threading N SNP algrithm DiBayes SNP caller Integrated small indel analysis Integrated large indel analysis Integrated Human CNV analysis N N 12
13 Integrated Inversin analysis Integrated Whle N Transcriptme analysis Output results frmat SAM/BAM utput. Fasta-like matching utput (including unique match and all matches), paired mates, Optinal GFF v0.2. SNP list text file Stats File New frmat: add extensin Old Stats file infrmatin and quality value Speed Optimized cmpute perfrmance fr cmplex Supprt cmplex genme analysis genme analysis Warranty N N AB supprt t end users Supprted OS Linux CentOS v4.x, PBS (Trque), PBS pr and SGE Linux, PBS, LSF, SGE 7. Data Visualizatin The BiScpe Sftware pipeline will generate reads results in BAM frmat including Base Space Sequence. It can be visualized in brwsers such as UCSC and Brad Institute s Integrative Genmics Viewer (IGV): 13
14 Fr research use nly. Nt intended fr any animal r human therapeutic r diagnstic use Life Technlgies Crpratin. All rights reserved. The trademarks mentined herein are the prperty f Life Technlgies Crpratin r their respective wners. 14
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