SeqScape Software Version 2.5 Comprehensive Analysis Solution for Resequencing Applications

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1 Product Bulletin Sequencing Software SeqScape Software Version 2.5 Comprehensive Analysis Solution for Resequencing Applications Comprehensive reference sequence handling Helps interpret the role of each polymorphism Accurate data analysis Detects heterozygous indels and provides quality values Two-tier analysis Reduces analysis time and complexity Powerful data visualization tools Allow you to focus on relevant data and create custom views of the same data Detailed reports Generates comprehensive reports with detailed results describing data quality Automation Integrated with Data Collection software to analyze raw data generated from Applied Biosystems genetic analyzers Introduction The goals of today s large resequencing studies are to detect SNPs, profile mutations, perform medical sequencing, identify haplotypes, subtype pathogens, and confirm clone constructs. For all of these applications, SeqScape software provides two levels of analysis SNP detection and the identification of sequences that most closely match a target sequence. Unlike tools initially designed for de novo analysis, SeqScape software is a comprehensive resequencing tool that analyzes data using full sequencefeature information, such as exon/ intron boundaries, translation frames, coding regions, and transcripts. No other software tool available manages gene complexity as well as SeqScape software, which provides accurate and reliable reports for all references, from the simplest to the most complex. Whether your research spans multiple genes or focuses on a single region of interest, SeqScape software supplies you with comprehensive referencesequence features, accurate polymorphism detection, powerful viewing features, and detailed reports that can be exported for use in other analyses. Flexibility to Configure a Reference The level of relevant information to be extracted from resequencing data depends not only on the quality of subject-sample sequences, but also on the reference sequence. The Reference Data Group (RDG), an advanced feature in SeqScape software, offers powerful capabilities for configuring reference sequence. For instance, you can directly import a GenBank SM file as a reference (Figure 1).

2 Figure 1. This figure shows a GenBank, format file downloaded from the NCBI public database. The sequence information from the GenBank file can be directly imported into the Reference Data Group (RDG) to use as a reference sequence. The software will automatically parse feature information from the file, including exon and intron boundaries, and translation information. This powerful feature allows the software to report mutations, nucleotides, or amino acids in the context of the genomic annotation. When studying a large gene, you can easily split the reference into smaller segments and delete any regions not required for a faster and easier data review. You can also configure alternate gene transcripts as layers, with different translation frames (Figure 2). You can even create a single template to use the same reference for multiple studies, and save time by avoiding repetitive steps. Complete Workflow The software lets you create a new project simply by selecting a previously configured project template and adding the subject sample sequences. A click on the Analysis button is all that is required to initiate automated basecalling, trimming, assembly, consensus alignment, and reference comparison (Figure 3). Figure 2. Complex references can be configured as shown in this example for the gene CDKN2A, with alternate transcripts. The RDG functionality in SeqScape software allows you to define unique layers with independent translation frames for each transcript. Analysis algorithms are designed and calibrated with Applied Biosystems instrument-specifications and reagent characteristics to provide accurate bases with quality values for each mutation. Distinctions between poor- and highquality data are clearly defined, as the KB Basecaller and SeqScape software algorithms filter out low-quality and anomalous data to provide a highquality assembly. Quality values allow you to review your data. Using software that provides a complete workflow eliminates working with multiple tools to get to your results.

3 Figure 3. SeqScape software workflow. for accurate detection of heterozygote calls (Figure 5). This ensures accurate detection of all types of mutations, including heterozygous insertion or deletion mutations (HIMs), which are often missed by other methods. First-tier Analysis Detects Mutations with Confidence The first-tier analysis detects all variations between the reference sequence and the subject sample sequence, including deletions, insertions, mismatches, heterozygous bases, and heterozygous insertion/deletions. The consensus caller in SeqScape software examines the quality of each trace for background noise as well as the orientation of the traces, and forms an accurate consensus call. Variations between the consensus and the reference sequence are reported as mutations in the Mutations report. Analysis at the consensus level corrects sequencing anomalies such as PCR noise or unincorporated dye terminators that are present in one strand and cause a basecalling error (Figure 4). Algorithms at the consensus level are essential to reduce the false-positive rate Second-tier Analysis Identifies Best Sequence Matches from an Allele Library Second-tier analysis compares each consensus sequence to a library of sequences and identifies the best matches, an important step in identification studies. Most labs must invest in additional software to perform this level of analysis. SeqScape software allows you to compare your sample data with a library of allelic sequences and perform a haploid or diploid search to identify the best matches. Results of alignment and identification are reported within the Library Search report (Figure 6). Figure 4. This figures shows an example of an accurate consensus generated using base quality information from the forward and reverse strands. The consensus calling algorithm corrects for miscalls in individual strands (indicated by red dots). Figure 5. A heterozygous deletion mutation is correctly identified and reported by SeqScape software algorithms.

4 Powerful Data Visualization Tools Sophisticated data visualization tools enable you to navigate effortlessly through large data sets and screen out non-relevant data. Thus, you can scan thousands of data points quickly and easily for mutations (Figure 7). The software allows you to view an assembly of the entire project or of each individual specimen (Figure 8). Detailed Reports Results reports from SeqScape software include hyperlinks to the data source to show the relationship between the results and the data (Figure 9). For each mutation detected, you can review the quality value provided, the classification of novel or known SNP, and the impact of the SNP on the amino acid translation level. Mutations can be sorted by each of these attributes for easy and fast review. You can also view the audit history of changes made to base pairs in the Audit report. Figure 6. For library searching, a library of pre-aligned sequences is imported into the Library Editor and aligned with the Reference Data Group (RDG). After analysis is complete, the results are reported in the Library Search report. Export Capabilities for Easy Integration SeqScape software provides a complete analysis, which usually requires several analysis tools. It also enables researchers to easily import results into other software packages or archival mechanisms. SeqScape software stores results in detailed reports and enables flexible exporting options. A variety of export functionalities are provided, including alignment of the complete project, assembly across a reference segment, and individual consensus, in standard FASTA format. You can export the reports in convenient formats, such as tab-delimited text, html, and xml for easy integration into downstream pipelines. Figure 7. In the Character/Dots view, base differences in the consensus are displayed against the reference as characters, and all bases common to the reference are represented as dots.

5 Security Features Requires user ID and password to enter the software Displays name when user is logged into the system Provides unique user ID and password for each user Figure 8. Specimen view illustrates the assembly of individual traces (forward and reverse) across the complete reference. Specifies password lifetime Prevents unauthorized user ID or password Audit Trail Features Generates an audit trail when bases are changed, added, or deleted Contains user name, time and date of each change, and the reason for each change Figure 9. Base changes reported in the Mutations Report can be directly linked to view the sample data. Automation Features Increase Productivity SeqScape software is the only mutation analysis software fully integrated with Applied Biosystems Data Collection software and reagents, which are designed expressly for our DNA sequencing instruments. SeqScape Software v2.5, together with Data Collection Software v3.0, automates data analysis, so you can concentrate on data review. By creating a SeqScape Software plate in Data Collection software, you can run an automated analysis pipeline that will generate project analysis reports in SeqScape software. Assists with 21 CFR Part 11 Requirements For researchers working in regulated environments, SeqScape software offers a full suite of features to provide data security, an audit trail of data changes, and electronic signatures. These features, which are designed to assist customers with 21CFR part 11 requirements and can be disabled or enabled at the time of software installation, include the following: Electronic Signatures Captures the printed name of the signer, the date and time of the signature, and the meaning associated with the signature Provides a unique electronic signature for each user Free 45-day Download and Webinars SeqScape Software v2.5 is available (/support/ software) as a 45-day free demo for use with your own data. For more information, contact your Applied Biosystems sales representative. Free Webinars are also available to provide introductory training on the software. For a schedule of upcoming Webinars, visit: info.appliedbiosystems.com/ webinars.

6 Ordering Information Description P/N SeqScape Software v2.5, Initial License SeqScape Software v2.5, 45-Day Demo and Tutorial,** 1 pack/5 users SeqScape Software v2.5 and DNA Sequencing Analysis Software v5.2, Initial License ABI PRISM 310 Genetic Analyzer Sequencing Analysis Software v5.2 and SeqScape Software v2.5, Initial License Sequencing Analysis Software v5.2, SeqScape Software v2.5, & GeneMapper SW v4.0, 1 license Applied Biosystems 3130/3130xl Genetic Analyzer Sequencing Analysis Software v5.2 and SeqScape Software v2.5, Initial License Sequencing Analysis v5.2, SeqScape v2.5, & GeneMapper Software v4.0, 1 license Upgrades from the 3100 Avant to the 3130/3130xl Genetic Analyzer Sequencing Analysis Software v5.2 and SeqScape Software v2.5, 1 license Applied Biosystems 3730/3730xl Genetic Analyzer Sequencing Analysis v5.2 and SeqScape Software v2.5, 3 licenses Sequencing Analysis v5.2, SeqScape v2.5, & GeneMapper Software v4.0, 3 licenses Additional License* SeqScape Software v Upgrades/Updates Upgrade to SeqScape Software v2.5 from v1.0/ Update to SeqScape Software v2.5 from v2.0/2.1.1** **User has an initial license. **Free download from For Research Use Only. Not for use in diagnostic procedures. Notice to Purchaser: License Disclaimer Purchase of this software product alone does not imply any license under any process, instrument or other apparatus, system, composition, reagent or kit rights under patent claims owned or otherwise controlled by Applera Corporation, either expressly, or by estoppel. SeqScape Software has not undergone specific developmental validation for human identification applications. Human identification laboratories that choose to use SeqScape Software for data analysis should perform their own developmental validation studies. ABI PRISM, Applied Biosystems, GeneMapper, and SeqScape are registered trademarks and AB (Design), Applera, and KB are trademarks of the Applera Corporation or its subsidiaries in the US and/or certain other countries. GenBank is a service mark of the US Department of Health and Human Services. Macintosh is a registered trademark of Apple Computer. Windows is a registered trademark of Microsoft Corporation. All other trademarks are the sole property of their respective owners Applied Biosystems. All Rights Reserved. Printed in the USA, 06/2005, LD Publication 133PB02-01 Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone Toll Free International Sales For our office locations, please call the division headquarters or refer to our Web site at /about/offices.cfm