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DNA Resequencing and Variant Identification on the Applied Biosystems 3130 Series Genetic Analyzers

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DNA Resequencing and Variant Identification on the Applied Biosystems 3130 Series Genetic Analyzers Application Note DNA Resequencing/Variant ID on the 3130 Series Systems DNA Resequencing and Variant Identification on the Applied Biosystems 3130 Series Genetic Analyzers Introduction Direct DNA sequencing is an accurate and proven technique for mutation detection. However, despite wide- spread use and accessibility, adapting this technique to gene resequencing projects can include challenges in primer design, PCR validation, data analysis, and data interpretation. In the course of designing more than 200,000 primer pairs and generating more than 18 million sequence reads, bioinformatics specialists and R&D scientists who worked on the Applera Genome Initiative gained valuable experience in primer design, as well as insight into the genomic complexities and technical challenges associated Figure 1. The Applied Biosystems 3130 Series Genetic Analyzers are robust, reproducible, systems that are fully with them. The experiences gained automated from the moment each 96- or 384-well plate is placed on the instrument and the run is initiated. The systems provide continuous, unattended operation, from automated polymer loading and sample injection to from the initiative revolutionized separation, detection, and data generation. The instrument also includes several new enhancements, such as a detection cell heater that improves sizing precision, thus facilitating better thermal control, an Automated Polymer DNA sequencing as a tool for the Delivery System that significantly reduces set-up time, and easy-to-use wizards for instrument operation. detection of human gene mutations. The 3130 and 3130xl Genetic sequencing as a tool for the detection Basecaller automatically processes Analyzers, when combined with the of human gene mutations. them and provides LOR (Length- Applied Biosystems VariantSEQr™ of-Read) greater than 500 base The 3130 Series Genetic Resequencing System, reagents, and Analyzers pairs (bp), with average base Quality SeqScape® Software v2.5 provide the Values greater than 20 (QV The 3130 Series Genetic Analyzers 20). most robust and efficient resequencing (Figure 1) are fully automated, high- Furthermore, the 3130xl system, using system for customers who require low performance, fluorescence-based, the UltraSeq36_ POP7 run module, to medium throughput. Together, the capillary electrophoresis systems that can efficiently sequence up to 41 runs components of this integrated system can analyze multiple samples simulta- (656 samples) in a 24-hour period, simplify the rate-limiting steps found neously. Samples are sequenced in 35 generating high-quality, high-resolu- in current resequencing protocols, tion data with minimal hands-on time. minutes on the 16-capillary 3130xl and offer a complete, cost-effective system, or on the 4-capillary 3130 sys- solution for laboratories performing The 3130 Series Systems are designed tem with 3130 POP-7™ Polymer and either large or small resequencing for ease-of-use to maximize laboratory the 36 cm capillary array. After the studies. It also revolutionizes DNA productivity while reducing the overall sequence data are collected, the KB™ www.appliedbiosystems.com the study of human genes and other target regions. The goal is to discover variants within genes that may corre- late to disease development and response to drug treatment. Experienced researchers identify primer design, validation, and downstream data analysis as the major bottlenecks in studies involving the resequencing of many genes. Researchers often spend weeks producing effective primers for both PCR and sequencing, especially when working with regions of high genomic complexity. The Applied Biosystems VariantSEQr™ Resequencing System, along with Figure 2. VariantSEQr Resequencing System includes ready-to-pipette M13-tailed PCR primers and universal protocols for PCR and sequencing, as well as project templates that incorporate relevant content and gene reference information. the 3130 Series Systems and SeqScape software, not only minimizes these bottlenecks but also eliminates the primer design and application validation cost per sample. Now more than ever, electrophoresis times than any other processes. The proven technology of researchers have the flexibility to choose capillary electrophoresis system this entire system provides simplified, one configuration for all their rese- available in the market today. robust protocols along with automatic quencing needs. The Automated The VariantSEQr Resequencing data analysis and reporting (Figure 3). Polymer Delivery System in the 3130 System for Low to Medium Series Systems allows automatic Throughput Advanced Design Pipeline polymer loading, which minimizes The Applied Biosystems VariantSEQr Applied Biosystems has developed an hands-on time and maintenance while Resequencing System consists of pre- advanced design pipeline that generates maximizing performance. The systems designed resequencing sets, streamlined resequencing primers for optimal enable the use of 3130 POP-7 Polymer, protocols, and a Project Template on performance. The pipeline also assigns not only for the 36 cm capillary array, a CD that contains the reference a confidence value to each primer but also for the 50 cm and 80 cm capil- sequence for integrated data analysis pair and annotates associated genome lary arrays. Run configurations, specific with SeqScape Software v2.5 (Figure 2). complexity and technical challenges. for the 3130 POP-7 Polymer, incorpo- This system is designed to work Gene coverage includes exons (coding rate a higher temperature through the seamlessly with Applied Biosystems or non-coding), splice junctions, and detection cell heater, which yields instruments, reagents, and software for regulatory regions (Figure 4). less run-to-run variability and faster PCR using Resolution of Automatic Genomic DNA Sequencing AmpliTaq Gold® sequencing reac- analysis of variants Genotype report plus using BigDye® PCR Master Mix tions on 3130 or using SeqScape® generation validated PCR Terminators and PCR 3130xl platforms Software v2.5 Figure 3. Workflow of the Applied Biosystems VariantSEQr™ Resequencing System illustrates intergration of the 3130 Series Systems and SeqScape® Software v2.5. The system has been designed to fit the workflow of a typical resequencing laboratory. An optimized protocol provides complete integration of each step, from PCR amplification using PCR primers validated by a combination of laboratory and computational systems, to the generation of genotype reports. An important new feature is the integration of the 3130 Series System and Data Collection Software v3.0 with SeqScape software. At the end of each run, the integration feature allows the sequence files not only to be automatically basecalled, but also trimmed, aligned, and assembled against a reference sequence within SeqScape software. Data analysis results and reports generated can be reviewed either locally or exported to separate desktops convenient to the scientists. www.appliedbiosystems.com Exons Intron/Exon Regulatory Region Junction RSA RSS Figure 4. Diagrammatic view of primers designed for a typical Resequencing Set (RSS). The blue regions at the top represent exons, and the lighter green regions represent either the promoter region or introns. The target region of the Resequencing Amplicons (RSA, shown in orange) have been designed to provide complete coverage of promoter regions, intron/exon junctions, and all exons. The regions flanking the target regions (represented in red) are part of the amplicons but may not always contain data of the desired quality. Each PCR primer is tailed with priming sites for either the M13 universal forward or reverse sequencing primer to permit robust and specific sequencing of the amplified regions. While a number of software packages have the ability to design primers for amplications, it has not been previously possible to know if sequences in the genome will interfere with the genera- tion of the high-quality sequence data necessary for resequencing projects. This Thermal Cycler uncertainty led us to develop a way to reliably predict resequencing amplicon VariantSEQr performance without the need to test every resequencing set in the laboratory. SeqScape® Software v2.5 SeqScape® Software v2.5 is a sequenc- BigDye Chemistry ing analysis software package designed expressly for resequencing applications (Figure 5). SeqScape software enables researchers to compare their sequence data to a reference sequence to identify variants. Each consensus sequence can 3130 Instrument be compared to a library of sequences to identify top matches. Unique to SeqScape software is the sequence fea- ture, which incorporates into the reference sequence information from SeqScape Software the sequence feature, such as exons, Figure 5. Applied Biosystems SeqScape® Software v2.5, designed expressly for mutation profiling, features robust algorithms, enhanced display capabilities, and detailed results reports. With a single mouse click, the software feeds the sequencing files into the analysis pipeline and generates detailed reports containing mutation informa- tion. Essential to the analysis pipeline, robust algorithms are integrated to ensure automated processing and accurate results from raw sequence data to the final mutation report. Unique to this tool are base-calling and con- sensus calling algorithms that provide quality values for each base pair, sample, and mutation to enable easy distinction between poor and high-quality data. Enhanced display capabilities, such as hyperlinks between
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