Targeted Resequencing

Open Slide Format and Barcoding Capability

Resequencing experiments are fundamental in basic and clinical research as scientists look for causative mutations within populations. Targeted resequencing involves the comparative analysis of candidate genes or regions and requires a high level of accuracy to identify low frequency SNPs and structural variants. Traditional capillary electrophoresis methods provide the highest accuracy and are best suited for analyzing a limited set of amplicons in a large number of patient samples. But this method becomes expensive and labor intensive when you need to analyze a large number of genes.

SOLiD™ System: Ideal for Large-Scale Resequencing

The SOLiD™ System offers the combination of throughput and accuracy necessary for assaying large numbers of candidate regions or when resequencing pooled or heterogeneous samples. The accuracy, throughput, and versatility of the SOLiD System is uniquely suited for large-scale resequencing experiments.

The system generates 100 Gb of mappable data in a single run, providing the coverage and statistical resolving power needed to correctly identify low frequency SNPs in a large number of amplicons.
The combination of 2 base encoding and dual interrogation of each base results in a system accuracy of greater than 99.94%, and a consensus accuracy at 15x coverage of 99.999%.
The open slide format and barcoding capability allows for multiplexing of up to 1,536 samples per run.

The TargetSeq™ Exome Enrichment System Provides Efficient Targeted Sequencing

Targeted—focused, high-density probe design
Economical—precapture sample multiplexing
Platform-optimized—superior performance on 5500 Series Genetic Analyzers and SOLiD® Systems
Integrated—streamlined data analysis with LifeScope™ Genomic Analysis Software

Exome sequencing is best performed using the TargetSeq™ Exome Enrichment System kits, which are based on an in-solution hybridization method for exome capture. The kits are optimally designed to seamlessly integrate into the 5500 Series Genetic Analysis Systems workflow.

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Products

Experimental Workflow: Find the Products You Need for Every Step

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Step 3: Construct DNA Library

You can prepare both fragment libraries and mate-paired libraries for targeted sequencing. Using mate-paired libraries enables you to detect structural rearrangementsas well as SNPs.

Products for Step 3: Construct DNA Library

SOLiD™ Fragment Library Oligos Kit: A set of oligonucleotide adaptors and primers for constructing fragment libraries. The kit contains P1 and P2 adaptors (double-stranded DNA) that are ready for ligation to library DNA and PCR primers for library amplification.

SOLiD™ Fragment Library Construction Kit with SizeSelect™ Gels: This kit provides a fully optimized complete set of reagents, columns, and our new SizeSelect™ gels to generate 10 fragment libraries using the SOLiD™ System standard fragment library protocol.

SOLiD™ Fragment Library Barcoding Kits: The SOLiD™ Fragment Library Barcoding Kits provides a complete set of 96 unique barcode sequences for the SOLiD™ 4 System for cost-effective next generation sequencing of fragment or paired-end libraries. When used in combination with the SOLiD™ Fragment Library Construction Kit, the SOLiD™ Fragment Library Barcoding Kits enables users to pool up to 96 fragment libraries prior to emulsion PCR and then conduct multiplexed sequencing analysis, simplifying the next generation sequencing workflow.

View more reagents, kits and bench-top devices on invitrogen.com

SOLiD™ Library TaqMan® Quantitation Kit

The SOLiD™ Library TaqMan® Quantitation Kit provides a complete qPCR solution to determine the appropriate amount of library to input into emulsion PCR for all library types.

E-Gel® SizeSelect™ Gels

Allows you to separate and recover DNA for short read fragment library construction in just 15 minutes.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.

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User Guide: SOLiD™ 4 System Library Preparation Quick Reference Card (PN 4445674B)

Step 4: Amplify & Sequence

After you construct your libraries, amplify the DNA using standarad emulsion PCR. Next, deposit the beads on a slide for sequencing on the SOLiD™ Analyzer.

Products for Step 4: Amplify & Sequence

Sequencing

SOLiD™ 4 System: The Applied Biosystems SOLiD™ 4 System is a revolutionary genetic analysis platform that enables massively parallel sequencing of clonally-amplified DNA fragments linked to beads. The sequencing methodology is based on sequential ligation with dye-labeled oligonucleotides. The SOLiD™ 4 System enables researchers to obtain higher-quality genomes at lower cost without the purchase of a new instrument.

Mate-Paired Library Sequencing Reagents for SOLiD™ 4 System, Main Kits: These kits contain sequencing master mixes in an easy-to-use strip tube format for ligation sequencing of mate-paired libraries on the SOLiD™ 4 System. Reagent mixing is not required, minimizing tube mix ups and providing a convenient automated method for sequencing reagent handling.

Fragment Library Sequencing Reagents for SOLiD™ 4 System, Main Kits: These kits contain sequencing master mixes in an easy-to-use strip tube format for ligation sequencing of fragment libraries on the SOLiD™ 4 System. Reagent mixing is not required, minimizing tube mix ups and providing a convenient automated method for sequencing reagent handling.

Instrument Buffers for SOLiD™ 4 System: This kit contains a set of buffers needed for ligation sequencing on the SOLiD™ 4 System and is sufficient for 110 cycles. The product is compatible only with the SOLiD™ 4 System. Store components at ambient, 2 °C to 8 °C, and at -15 °C to -25 °C.

Workflow Analysis Reagents for SOLID™ 4 System: This kit is for accurate qualification and quantitation of templated beads prior to your sequencing run. Use this kit to perform a workflow analysis (WFA) run on the SOLiD™ 4 Analyzer.

Emulsion PCR (ePCR)

SOLiD™ EZ Bead™ System: The new SOLiD™ EZ Bead™ System simplifies next-generation sequencing workflows by providing an automated solution for reproducible templated bead preparation with less than 1 hour of hands-on time. The system is specifically designed with system modularity and quality control check points for greater experimental control. The SOLiD™ EZ Bead™ System streamlines the sequencing workflow through significant time savings and support for up to 96 multiplexed libraries. With automation, minimal costs, and seamless effort, the SOLiD™ EZ Bead™ System dramatically increases sequencing productivity to further accelerate scientific discovery.

SOLiD™ EZ Bead™ System Reagents and Accessory Kits: The new SOLiD™ EZ Bead™ System simplifies next-generation sequencing workflows by providing an automated solution for reproducible templated bead preparation with less than 1 hour of hands-on time. The system is specifically designed with system modularity and quality control check points for greater experimental control. SOLiD™ EZ Bead™ System streamlines the sequencing workflow through significant time savings and support for up to 96 multiplexed libraries.

SOLiD™ EZ Bead™ Emulsifier Oil Pack Kit

Bead Deposition

SOLiD™ Bead Deposition Kits: These kits contains components for depositing templated beads onto a slide for ligation sequencing on the SOLiD™ Analyzer. The kits contain reagents for performing a 3’ end modification and buffers. These kits are available in a 10 full-scale reaction size and a 20 mini-reaction size.

SOLiD™ Bead Enrichment Kits: These kits contain components to enrich templated beads amplified by emulsion PCR. The kit includes enrichment beads and reagents. These kits are available in a 10 full-scale reaction size and a 20 mini-reaction size.

SOLiD™ Slide Kits: These kits contain activated slides and storage buffer for ligation sequencing on the SOLiD™ Analyzer.

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Step 5: Analyze Data

Using the data analysis tools listed below:

Align reads to reference sequence
Identify SNPs
Translate to base space
Visualize annotated data

Products for Step 5: Analyze Data

Data Analysis Tools

The tools you need for each step in the targeted sequencing workflow:


Data Analysis Step	Applied Biosystems Tools	Applied Biosystems Software	3rd-Party Software
1. Align reads to reference in color space	Resequencing Analysis Pipeline	SETS* SOLiD BioScope**	SHRiMP BFAST SOCS Geospiza Genomatix
2. Generate quality metrics and perform mate pair rescue	Resequencing Analysis Pipeline	SOLiD BioScope**
3. Generate sequencing and alignment statistics	Resequencing Analysis Pipeline	SETS*
4. Identify Polymorphisms	Resequencing Analysis Pipeline Small Indel Inversion Tool Large Indel CNV SNP Tool	SOLiD BioScope**	CNV-Seq Genomatix Partek
5. Translate color space to base space	GFF Conversion Tool Base QV Tool SAM Conversion Tool	SOLiD BioScope**
6. Visualize in context of annotation		SAB	Biotique Genomatix GenomeQuest NextGENe Partek UCSC Browser
7. Convert to SRF for publishing	SRF Conversion Tools

*on-instrument **off-instrument

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Publications & Literature
Application Note
Posters
Publications
- Targeted resequencing of candidate genes using selector probes Publication: Nucleic Acids Research (2010) Authors: Johansson, et al.
- De novo mutations of SETBP1 cause Schinzel-Giedion syndrome. Publication: Nature Genetics, May 2010 Authors: Hoischen, et al.
- PHF6 mutations in T-cell acute lymphoblastic leukemia. Publication: Nature Genetics 42, 338-342 (2010) Authors: Van Vlierberghe, et al.
- Accurate SNP and mutation detection by targeted custom microarray-based genomic enrichmentof short-fragment sequencing libraries. Publication: Nucleic Acids Research, 2010, 1–9 Authors: Mokry, et al.
- Comparison of Three Targeted Enrichment Strategies on the SOLiD Sequencing Platform Publication: Plos One (2011) Authors: Hedges D.J. et al.
- Population genetics in non- model organisms: II. Natural selection in marginal habitats revealed by deep sequencing on dual platforms Publication: Molecular Biology and Evolution (2011) Authors: Zhou R. et al.
- De novo nonsense mutations in ASXL1 cause Bohring-Opitz syndrome Publication: Nature Genetics (2011) Authors: Hoischen A. et al
- Integrated genomic analyses of ovarian carcinoma Publication: Nature (2011) Authors: Cancer Genome Atlas Research Network

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Targeted Resequencing

SOLiD™ System: Ideal for Large-Scale Resequencing

The TargetSeq™ Exome Enrichment System Provides Efficient Targeted Sequencing