PCR Re-Sequencing
PCR Re-Sequencing using the AB 3730xl Platform
The AGRF provides a comprehensive PCR re-sequencing service, from assay design to analysis. PCR re-sequencing is commonly used for the identification of somatic mutations and genetic variability, such as SNPs. This is a large scale service with automated pipelines as shown below in the PCR re-sequencing Service Process.
This NATA accredited service is comprised of three main components:
PCR Assay Design & Validation
Through our integrated Bioinformatics section, the AGRF is able to provide primer design for the generation of amplimer targets for any gene/region of interest, for which valid accession numbers are provided. AGRF uses a robust optimization and validation process that produces amplicon success rates that average between 80-90%. All amplicons are PCR validated prior to full project screening.
Quality Amplification & Sequencing
PCR amplification reactions are set up in a dedicated amplicon free room using fully automated robotic platforms. All samples undergo DNA sequencing for either single-stranded or double stranded, depending on the client’s needs.
Analysis
AGRF Bioinformatics has several tools available for highly accurate mutation identification and SNP identification.
Our Bioinformatics team can provide various analysis & results files (see Results Formats), made available in electronic format via our secure website.
PCR re-Sequencing: Sample Submission Requirements
PCR Re-Sequencing AB 3730xl Service Process
Amplicon Re-Sequencing using the Roche GS FLX (454)
The AGRF provides a comprehensive Amplicon re-sequencing service utilising the Roche GS FLX platform. This platform supports “Ultra Deep Sequencing” of one or multiple target sequences of interest, allowing the process of detection, identification and quantification of low-level (≥5%) DNA variants in a population. Typical applications of Amplicon re-sequencing include the investigation of;
- correlations between specific mutations and disease
- diversity in clinical samples
- diversity in environmental samples
- population studies to find rare alleles
The GS FLX provides several options for DNA sample preparation for Amplicon re-sequencing.
General DNA Sample Preparation - The DNA sample preparation procedure for Amplicon re-sequencing consists of a simple PCR amplification reaction, but uses special Fusion Primers (sequences available upon request). The 3’-part of each primer is designed to anneal with a specific sequence on either side of the target of interest, on the DNA sample amplified. The amplicon provided should be no longer than 500 bp. This requires detailed knowledge of the target sequence, in particular the sites targeted by the primers. The 5’-part of the primers exists in two types, “A” and “B”, to match other components of the GS-FLX system. Click here to link to the Roche website outlining more technical information.
Amplicon Library Preparation – This feature of the GS FLX allows rapid amplicon library preparation, allowing for single molecule sequencing without cloning the target sequences into bacteria. The GS FLX provides reads of approximately 200-300 nucleotides in length, full coverage of amplicons up to 500bp can be obtained by reading an amplicon library. Click here to link in to the Roche website outlining more technical information.
Amplicon Re-Sequencing Roche GS FLX Service Process
