SNP Discovery and Resequencing

Beckman Coulter Genomics’ high throughput SNP Discovery and Resequencing pipeline has set Beckman Coulter Genomics apart as a premier service provider.  Now the same amplicon-based targeted resequencing approach for accurate polymorphism identification with Sanger sequencing can also be leveraged for rare SNP detection using Next Generation sequencing technologies.

Assay Design

Beckman Coulter Genomics uses fully-automated and optimized assay design software for gene-driven amplicon modeling. A unique automated assay validation process produces amplicon success rates that average >95%.

Validation

PCR^† and sequence-validation are available for amplicons prior to full project screening. Reactions that do not pass undergo further rounds of assay development using techniques such as alternative protocols, additives, nested reactions, and redesigned primer sets. Years of assay development experience allow the design of amplicons spanning genomic regions of varying degrees of sequence difficulty.

Quality Amplification and Sequencing

PCR amplification reactions are set up using fully-automated, high throughput robotic platforms. The Agencourt SPRI technology is used to purify PCR reactions resulting in pristine templates that have proven to reliably generate long, high quality Sanger reads. All samples undergo bi-directional DNA sequencing to increase quality and resolve homopolymers, insertions, deletions and microsatellites.

Flexible Analysis Packages

Proprietary enhancements of Beckman Coulter Genomics automated SNP calling software minimize both false positive and false negative rates. Beckman Coulter Genomics’ proprietary SNP Scoring Software allows rapid identification of:

• Intronic and exonic SNPs
• Amino acid changing SNPs
• dbSNP novelty of SNPs
• Patient genotypes

Beckman Coulter Genomics offers additional options for manual data review for annotation of Sanger SNP results. Basic manual analysis includes identification of insertion and deletion start sites, while advanced manual analysis also includes annotation of inserted and deleted sequences within each sample.

Proven Experience: SNP Detection in Cancer Research

Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.
Science. 2008 Sep 26;321(5897):1801-6.

An integrated genomic analysis of human glioblastoma multiforme.
Science. 2008 Sep 26;321(5897):1807-12.

The genomic landscapes of human breast and colorectal cancers.
Science. 2007 Nov 16;318(5853):1108-13.

The consensus coding sequences of human breast and colorectal cancers.
Science. 2006 Oct 13;314(5797):268-74.

Rare Mutation Detection with Next Generation Sequencing

The goal of a rare mutation detection project may be to detect a rare SNP in 5% of a population of samples or to detect a cancer causing SNP in a tumor biopsy sample largely contaminated by normal tissue. Ultra-deep sequencing of PCR amplicons for rare mutation detection is uniquely enabled by the clonal reads and massively parallel throughput of Next Generation sequencing. The read lengths and multiplexing capabilities of 454 Life Sciences* GS FLX Titanium sequencing particularly lend themselves to PCR product sequencing.

Typical ultra-deep, rare mutation detection projects are intricate multiplexes of amplicons and samples. Beckman Coulter Genomics has successfully coupled the highly successful, high-throughput upstream assay design and PCR amplification process to complex SNP detection projects with 454 GS FLX Titanium sequencing. It is critical in such projects to fully understand the associated research goal and experimental parameters to recommend appropriate multiplexing and sequence coverage to achieve the desired assay sensitivity. If you have a rare mutation detection sequencing project please contact your local Beckman Coulter Genomics representative to arrange an expert consultation.