Highly multiplexed molecular inversion probe genotyping: Over 10,000 targeted SNPs genotyped in a single tube assay

Abstract

Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of ∼90% with concordance rates >99.6% and completeness levels >98% for assays multiplexed up to 12,000plex levels. Furthermore, these individual metrics can be "traded off" and, by sacrificing a small fraction of the conversion rate, the accuracy can be increased to very high levels. No loss of performance is seen when scaling from 6,000plex to 12,000plex assays, strongly validating the ability of the technology to suppress cross-reactivity at high multiplex levels. The results of this study demonstrate the suitability of this technology for comprehensive association studies that use targeted SNPs in indirect linkage disequilibrium studies or that directly screen for causative mutations.

Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements

“…For example, the methylation status of 1534 CpG sites was assessed using a mixture of ∼6000 primers (Bibikova et al 2005). Another example is the use of highly multiplexed LMA with up to 20,000 Molecular Inversion Probes in a single reaction to detect single nucleotide polymorphisms (SNPs) (Hardenbol et al 2005;Wang et al 2005). When 5C is performed at a similar level of multiplexing, e.g., using 10,000 5C primers in a single experi- Table 7.…”

“…Inclusion of universal tails at the ends of 5C primers allows subsequent amplification of ligated primers. LMA-based approaches are quantitative and can be performed at high levels of multiplexing using thousands of primers in a single reaction (Fan et al 2004;Bibikova et al 2005;Hardenbol et al 2005;Wang et al 2005).…”

Introduction to Microarray‐Based Detection Methods

“…MIP technology has been shown to work well for multiplexing, i.e. massive parallel processing (12000 MIPs in the same reaction tube) [77]. The power and versatility of MIP technology makes it perfectly suited for the identification and quantification of microbes.…”

“…The power and versatility of MIP technology makes it perfectly suited for the identification and quantification of microbes. MIP s high sensitivity and specificity in detecting large numbers of SNPs [76,77] should allow us to harness this technology to detect a large number of pathogens and to identify multiple infections in an individual sample. A MIP is comprised of genomic recognition sequences, common amplification sequences and a molecular barcode for each genotype assigned to a specific gene.…”

High‐Resolution Analysis of Genomic Copy Number Changes

“…The 1 bp gap is filled; subsequent ligation seals the nick and generates a circular probe. Restriction digestion then releases the circularized probe and the resultant product is PCR-amplified using common primers [24]. The four nucleotide reactions are labeled in different colors and pooled.…”