Abstract
Only a small fraction of large genomes such as that of the human contains the functional regions such as the exons, promoters, and polyA sites. A platform technique for selective enrichment of functional genomic regions will enable several next-generation sequencing applications that include the discovery of causal mutations for disease and drug response. Here, we describe a powerful platform technique, termed “functional genomic fingerprinting” (FGF), for the multiplexed genomewide isolation and analysis of targeted regions such as the exome, promoterome, or exon splice enhancers. The technique employs a fixed part of a uniquely designed Fixed-Randomized primer, while the randomized part contains all the possible sequence permutations. The Fixed-Randomized primers bind with full sequence complementarity at multiple sites where the fixed sequence (such as the splice signals) occurs within the genome, and multiplex amplify many regions bounded by the fixed sequences (e.g., exons). Notably, validation of this technique using cardiac myosin binding protein-C (MYBPC3) gene as an example strongly supports the application and efficacy of this method. Further, assisted by genomewide computational analyses of such sequences, the FGF technique may provide a unique platform for high-throughput sample production and analysis of targeted genomic regions by the next-generation sequencing techniques, with powerful applications in discovering disease and drug response genes.
Highlights
The large scale sequencing of thousands of genomes from disease cohorts by the generation sequencing (NGS) techniques are expected to uncover causal gene mutations [1,2,3,4,5]
The scalable technique we describe in this report, termed Functional Genomic Fingerprinting (FGF), is capable of selectively amplifying and screening any genomic functional regions bounded by short repeated sequences such as the exons that are bordered by splice signals, or regions surrounding the regulatory elements within a genome
The R part contains all possible sequence combinations, allowing for complete complementary base pairing over the entire length of the FR primers at multiple locations wherever the fixed sequence occurs in a template DNA
Summary
The large scale sequencing of thousands of genomes from disease cohorts by the generation sequencing (NGS) techniques are expected to uncover causal gene mutations [1,2,3,4,5]. The scalable technique we describe in this report, termed Functional Genomic Fingerprinting (FGF), is capable of selectively amplifying and screening any genomic functional regions bounded by short repeated sequences such as the exons that are bordered by splice signals, or regions surrounding the regulatory elements (e.g., promoters and polyA sites) within a genome. Automation of FGF method might be a powerful target enrichment strategy for selective genomic functional regions, which may have potential applications in medical, agricultural and biotechnology fields. If this technique is demonstrated, it should be currently feasible to scale it to sequence selective functional regions of the genome, such as the exome or promoterome, on NGS platforms, and compare them between cohorts of 1000 patient versus 1000 normal individuals, enabling the identification of causal genes. As a targeted polymerase chain reaction (PCR) based technique that largely avoids nonspecificity, FGF may be a viable and cost-effective alternative to array and solution based hybridization enrichment technologies
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