Abstract
Molecular inversion probe (MIP)-based capture is a scalable and effective target-enrichment technology that can use synthetic single-stranded oligonucleotides as probes. Unlike the straightforward use of synthetic oligonucleotides for low-throughput target capture, high-throughput MIP capture has required laborious protocols to generate thousands of single-stranded probes from DNA microarray because of multiple enzymatic steps, gel purifications and extensive PCR amplifications. Here, we developed a simple and efficient microarray-based MIP preparation protocol using only one enzyme with double-stranded probes and improved target capture yields by designing probes with overlapping targets and unique barcodes. To test our strategy, we produced 11 510 microarray-based duplex MIPs (microDuMIPs) and captured 3554 exons of 228 genes in a HapMap genomic DNA sample (NA12878). Under our protocol, capture performance and precision of calling were compatible to conventional MIP capture methods, yet overlapping targets and unique barcodes allowed us to precisely genotype with as little as 50 ng of input genomic DNA without library preparation. microDuMIP method is simpler and cheaper, allowing broader applications and accurate target sequencing with a scalable number of targets.
Highlights
The advent of massively parallel sequencing, so-called nextgeneration sequencing (NGS), has enabled researchers to sequence the human genome in cost- and time-efficient manner [1]
After the Molecular inversion probe (MIP) assay was developed for multiplexed genotyping of single nucleotide polymorphisms (SNPs) [9,10,11,12], it has become widely applied to detect copy number variations (CNVs) [8], RNA editing [13], methylation profiles [14,15], germline mutations [16,17], somatic mutations [18] and genotypes in duplicated genes [19]
With our preparation protocol producing high quantity of probes, microDuMIPs can be added at a higher ratio; the captured products were saturated at 1:500 genomic DNA (gDNA):probe ratio (Figure 3A)
Summary
The advent of massively parallel sequencing, so-called nextgeneration sequencing (NGS), has enabled researchers to sequence the human genome in cost- and time-efficient manner [1]. Molecular inversion probe (MIP) capture, one of solutionphase captures, is using single-stranded oligonucleotides consisting of a backbone sequence flanked by annealing arms, which complement the genomic sequences next to the target [4]. Compared with other target-enrichment methods, MIP-based methods have several advantages. They show high target specificity, require less genomic DNA (gDNA), have scalable number of targets and do not require preprocessing steps such as DNA fragmentation prior to capture experiments [6,7,8]. After the MIP assay was developed for multiplexed genotyping of single nucleotide polymorphisms (SNPs) [9,10,11,12], it has become widely applied to detect copy number variations (CNVs) [8], RNA editing [13], methylation profiles [14,15], germline mutations [16,17], somatic mutations [18] and genotypes in duplicated genes [19]
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