Targeted next-generation sequencing by specific capture of multiple genomic loci using low-volume microfluidic DNA arrays

Abstract

We report a flexible method for selective capture of sequence fragments from complex, eukaryotic genome libraries for next-generation sequencing based on hybridization to DNA microarrays. Using microfluidic array architecture and integrated hardware, the process is amenable to complete automation and does not introduce amplification steps into the standard library preparation workflow, thereby avoiding bias of sequence distribution and fragment lengths. We captured a discontiguous human genomic target region of 185 kb using a tiling design with 50mer probes. Analysis by high-throughput sequencing using an Illumina/Solexa 1G Genome Analyzer revealed 2150-fold enrichment with mean per base coverage between 4.6 and 107.5-fold for the individual target regions. This method represents a flexible and cost-effective approach for large-scale resequencing of complex genomes.

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing

“…The average depth of coverage was between 2.9-and 5.0-fold for all target regions for both experiments (Table 1). Overall, the data suggest similar or better reproducibility than previously reported for microarray-based sequence capture Hodges et al 2007;Okou et al 2007;Bau et al 2009). Importantly, analysis of the uniqueness of obtained read pairs revealed that more than 98% for both runs, were unique, which is higher than previously reported for standard Illumina GAII sequencing without any enrichment method (Quail et al 2008).…”

“…Recently, sequence enrichment using solid-phase hybridization to DNA microarrays with flexible content has been described Hodges et al 2007;Okou et al 2007;Bau et al 2009). For several projects targeting different regions, enrichment factors of several hundred-to a 1000-fold have been reported, resulting in good depth of coverage for at least a fraction of the target region.…”

Evaluation of Oligonucleotide Sequence Capture Arrays and Comparison of Next-Generation Sequencing Platforms for Use in Molecular Diagnostics

“…Among these methods are Agilent's SureSelect in-solution hybrid capture method (8 ), RainDance Technologies' microdroplet PCR-based enrichment (9 ), and Febit's microfluidicsbased HybSelect technology (10 ). The Agilent and Febit methodologies for sequence capture are similar to NimbleGen arrays in that they rely on hybrid selection, whereas the RainDance methodology is a highly multiplexed PCR reaction.…”

DNA Sequence Capture and Enrichment by Microarray Followed by Next-Generation Sequencing for Targeted Resequencing: Neurofibromatosis Type 1 Gene as a Model

“…Currently, PCR-based methods are the most widely used for preparing a genomic sample for sequencing purposes (11)(12)(13)(14); however, PCR methods fall short of meeting the technological needs for sequencing highly complex genomic regions and in large-scale genomic studies. PCR methods cannot amplify very long sequences reliably, and multiplex enrichment of thousands of sequences is difficult (14 ). Several technologies have been developed for targeted enrichment by sequence capture, including solid phase-based microarrays and solution phase-based methods (15)(16)(17)(18).…”

“…Owing to the limited availability of the SureSelect™ system (Agilent Technologies) and the complexity of synthesizing padlock probes, we used the microarray method in this study (16 ). This recently developed technology selects targeted sequences by hybridization to an oligonucleotide microarray and demonstrates great potential for the efficient enrichment of specific, large high-complexity genomic regions of interest (14,15,19,20 ). Combining this technology with NGS produces a powerful sequencing tool that has the potential to be implemented in a clinical diagnostics laboratory.…”