Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing

Abstract

The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3-5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5-6 d for array-based enrichment and 3-4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.

Joubert syndrome: genotyping a Northern European patient cohort

“…Following SOLiD sequencing, color space reads were mapped against GRCH37/hg19 reference genome using a custom pipeline based on the BWA software (http://bio-bwa.sourceforge.net), and variants and small indels were annotated as described previously. 17 Average sample coverage was 147X and 136X, and 92 and 89% of requested sequences were covered by 420 reads for run 1 and 2 respectively. Run 2 had a higher fraction of duplicated reads compared with run 1, probably generated during PCR amplification steps post enrichment.…”

“…Fragment library preparation and genomic enrichment on a 1 M custom microarray (Agilent Technologies, Santa Clara, CA, USA) was performed as previously described. 17 Briefly, 2 μg of purified gDNA (Qiagen, Hilden, Germany) was sheared into 150 bp fragments with a Covaris S2 sonicator (Covaris, Woburn, MA, USA), then blunt-ended and 5′-phosphorylated, and finally shortened double-stranded adaptors complementary with the SOLiD next-generation sequencing platform were ligated to the ends. Next, the nonphosphorylated and nonligated 3′ ends were nick-translated, and bar-coded sequences unique to each sample were added using seven PCR cycles.…”

“…Amplified library fragments were pooled into two pools of 32 and 34 samples, respectively, and a region of 150-225 bp was excised from a 2% agarose gel. Following gel purification (Qiagen), samples were enriched using a custom protocol 17 and amplified in 13 PCR cycles with primers complementary to the full SOLiD oligo sequences. The enriched pool was then used for emulsion PCR following the manufacturer's instructions (Life Technologies, Carlsbad, CA, USA).…”

Preserved genetic diversity in organoids cultured from biopsies of human colorectal cancer metastases

“…Tumor cell percentage and percentage of necrosis were determined by a trained pathologist based on H&E staining. Six hundred nanograms of genomic DNA were required per sample to generate barcoded fragment libraries as described by Harakalova et al (11). Samples were enriched using SureSelect technology (Agilent Technologies) for the actionable cancer genome consisting of 1,977 genes, as previously described by Hoogstraat et al (12) and Vermaat et al (13).…”

Genomic breeding for food, environment and livelihoods

“…The cost, in time and money, of genetic sequencing has dropped precipitously in the last few years through combined innovations in chemistry, robotics and computing (Smith et al 2010;Harakalova et al 2011). Genomic sequencing is faster, cheaper and more reliable than ever before.…”