Experiences with array-based sequence capture; toward clinical applications

Abstract

Although sequencing of a human genome gradually becomes an option, zooming in on the region of interest remains attractive and cost saving. We performed array-based sequence capture using 385K Roche NimbleGen, Inc. arrays to zoom in on the protein-coding and immediate intron-flanking sequences of 112 genes, potentially involved in mental retardation and congenital malformation. Captured material was sequenced using Illumina technology. A data analysis pipeline was built that detects sequence variants, positions them in relation to the gene, checks for presence in databases (eg, db single-nucleotide polymorphism (SNP)) and predicts the potential consequences at the level of RNA splicing and protein translation. In the samples analyzed, all known variants were reliably detected, including pathogenic variants from control cases and SNPs derived from array experiments. Although overall coverage varied considerably, it was reproducible per region and facilitated the detection of large deletions and duplications (copy number variations), including a partial deletion in the B3GALTL gene from a patient sample. For ultimate diagnostic application, overall results need to be improved. Future arrays should contain probes from both DNA strands, and to obtain a more even coverage, one could add fewer probes from densely and more probes from sparsely covered regions.

High prevalence of mutations in LCAT in patients with low HDL cholesterol levels in the Netherlands: Identification and characterization of eight novel mutations

“…It is likely that in the advent of next generation sequencing and its application in patient diagnostics [Almomani et al, 2011] that future studies will unequivocally reveal to what extent LCAT mutations cause low HDL-c in patient and general population studies. In this study, we characterized nine novel missense mutations to study whether the respective amino acid changes had an effect on LCAT secretion and/or LCAT activity.…”

Targeted sequence capture and GS-FLX Titanium sequencing of 23 hypertrophic and dilated cardiomyopathy genes: implementation into diagnostics

“…A balanced representation of all targeted exons would reduce the average coverage needed to detect variants with high confidentiality, consequently lowering the false negative rate. Therefore, we have designed arrays with a more balanced coverage as has previously been proposed by others 47 48. The rebalanced capture array has been used to analyse nine HCM patients and 19 DCM patients.…”

Improved coverage and accuracy with strand-conserving sequence enrichment

“…Various methods have been developed to enable whole-exome sequencing and targeted-region sequencing. Early on, solid-state capture arrays were used, but these were expensive and had relatively complex protocols [3]. In-solution capture and PCR-based enrichment methods have reduced the cost and complexity of protocols considerably [4].…”