Massively parallel sequencing of ataxia genes after array-based enrichment

Abstract

Massively parallel sequencing has tremendous diagnostic potential but requires enriched templates for sequencing. Here we report the validation of an array-based sequence capture method in genetically heterogeneous disorders. The model disorder selected was AR ataxia, using five subjects with known mutations and two unaffected controls. The genomic sequences of seven disease genes, together with two control loci were targeted on a 2-Mb sequence-capture array. After enrichment, the patients' DNA samples were analyzed using one-quarter Roche GS FLX Titanium sequencing run, resulting in an average of 65 Mb of sequence data per patient. This was sufficient for an average 25-fold coverage/base in all targeted regions. Enrichment showed high specificity; on average, 80% of uniquely mapped reads were on target. Importantly, this approach enabled automated detection of deletions and hetero- and homozygous point mutations for 6/7 mutant alleles, and greater than 99% accuracy for known SNP variants. Our results also clearly show reduced coverage for sequences in repeat-rich regions, which significantly impacts the reliable detection of genomic variants. Based on these findings we recommend a minimal coverage of 15-fold for diagnostic implementation of this technology. We conclude that massive parallel sequencing of enriched samples enables personalized diagnosis of heterogeneous genetic disorders and qualifies for rapid diagnostic implementation.

Targeted next-generation sequencing detects point mutations, insertions, deletions and balanced chromosomal rearrangements as well as identifies novel leukemia-specific fusion genes in a single procedure

“…Overall, the sequencing output is in line with recently published data from a similar design strategy, in which a 2 Mb sequence capture array was used to study autosomal recessive ataxia. 37 In this study, an analysis pipeline was used to map the obtained reads both exactly against the human genome to detect point mutations, small deletions and insertions, but also to identify chimeric sequences mapping to different regions in the genome. Focusing on the first five patients, analyzed using the 1.9 Mb capture array for 95 gene targets, after stringent filtering a median of 247 exonic variants was observed.…”

GENETICS IN ENDOCRINOLOGY: The genetics of phaeochromocytoma: using clinical features to guide genetic testing

“…exon capture and targeted resequencing by second generation massively parallel sequencing) will reduce greatly the cost of genetic diagnosis and so allow comprehensive analysis for all (i.e. currently known and those yet to be identified) inherited phaeochromocytoma genes (60). Hence, clinicians should bear in mind that individuals who appear likely to have inherited disease but do not have a mutation in a currently known gene should have DNA banked to allow for future mutation analysis.…”

A compound heterozygous mutation in DPAGT1 results in a congenital disorder of glycosylation with a relatively mild phenotype

“…Next-generation sequencing and analysis was carried out, as described before. 11 In brief, exome enrichment was performed using the SureSelect Human All Exon 50 Mb Kit (Agilent Technologies, Santa Clara, CA, USA), covering B21 000 genes. The enriched exome library was equimolarly pooled in a set of four samples, including three other unrelated samples.…”