Bioinformatický seminár

Tue 30 Nov. 2010, 17:20
I-9

Title: Raca et al. Next generation sequencing in research and diagnostics of ocular birth defects. Mol. Genet. Metab. 2010
Speaker: Dávid Porubský

Sequence capture enrichment (SCE) strategies and massively
parallel next generation sequencing (NGS) are expected to
increase the rate of gene discovery for genetically heterogeneous
hereditary diseases, but at present, there are very few examples
of successful application of these technologic advances in
translational research and clinical testing. Our study assessed
whether array based target enrichment followed by re-sequencing
on the Roche Genome Sequencer FLX (GS FLX) system could be used
for novel mutation identification in more than 1000 exons
representing 100 candidate genes for ocular birth defects, and as
a control, whether these methods could detect two known mutations
in the PAX2 gene. We assayed two samples with heterozygous
sequence changes in PAX2 that were previously identified by
conventional Sanger sequencing. These changes were a
c.527G>C (S176T) substitution and a single basepair deletion
c.77delG. The nucleotide substitution c.527G>C was easily
identified by NGS. A deletion of one base in a long polyG
stretch (c.77delG) was not registered initially by the GS
Reference Mapper, but was detected in repeated analysis using two
different software packages. Different approaches were evaluated
for distinguishing false positives (sequencing errors) and benign
polymorphisms from potentially pathogenic sequence changes that
require further follow-up. Although improvements will be
necessary in accuracy, speed, ease of data analysis and cost, our
study confirms that NGS can be used in research and diagnostic
settings to screen for mutations in hundreds of loci in
genetically heterogeneous human diseases.