High-throughput re-sequencing to identify rare allelic variants

Recent advances on genomic studies allow to re-sequence genes with high accuracy and in high throughput procedure. Rare allelic variants are important to be analyzed because are often the molecular basis of human disease. Indeed, numerous syndromes are associated to mutations in rare allele, but a lot of work have to be done yet.

A new protocol has been published to apply an array to high throughput re-sequencing. Two problems were encountered at the beginning of the study: the upstream target preparation techniques available until now were not able to produce thousands of samples simultaneously and the accuracy was too low to distinguish rare variant to false positives. Scientists from Genentech and Stanford Genome Technology Center developed a method for target amplification by capture and ligation (TACL) based of novel probes for genomic DNA that are amplified by PCR, incorporate deoxyuridine and are purified. TACL method provided high reproducibility ad specificity in terms of capturing of the target regions also when the starting sample concentration was lower than 15 nanograms. Then, they cloned TACL probes into bacteria and used them to hybridize to probes previously captured. The bacterial growth in selective media allowed to recognize where the mismatched was preset, thus enriching the rare alleles. This technique seems promising and user-friendly because doesn’t need of particular instrumentation to be successfully employed.