Abstract
We developed a targeted DNA sequencing method that is capable of detecting a comprehensive panel of DNA mutations including small DNA mutations and large DNA deletions with unknown/flexible boundaries. The method directly identifies the large DNA deletions (Cat-D) without relying on sequencing coverage to make the genotype calls. We performed the method to simultaneously detect 10 small DNA mutations in β-thalassemia and 2 large genomic deletions in α-thalassemia from 10 genomic DNA samples. Cat-D was performed on 8 genomic DNA samples in duplicate. The 18 Cat-D samples were combined in one sequencing run. In total, 216 genotype calls were made, and 215 of the genotype calls were accurate. No false negative genotype calls were made. One false positive genotype call was made on one target mutation in one experimental duplicate from a genomic DNA sample. In summary, Cat-D can be developed into a robust, high-throughput and cost-effective method suitable for population-based carrier screens.
Highlights
Deep sequencing technologies have made personal genome sequencing possible, the wide use of the technology on population-based carrier screens for genetic disorders is limited by the lack of a robust and cost-effective targeted sequencing method capable of detecting large DNA deletions
Compared with other available methods for targeted sequencing, padlock capture is more suitable for population-based carrier screens, as once synthesized, the padlock library can be regenerated by PCR, whereby microarrays or RNA baits used for target enrichment in other methods are expensive and non-reusable[4]
In contrast to the point mutations commonly seen in β-thalassemia[8,9], the common mutations found in α-thalassemia are a series of large DNA deletions (~3–40 kb)[10] (Supplementary Fig. S1)
Summary
Deep sequencing technologies have made personal genome sequencing possible, the wide use of the technology on population-based carrier screens for genetic disorders is limited by the lack of a robust and cost-effective targeted sequencing method capable of detecting large DNA deletions. The experimental techniques being used in clinical labs for detecting large DNA deletions in thalassemia[10], such as gap-PCR, are low throughput (one test for one patient sample) and not comprehensive (one test for one specific mutation) These techniques are only used for patient DNA diagnosis and are unsuitable for population-based carrier screens. Both methods require a suitable target enrichment step if they are to be used for population-based mutation carrier screens Both methods are not suitable for the clinical detection of small DNA mutations. The large genomic deletions observed in thalassemia represent a special type of mutation that is frequently observed in human genetic disorders but is difficult to detect using conventional sequencing approaches
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