GLA gene variants were searched for in pooled DNA using massively parallel sequencing21 (link) or in individual genomic DNA samples using Illumina MySeq platform. Briefly, The SmartChip MyDesign TE system is designed to enrich multiple samples for multiple resequencing targets simultaneously using a 4-PCR-primer amplification strategy. The system consists of the SmartChip MyDesign Chips that contain 5184 nanowells. The separate, 100-nL reactions are then amplified using the Techne Prime Thermal Cycler, which is preprogrammed with the recommended thermal cycling program and equipped with an adapter plate for cycling 1 or 2 chips. After the PCR, pooled amplicons are collected from the chip(s) via centrifugation using single-use components supplied in the SmartChip TE Collection Kit. The library was then loaded on Miseq platform. Raw image files were processed by base calling Software for base calling with default parameters and the sequences of each individual were generated as 250 base pairs paired-end reads. Conventional Sanger sequencing was used to further analyze individual samples from selected patient DNA pools.
Protocol detail
Targeted Sequencing of GLA Gene Variants
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Corresponding Organization : Baylor Medical Center at Garland
Other organizations : Amicus Therapeutics (United States)
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Variable analysis
independent variables
- GLA gene variants
- DNA sequencing results
- Pooled DNA samples
- Individual genomic DNA samples
- 4-PCR-primer amplification strategy using SmartChip MyDesign TE system
- Thermal cycling program using Techne Prime Thermal Cycler
- Sanger sequencing for selected patient DNA pools
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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