Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10

Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)(n) or a (ATCCT)(n)/(ATCCC)(n) interruption within the (ATTCT)(n) repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)(n) may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies.