Abstract
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.
Highlights
Many human diseases are attributed to microsatellite repeat expansions
We demonstrate the use of a combination of repeatprimed PCR in conjunction with the high-resolution pulse field capillary electrophoresis analysis using the FEMTO Pulse Automated Pulsed-Field CE Instrument (Agilent, Santa Clara, CA) to successfully determine sequence content of large expansions of pentanucleotide repeats in spinocerebellar ataxia type 10 (SCA10) patients
We demonstrated that the RP-FEMTO provides a fast and economical analysis of interrupting repeat sequence structures in large expanded repeats of SCA10
Summary
The mutation of spinocerebellar ataxia type 10 (SCA10) is a complex ATTCT pentanucleotide repeat within intron. RP-PCR and Pulse Field capillary electrophoresis of large repeats in SCA10 benefits between academic investigators and PacBio investigators. PacBio provided support in the form of salaries for authors [TC and YCT], but did not have any additional role in the study design, decision to publish, or preparation of the manuscript. They performed No-Amp SMRT sequencing of the DNA samples that we provided and performed computational analyses of the data to generate circular consensus sequences of the repeat. The specific roles of these authors are articulated in the ‘author contributions’ section
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