C08 Determination of huntington’s disease haplotype by single molecule, nanopore sequencing

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Abstract

Background

Huntington’s disease (HD) is characterized by a pathogenic expansion of the (CAG)n repeat in the Huntingtin (HTT) gene. Genetic epidemiology studies have shown the occurrence of different HTT haplotypes with varying prevalence across the world. Currently, there exists no reliable prevalence estimates for the Indian population. Conventional phasing of SNP genotypes with disease allele (expanded CAG triplet repeat) in Huntington’s disease for haplotype classification is carried out using parental genotypes. In the absence of parental genotype availability, patient DNA can be studied if long read sequencing of the entire HTT region is feasible. Nanopore sequencing technology enables sequencing of long reads including stretches of triplet repeats.

Aim

To determine the genetic background of Huntington’s disease subjects by phasing SNP genotypes with disease allele (expanded CAG triplet repeat) using Nanopore sequencing technology.

Methods and Results

Six DNA samples with varying CAG repeat length were selected. A 7 kb product of interest encompassing the HTT gene was enriched with specific primers. The sequencing library was prepared by 1D Native barcoding genomic DNA technique, followed by sequencing on MinION. Basecalled reads were subjected to Burrows-Wheeler Aligner (BWA) against chromosome 4 (NC_000004.12) and aligned reads were assembled using CANU assembler. Preliminary results showed 50% of reads mapping to Chr 4 in one of the samples, indicating enrichment of the desired region. Post assembly, the SNPs in the assembled sequence will be phased to the pathogenic allele. The results of the analysis will be presented and discussed.

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