Characterising tandem repeats in dementia case study

Tandem repeats (TRs) have been implicated in a number of diseases, including Huntington’s, frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Such repeats can cause disease through their length, sequence or base modifications; however, these features are often difficult, if not impossible, to assess with traditional analysis technologies¹. In addition, existing techniques only target a single tandem repeat locus at a time and have long turnaround times. Direct, long-read nanopore sequencing is capable of spanning large tandem repeats, revealing their size, nucleotide composition and the presence of base modifications. Furthermore, with the high-throughput, high-yield PromethION platform, this can be achieved at scale — allowing complete analysis of tandem repeats across multiple genomes.

To assess the facility of nanopore sequencing to fully resolve tandem repeats, researchers at VIB - University of Antwerp performed whole genome sequencing of 11 samples using the PromethION^1,2. Uniformly high yields were obtained for all samples, with a maximum yield per flow cell of 98 Gb* (30x genome coverage). The team focused on the analysis of a variable number tandem repeat (VNTR) within the ABCA7 gene which they had recently discovered results in a >4-fold increased risk of Alzheimer’s disease.

All ABCA7 VNTR alleles, including those containing up to 10,000 bp expansions, were spanned by the long sequencing reads; however, existing nucleotide sequence aligners struggled to accurately resolve the length of these long tandem repeats. To circumvent these challenges the team developed NanoSatellite³, a novel algorithm that utilises raw nanopore “squiggle” data rather than basecalled sequence to characterise the repeats. This method allowed the detection of all clinically-relevant repeat expansions and was shown to deliver read length estimations commensurate with Southern blotting — the current ‘goldstandard’ analysis technique (Figure 1). Importantly, NanoSatellite also allowed the identification of two VNTR alleles for two individuals that appeared to be homozygous when using Southern blotting, emphasising the increased sensitivity of this technique.

Highlighting the benefits of the PromethION platform, the researchers commented:

'Many tandem repeats in the human genome — some of which are currently uncharacterized — can be studied at once with a single sequencing run and somatic differences of unstable (expanded) TRs could be evaluated, which eventually will lead to the identification of novel disease-associated TRs and improved diagnostics’¹.

Figure 1: Tandem repeat lengths called from raw nanopore signal data using NanoSatellite or nucleotide sequence using tandem-genotypes repeat caller (basecalled using either Albacore, Scrappie events, and Scrappie raw) were compared. In all cases, NanoSatellite delivered higher concordance with Southern blotting (dotted line). Positive strand (red); negative strand (blue). TG = tandem-genotypes repeat caller. Image adapted from De Roeck et al.¹

The use of raw nanopore signal to characterise short tandem repeats has also been demonstrated by Gießelmann et al⁴. Employing a CRISPR-Cas12a enrichment approach, the team selectively targeted repeats within the C9orf72 gene (causative of frontotemporal dementia [FTD] and amyotrophic lateral sclerosis [ALS]) and the FMR1 gene (implicated in fragile X syndrome). The in-house developed nanoSTRique analysis tool allowed precise characterisation of repeat length, while the amplification free enrichment strategy further enabled analysis of epigenetic modifications — revealing significantly increased methylation at gene promoter CpG islands for the expanded alleles of both genes.

This case study is taken from the clinical white paper.