Genetics
A new nanopore sequencing method resolves the notoriously complex LPA KIV-2 repeat region with near-ddPCR accuracy across five ancestries (Genome Med 2024)
Original title: Nanopore sequencing with unique molecular identifiers enables accurate mutation analysis and haplotyping in the complex lipoprotein(a) KIV-2 VNTR
The LPA kringle IV type-2 (KIV-2) region is a repetitive, 1-40-copy, 5.6 kb-per-unit genomic segment that is the primary regulator of Lp(a) concentration but has long resisted accurate sequencing due to its intricate structure, multiple haplotypes, internal homologies, and an embedded short tandem repeat. The authors developed UMI-ONT-Seq, an amplicon-based nanopore sequencing method using unique molecular identifiers, and validated it on 15 human samples plus low-level plasmid mixtures (0.5% to 5%), achieving high sensitivity, specificity and precision for SNP detection (0.977, 1.000, 0.993 respectively) and full-length haplotype retrieval, with results highly correlated with next-generation sequencing (R-squared 0.983). KIV-2 copy number determined by the method agreed closely with droplet digital PCR, with 70% of samples falling within the confidence interval of ddPCR itself. Applying the method to 48 multi-ancestry 1000 Genomes samples, the authors characterised 62,679 intra-KIV-2 short tandem repeat sequences and mapped KIV-2 SNP haplotype patterns across five ancestries, providing a powerful new tool for large-scale, ancestry-inclusive study of this key Lp(a)-determining genomic region.
Original abstract
Background: Repetitive genome regions, such as variable number of tandem repeats (VNTR) or short tandem repeats (STR), are major constituents of the uncharted dark genome and evade conventional sequencing approaches. The protein-coding LPA kringle IV type-2 (KIV-2) VNTR (5.6 kb per unit, 1-40 units per allele) is a medically highly relevant example with a particularly intricate structure, multiple haplotypes, intragenic homologies, and an intra-VNTR STR. It is the primary regulator of plasma lipoprotein(a) [Lp(a)] concentrations, an important cardiovascular risk factor. Lp(a) concentrations vary widely between individuals and ancestries. Multiple variants and functional haplotypes in the LPA gene and especially in the KIV-2 VNTR strongly contribute to this variance.
Methods: We evaluated the performance of amplicon-based nanopore sequencing with unique molecular identifiers (UMI-ONT-Seq) for SNP detection, haplotype mapping, VNTR unit consensus sequence generation, and copy number estimation via coverage-corrected haplotypes quantification in the KIV-2 VNTR. We used 15 human samples and low-level mixtures (0.5 to 5%) of KIV-2 plasmids as a validation set. We then applied UMI-ONT-Seq to extract KIV-2 VNTR haplotypes in 48 multi-ancestry 1000 Genome samples and analyzed at scale a poorly characterized STR within the KIV-2 VNTR.
Results: UMI-ONT-Seq detected KIV-2 SNPs down to 1% variant level with high sensitivity, specificity, and precision (0.977 ± 0.018; 1.000 ± 0.0005; 0.993 ± 0.02) and accurately retrieved the full-length haplotype of each VNTR unit. Human variant levels were highly correlated with next-generation sequencing (R2 = 0.983) without bias across the whole variant level range. Six reads per UMI produced sequences of each KIV-2 unit with Q40 quality. The KIV-2 repeat number determined by coverage-corrected unique haplotype counting was in close agreement with droplet digital PCR (ddPCR), with 70% of the samples falling even within the narrow confidence interval of ddPCR. We then analyzed 62,679 intra-KIV-2 STR sequences and explored KIV-2 SNP haplotype patterns across five ancestries.
Conclusions: UMI-ONT-Seq accurately retrieves the SNP haplotype and precisely quantifies the VNTR copy number of each repeat unit of the complex KIV-2 VNTR region across multiple ancestries. This study utilizes the KIV-2 VNTR, presenting a novel and potent tool for comprehensive characterization of medically relevant complex genome regions at scale.
Summary written by lp-a.org from the published abstract; figures as published. Page updated 18 August 2026. Methods.