Targeted haplotyping in pharmacogenomics using Oxford Nanopore Technologies’ adaptive sampling

Deserranno, Koen; Tilleman, Laurentijn; Rubben, Kaat; Deforce, Dieter; Van Nieuwerburgh, Filip

doi:10.3389/fphar.2023.1286764

Cited by 3 publications

(1 citation statement)

References 52 publications

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“…Using R10.4 flow cell, the simplex sequencing mode demonstrates an average read quality of 98%, while duplex reads exhibit 99% accuracy 58 . Moreover, by implementing the latest base-calling algorithm (dorado) for processing data produced from the flow cell (R10.4.1) together with Q20+ reagent, this combination claims an overall 99% reads accuracy 60, 61 .…”

Section: Introductionmentioning

confidence: 99%

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing

Su,

Chandradoss,

Malachowski

et al. 2024

Preprint

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More than 60 human disorders have been linked to unstable expansion of short tandem repeat (STR) tracts. STR length and the extent of DNA methylation is linked to disease pathology and can be mosaic in a cell type-specific manner in several repeat expansion disorders. Mosaic phenomenon have been difficult to study to date due to technical bias intrinsic to repeat sequences and the need for multi-modal measurements at single-allele resolution. Nanopore long-read sequencing accurately measures STR length and DNA methylation in the same single molecule but is cost prohibitive for studies assessing a target locus across multiple experimental conditions or patient samples. Here, we describe MASTR-seq, Multiplexed Analysis of Short Tandem Repeats, for cost-effective, high-throughput, accurate, multi-modal measurements of DNA methylation and STR genotype at single-allele resolution. MASTR-seq couples long-read sequencing, Cas9-mediated target enrichment, and PCR-free multiplexed barcoding to achieve a >ten-fold increase in on-target read mapping for 8-12 pooled samples in a single MinION flow cell. We provide a detailed experimental protocol and computational tools and present evidence that MASTR-seq quantifies tract length and DNA methylation status for CGG and CAG STR loci in normal-length and mutation-length human cell lines. The MASTR-seq protocol takes approximately eight days for experiments and one additional day for data processing and analyses.

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Section: Introductionmentioning

confidence: 99%

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing

Su,

Chandradoss,

Malachowski

et al. 2024

Preprint

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Haplotype Inference Using Long-Read Nanopore Sequencing: Application to GSTA1 Promoter

Mlakar,

Dupanloup,

Gloor

et al. 2024

Mol Biotechnol

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Recovering true haplotypes can have important clinical consequences. The laboratory process is difficult and is, therefore, most often done through inference. In this paper, we show that when using the Oxford nanopore sequencing technology, we could recover the true haplotypes of the GSTA1 promoter region. Eight LCL cell lines with potentially ambiguous haplotypes were used to characterize the efficacy of Oxford nanopore sequencing to phase the correct GSTA1 promoter haplotypes. The results were compared to Sanger sequencing and inferred haplotypes in the 1000 genomes project. The average read length was 813 bp out of a total PCR length of 1336 bp. The best coverage of sequencing was in the middle of the PCR product and decreased to 50% at the PCR ends. SNPs separated by less than 200 bp showed > 90% of correct haplotypes, while at the distance of 1089 bp, this proportion still exceeded 58%. The number of cycles influences the generation of hybrid haplotypes but not extension or annealing time. The results demonstrate that this long sequencing reads methodology, can accurately determine the haplotypes without the need for inference. The technology proved to be robust but the success of phasing nonetheless depends on the distances and frequencies of SNPs.

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Cas9-targeted-based long-read sequencing for genetic screening of RPE65 locus

Rodilla,

Núñez-Moreno,

Benitez

et al. 2024

Front. Genet.

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IntroductionLong-read sequencing (LRS) enables accurate structural variant detection and variant phasing. When a molecular diagnosis is suspected, target enrichment can reduce the cost and duration of sequencing.MethodsLRS was conducted in five inherited retinal dystrophy (IRD) patients harboring a monoallelic variant in RPE65 that remained uncharacterized after clinical exome sequencing (CES). CRISPR-Cas9 guide RNA probes were designed to target a 31 kb region, including the entire RPE65 locus. The DNA was sequenced on a MinION platform. Short-read ×30 whole-genome sequencing (WGS) was performed for five patients to validate nanopore results.ResultsThe nanopore sequencing process yielded a median of 271 reads within the targeted region, with a mean depth of 109 and a median read size of 8 kb. All variants identified by CES have been detected using this approach, and no additional RPE65 gene causative variants were found. Nanopore variant detection demonstrated performance akin to short-read WGS at similar coverage levels, although exhibiting increased false positive calls at lower coverage.DiscussionIn this study, we explore the advantages of using a targeted approach together with long-read sequencing to identify variants associated with IRD. The results underscore the utility of targeted long reads for characterizing patients affected by rare diseases when first-tier diagnostic tests are non-conclusive.

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Targeted haplotyping in pharmacogenomics using Oxford Nanopore Technologies’ adaptive sampling

Cited by 3 publications

References 52 publications

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing

Haplotype Inference Using Long-Read Nanopore Sequencing: Application to GSTA1 Promoter

Cas9-targeted-based long-read sequencing for genetic screening of RPE65 locus

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