Haplotyping Using Long-Range PCR and Nanopore Sequencing to Phase Variants: Lessons Learned From the ABCA4 Locus

Clinton, Benjamin Mc; Watson, Christopher M.; Crinnion, Laura A.; McKibbin, Martin; Ali, Manir; Inglehearn, Chris F.; Toomes, Carmel

doi:10.1016/j.labinv.2023.100160

Cited by 2 publications

(1 citation statement)

References 23 publications

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“…This has the benefit of requiring less input DNA and sequencing of only the specific PCR products (i.e., the amplified target gene). However, it requires prior knowledge of the location of the insert for designing primers, additional laboratory work, and may create challenges for the data analysis [51,52]. On the other hand, the drawback of targeted adaptive nanopore sequencing is the reduced performance of the flow cell over time.…”

Section: Discussionmentioning

confidence: 99%

Characterisation of a LINE-1 Insertion in the RP1 Gene by Targeted Adaptive Nanopore Sequencing in a Family with Retinitis Pigmentosa

Backlund,

Repo,

Kangas

et al. 2024

Human Mutation

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Retinitis pigmentosa (RP) is a group of inherited degenerative retinal disorders affecting more than 1.5 million people worldwide. For 30-50% of individuals with RP, the genetic cause remains unresolved by current clinical diagnostic gene panels. It is likely explained by variants in novel RP-associated genes or noncoding regulatory regions, or by complex genetic alterations such as large structural variants. Recent developments in long-read sequencing techniques have opened an opportunity for efficient analysis of complex genetic variants. We analysed a Finnish family with dominantly inherited RP affecting six individuals in three generations. Two affected individuals underwent a comprehensive clinical examination in combination with a clinical diagnostic gene panel, followed by whole exome sequencing in our laboratory. They exhibited typical signs of RP, yet initial sequence analysis found no causative variants. Reanalysis of the sequencing data detected a LINE-1 (L1) retrotransposon insertion of unknown size in exon 4 of the RP1 axonemal microtubule-associated (RP1) gene. The large chimeric L1 insertion that segregated with the disease was further characterised using targeted adaptive nanopore sequencing of RP1, allowing us to identify a 5.6 kb L1 transposable element insertion in RP1 as the cause of RP in this family with dominantly inherited RP.

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Section: Discussionmentioning

confidence: 99%

Characterisation of a LINE-1 Insertion in the RP1 Gene by Targeted Adaptive Nanopore Sequencing in a Family with Retinitis Pigmentosa

Backlund,

Repo,

Kangas

et al. 2024

Human Mutation

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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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Haplotyping Using Long-Range PCR and Nanopore Sequencing to Phase Variants: Lessons Learned From the ABCA4 Locus

Cited by 2 publications

References 23 publications

Characterisation of a LINE-1 Insertion in the RP1 Gene by Targeted Adaptive Nanopore Sequencing in a Family with Retinitis Pigmentosa

Characterisation of a LINE-1 Insertion in the RP1 Gene by Targeted Adaptive Nanopore Sequencing in a Family with Retinitis Pigmentosa

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

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