Long-Read Nanopore Sequencing of RPGR ORF15 is Enhanced Following DNase I Treatment of MinION Flow Cells

Abstract

Introduction RPGR ORF15 is an exon present almost exclusively in the retinal transcript of RPGR . It is purine-rich, repetitive and notoriously hard to sequence, but is a hotspot for mutations causing X-linked retinitis pigmentosa. Methods Long-read nanopore sequencing on MinION and Flongle flow cells was used to sequence RPGR ORF15 in genomic DNA from patients with inherited retinal dystrophy. A flow cell wash kit was used on a MinION flow cell to increase yield. Findings were confirmed by PacBio SMRT long-read sequencing. Results We showed that long-read nanopore sequencing successfully reads through a 2 kb PCR-amplified fragment containing ORF15 . We generated reads of sufficient quality and cumulative read-depth to detect pathogenic RP-causing variants. However, we observed that this G-rich, repetitive DNA segment rapidly blocks the available pores, resulting in sequence yields less than 5% of the expected output. This limited the extent to which samples could be pooled, increasing cost. We tested the utility of a MinION wash kit containing DNase I to digest DNA fragments remaining on the flow cell, regenerating the pores. Use of the DNase I treatment allowed repeated re-loading, increasing the sequence reads obtained. Our customised workflow was used to screen pooled amplification products from previously unsolved inherited retinal disease (IRD) in patients, identifying two new cases with pathogenic ORF15 variants. Discussion We report the novel finding that long-read nanopore sequencing can read through RPGR-ORF15 , a DNA sequence not captured by short-read next-generation sequencing (NGS), but with a more reduced yield. Use of a flow cell wash kit containing DNase I unblocks the pores, allowing reloading of further library aliquots over a 72-h period, increasing yield. The workflow we describe provides a novel solution to the need for a rapid, robust, scalable, cost-effective ORF15 screening protocol. Supplementary Information The online version contains supplementary material available at 10.1007/s40291-023-00656-z.

PHYH c.678+5G>T Leads to In-Frame Exon Skipping and Is Associated With Attenuated Refsum Disease

“…Furthermore, this report constitutes an additional case in which long-read sequencing has provided key information to characterize a complex variant. Long-read or third-generation sequencing methods such as Pacific Biosciences (PacBio) and ONT have shown promising results in detecting structural variants and disease-causing variants in repeat expansions, 41 , 42 as well as characterizing RNA splicing isoforms. 43 , 44 The increasing accuracy and decreasing cost are making these techniques a viable option to possibly overcome some of the difficulties faced with short-read sequencing.…”