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. 2021 Aug 5;108(8):1436-1449.
doi: 10.1016/j.ajhg.2021.06.006. Epub 2021 Jul 2.

Targeted long-read sequencing identifies missing disease-causing variation

Danny E Miller  1 , Arvis Sulovari  2 , Tianyun Wang  2 , Hailey Loucks  3 , Kendra Hoekzema  2 , Katherine M Munson  2 , Alexandra P Lewis  2 , Edith P Almanza Fuerte  3 , Catherine R Paschal  4 , Tom Walsh  5 , Jenny Thies  3 , James T Bennett  6 , Ian Glass  3 , Katrina M Dipple  7 , Karynne Patterson  2 , Emily S Bonkowski  3 , Zoe Nelson  3 , Audrey Squire  3 , Megan Sikes  3 , Erika Beckman  3 , Robin L Bennett  8 , Dawn Earl  3 , Winston Lee  9 , Rando Allikmets  10 , Seth J Perlman  11 , Penny Chow  12 , Anne V Hing  12 , Tara L Wenger  3 , Margaret P Adam  3 , Angela Sun  13 , Christina Lam  14 , Irene Chang  3 , Xue Zou  15 , Stephanie L Austin  16 , Erin Huggins  16 , Alexias Safi  16 , Apoorva K Iyengar  17 , Timothy E Reddy  18 , William H Majoros  18 , Andrew S Allen  18 , Gregory E Crawford  16 , Priya S Kishnani  16 ; University of Washington Center for Mendelian Genomics; Mary-Claire King  5 , Tim Cherry  19 , Jessica X Chong  20 , Michael J Bamshad  21 , Deborah A Nickerson  22 , Heather C Mefford  20 , Dan Doherty  23 , Evan E Eichler  24
Affiliations

Targeted long-read sequencing identifies missing disease-causing variation

Danny E Miller et al. Am J Hum Genet. .

Abstract

Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.

Keywords: long-read sequencing, adaptive sampling, nanopore sequencing, targeted long-read sequencing.

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Conflict of interest statement

P.S.K. reports receiving consulting fees from Sanofi Genzyme, Amicus Therapeutics, Maze Therapeutics, JCR Pharmaceutical, and Asklepios Biopharmaceutical, Inc; research and grant support from Sanofi Genzyme, Valerion Therapeutics, and Amicus Therapeutics; has equity in Asklepios Biopharmaceutical, Inc., and Maze Therapeutics; and is a member of the Pompe and Gaucher Disease Registry Advisory Board for Sanofi Genzyme, Amicus Therapeutics, and Baebies.

Figures

Figure 1
Figure 1
Targeted long-read sequencing simultaneously detects repeat expansion and methylation status Expansion and methylation of a GGC repeat in the 5′ UTR of XYLT1 is a common cause of Baratela-Scott syndrome. (A) Southern blot of family 04 reported by LaCroix and colleagues demonstrates that the proband (04-01) carries an expansion (1) of a region defined by two KpnI restriction enzyme sites containing a GGC repeat, the mother (04-02) carries one premutation (2) and one wild-type allele (3), and the father (04-03) carries two wild-type alleles (4). Both panels are from the same Southern blot on day 6 of exposure. (B) T-LRS of the trio revealed that the length of fragments from single reads spanning both KpnI cut sites used in (A) was consistent with the results from the Southern blot. Colored dots in (B) correspond to methylated (red) and non-methylated (blue) reads shown in (C); gray represents reads where methylation status was not determined. (C) Expansion of the GGC repeat in the proband results in methylation of the 5′ UTR and exon 1. Two reads in the mother are methylated (red), one of which spans the region between the KpnI cut sites and whose length is consistent with a premutation allele as shown in (B). The second methylated read terminates within the repeat and the length cannot be assayed.
Figure 2
Figure 2
Targeted long-read sequencing identifies additional structural differences not observed by standard clinical testing (A) T-LRS of individual S014 revealed two additional deletions and one rearrangement (inversion) not reported by CMA. Reanalysis of the CMA data confirmed deletion L. The “subway” plot shows how each region is connected and allows for reconstruction of the new DNA sequence and gene order in the individual. (B) Clinical CMA of individual S020 identified three deletions on chromosomes 4 and 14 and the subsequent karyotype revealed a complex translocation involving chromosomes 2, 4, 10, and 14. T-LRS identified 11 translocations, 13 rearrangements, and 6 deletions directly affecting 12 genes. Reconstruction of each derivative chromosome estimates the size of each event, as represented by the boxes surrounding part of the derivative chromosomes on the karyotype and is consistent with expected sizes based on karyotype.
Figure 3
Figure 3
Targeted long-read sequencing identifies variants not detected by clinical testing Pathogenic, likely pathogenic, or variants of uncertain significance (VUSs) identified by T-LRS along with variants identified by prior clinical testing (denoted by an asterisk). (A) T-LRS detected a candidate intronic splice acceptor variant as well as the known paternally inherited stop-gain. Long-read phasing demonstrates that these variants are in trans. (B) A 1,900 bp deletion within HPS1 removes exon 3; phasing revealed that this variant and the previously known paternally inherited stop-gain occur on different haplotypes. Clinical testing with an exon-level array confirmed the deletion. (C) T-LRS reveals a previously known paternally inherited stop-gain as well as a novel Alu insertion in exon 20 of ALMS1. Subsequent clinical testing confirmed the Alu was pathogenic and maternally inherited. (D) A 187 bp deletion and 17 Mbp inversion disrupts HPRT1. Clinical testing confirmed the presence of an inversion. (E) Insertion of a 1,500 bp composite retrotransposable element is predicted to create multiple splice acceptor and donor sites and represents a candidate second hit. Linkage disequilibrium phasing suggests the variants are on different haplotypes. (F) Expansion of an AGAA repeat within DMD represents a VUS in an individual with Duchenne muscular dystrophy and a family history lacking a genetic diagnosis.
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