Diagnostic analysis of the highly complex OPN1LW/OPN1MW gene cluster using long-read sequencing and MLPA

DNA samples from clinical archive

A diagnostic laboratory can use (de-identified) archived clinical samples to validate and implement novel diagnostic assays. The derived clinically relevant variants can be shared, but in absence of explicit data-sharing consent at individual patient level, FASTQ, BAM and VCFs cannot be disclosed. These methods are also in accordance with relevant guidelines and regulations and approved by the institutional review board of the Radboud University Medical Center (2020-7142) and the Declaration of Helsinki.

All clinical samples (n = 50) were received between 2015 and 2020 by the Department of Human Genetics of the Radboud University Medical Center. In 43 male samples the genetic test was requested to genetically confirm the diagnosis of protanopia (n = 1), BED (n = 32), BCM (n = 8) or cone dystrophy (n = 2). The type of visual impairment was diagnosed by expert ophthalmologists based on appropriate clinical examinations. In seven female samples the genetic test was requested for carrier status analysis, because of multiple affected children (n = 1), affected child without a positive family history (n = 4) or affected or carrier sibling (n = 2). Genomic DNA isolation of peripheral blood was performed in an automated manner as described previously32. In short, genomic DNA isolation was performed automatically using a HamiltonMicrolab STARautoload system with an integrated Chemagen MSM I separation module (Hamilton Robotics GmbH). DNA isolation was performed using the Chemagic DNA blood kit special (PerkinElmer) according to the manufacturer’s instructions. For the purpose of this study, the samples were anonymized and coded with a unique study number (USN).

MLPA

A MLPA test specific for the OPN1LW/OPN1MW gene cluster was developed together with MRC Holland. The SALSA MLPA Probemix X080 Opsin consists of 9 control probes and 16 probes targeting the OPN1LW/OPN1MW gene cluster, all located on the X-chromosome. Two probes target the LCR. Six probes target OPN1LW (NM_020061.5): c.-77A in exon 1, c.300A in exon 2, c.457C in exon 3, c.706A in exon 4, and c.830 A and c.926A in exon 5. Six probes target OPN1MW (NM_000153.2): c.-62A in exon 1, c.300G in exon 2, c.457A in exon 3, c.706 G in exon 4, and c.830T and c.926T in exon 5. Two probes target both OPN1LW and OPN1MW: c.607T in exon 4, and c.1083A in exon 6 (Fig. 1a). Probe sequences are stated in Supplementary Table 4. For the probes targeting c.457 and c.706 of both OPN1LW and OPN1MW single nucleotide variants have been described within 10 nucleotides of the target. Accordingly, there is a possibility that when such a variant is present this will affect the performance of the probe.

The MLPA reaction was performed according to the manufacturer’s protocol (MRC Holland) on a thermocycler (Geneamp PCR system 9700 (ThermoFisher) or Veriti 96 well thermal cycler (Applied Biosystems)). In short, 50–100 ng DNA was denatured and mixed with 1.5 µl SALSA MLPA buffer and 1.5 µl probe mix. After 16 to 20 hours of hybridization at 60 °C, 3 µl SALSA ligase buffer A, 3 µl SALSA ligase buffer B, 1 µl SALSA ligase-65 and 25 µl H2O was added and incubated for 15 min at 54 °C. Finally, a polymerase chain reaction (PCR) was performed by adding 2 µl SALSA PCR primer mix, 0.5 µl SALSA polymerase, and 7.5 µl H2O. The PCR protocol was: 35 cycles of 30 seconds at 95 °C, 30 s at 60 °C and 1 min at 72 °C, followed by a final elongation step of 20 min at 72 °C. A mixture of 1 µl MLPA sample and 8.8 µl formamide (Hi-Di, Applied Biosystems) and 0.2 µl size standard (GeneScan 500-Liz, Applied Biosystems) was analyzed on a fragment analyzer (Model 3130, Applied Biosystems). Genemarker (V2.6.7, Softgenetics) was used for data analysis.

Long-range PCR

Four long-range PCRs (LR-PCRs) were designed to encompass the OPN1LW/OPN1MW gene cluster (Fig. 1a). One amplicon of 14,374 bp was designed specific for the LCR region (primers 099-692 GCAAAGGCTCTTCCTTTGTG and 099-693 AGGTGAAGGCAGGGTAAGGT). One amplicon of 15,634 bp specifically spans the first opsin gene copy (most often a (hybrid) OPN1LW) in the cluster (primers 044-982 GAGGCGAGGCTACGGAGT and 044-984 GCAGTGAAAGCCTCTGTGACT). One amplicon of 14,034 bp spans the second and if present consecutive opsin gene copies (most often (hybrid) OPN1MW) in the cluster (primers 080-273 AGCTGGGAGTACAGGTATTTG and 044-984). Lastly, one amplicon of 16,589 bp was designed to identify part of the last opsin gene (most often a (hybrid) OPN1MW) in the cluster (primers 099-820 AGGTGTAGAGCCCTAGCAAAC and 099-821 TCTCATTCATAAATTGCTGGTA).

PCR was as follows: 100 ng DNA, 12.5 µl LongAmp Hot Start Taq 2x Master Mix (Bioke), 2 µl 10 µM forward and reverse primer, 10.5 µl H2O. The PCR protocol for LCR, first gene, and second and consecutive genes was: 94 °C for 1 min, followed by 30 cycles of 30 s at 94 °C and 14 min at 65 °C, followed by a final elongation step of 10 min at 65 °C. The PCR protocol for the last gene was: 94 °C for 1 min, followed by 30 cycles of 30 s at 94 °C, 1 min 56 °C, and 15 min at 65 °C, followed by a final elongation step of 10 min at 65 °C. All amplicons were checked on agarose gel or DNA ScreenTape Analysis (TapeStation, Agilent).

Library preparation

LR amplicons were purified by AMPure PB beads (Pacific Biosciences), using a bead ratio of 1.5x. Library preparation was done according to protocol ‘Procedure and Checklist—Preparing SMRTbell Libraries using PacBio Barcoded Adapters for Multiplex SMRT Sequencing’ (Pacific Biosciences, Part Number 100-538-700-02). In brief, sample concentrations were measured with Qubit 3.0 (ThermoFisher Scientific). Equimolar amounts of amplicons, adding up to a total input of 200 ng to 1 ug DNA, were used to perform one-step end repair and adapter ligation (using barcoded hairpin adapters), followed by equimolar pooling. The pool was purified with AMPure PB beads, followed by DNA damage repair, exonuclease digestion, and an additional two rounds of AMPure PB beads purification.

Generation of polymerase-bound SMRTbell complexes and SMRT sequencing

Generation of polymerase-bound SMRTbell complexes was performed using the Sample Setup option in SMRTLink (Pacific Biosciences). In brief, sequencing primers were conditioned and annealed to the SMRTbell library, followed by dilution and binding of the sequencing polymerase. The polymerase bound complex was purified using AMPure PB beads, and concentration was measured via Qubit. An internal control sample was diluted and added to the polymerase-bound complex, together with DTT, Sequel additive, and complex dilution buffer.

Sequencing was performed using the Run Design option in SMRTLink. Libraries were loaded using diffusion loading with an on plate concentration of 4.5 pM. All runs were sequenced using a movie time of 20 h per SMRTcell and included pre-extension. Sequencing was performed on a Sequel I system (Pacific Biosciences) with ICS version 6.0. The amplicons of the OPN1LW/OPN1MW gene cluster were sequenced together with other types of long-read amplicons. Sequencing runs were performed twice a week, irrespective of the number of samples available, with a maximum of 24 different barcodes per sequencing run, a barcode can contain more than one amplicon. Accordingly, coverage varies between the different samples. The low number of barcodes however guarantees sufficient coverage for each analysis. The minimum coverage required for data analysis was 20x.

Data analysis and variant calling

Following sequencing, raw data was analyzed using CCS mapping in SMRTLink. Analysis was performed using default settings with minor modifications: only reads longer than 10 kb were mapped and for CCSmapping the selected reference was an adapted GRCh37 with only one copy of OPN1MW. The additional setting ‘PlaceGapConsistently’ was added to the algorithm options in order to allow left alignment, and ‘consolidate bam’ was set to ‘ON’ (default: OFF). Generated bam files were uploaded into SeqNext (JSI Medical systems) and variant calling was performed using default settings on OPN1LW (NM_020061.6) and OPN1MW (NM_000513.2).

Optical genome mapping

Optical genome mapping of control samples S3.1 to S3.4 was performed as described previously29, with minor modifications. In brief, ultra-high molecular weight DNA was isolated from 650 ul peripheral blood (EDTA) using the SP Blood and Cell Culture DNA Isolation Kit according to manufacturers’ instructions (Bionano Genomics). Per sample, 750 ng of DNA was labeled with the DLS (Direct Label and Stain) DNA Labeling Kit (Bionano Genomics), and labeled DNA was imaged on the Saphyr System (Bionano Genomics) using ICS version 5.2. The annotated de novo assembly pipeline was executed with Bionano Solve software 3.6.1. Data of genome in a bottle sample NA1287828 is provided by courtesy of Bionano Genomics. Quality statistics of the samples are stated in Supplementary Table 5.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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