De Novo Assembly-Based Analysis of RPGR Exon ORF15 in an Indigenous African Cohort Overcomes Limitations of a Standard Next-Generation Sequencing (NGS) Data Analysis Pipeline

Abstract

RPGR exon ORF15 variants are one of the most frequent causes for inherited retinal disorders (IRDs), in particular retinitis pigmentosa. The low sequence complexity of this mutation hotspot makes it prone to indels and challenging for sequence data analysis. Whole-exome sequencing generally fails to provide adequate coverage in this region. Therefore, complementary methods are needed to avoid false positives as well as negative results. In this study, next-generation sequencing (NGS) was used to sequence long-range PCR amplicons for an IRD cohort of African ancestry. By developing a novel secondary analysis pipeline based on de novo assembly, we were able to avoid the miscalling of variants generated by standard NGS analysis tools. We identified pathogenic variants in 11 patients (13% of the cohort), two of which have not been reported previously. We provide a novel and alternative end-to-end secondary analysis pipeline for targeted NGS of ORF15 that is less prone to false positive and negative variant calls.

Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

“…Furthermore, the protocol can be useful in analyzing challenging regions that are typically not covered by other methods, such as RPGR ’s exon ORF15. It has been shown previously that NGS of a LR PCR over this region provides good coverage and we have developed a secondary data analysis pipeline to improve sensitivity and specificity [ 28 , 29 ]. This strategy permitted the identification of a novel 20 bp insertion (NM_001034853.1:c.2819_2838dup, Table 3 , Table A1 ) that was not detected by standard analysis pipelines.…”

“…Primers for LR PCR were designed on NCBI's Primer-Blast to be 28 bp (range[26][27][28][29][30] long and to have a melting temperature (Tm) of 67 • C (range 65-68 • C) under default settings[31]. The size of PCR products depended on the target locus and ranged from 4.3 to 19.9 kb (mean size = 15.8 kb, median size = 17.6 kb).…”

Long-range PCR-based NGS applications to diagnose Mendelian retinal diseases

“…Primer for LR PCR were designed on NCBI's Primer-Blast to be 28 bp (range [26][27][28][29][30] long and to have a melting temperature (Tm) of 67° C (range 65°-68°C) under default settings 22 . The size of PCR products depended on the target locus, and ranged from 4.3 to 19.9 kb (mean size = 15.8 kb, median size = 17.6 kb).…”

“…CNVs analysis on target-capture sequencing data (ABCA4 capture and ES results) was performed using panelcn.mops 25 LR PCR sequencing data of RPGR's ORF15 was assembled de novo using SPAdes and the resulting contig was aligned to the reference sequence, as previously described. 26,27 Table S2. Briefly, primers were designed based on the estimated breakpoints location, LR PCR was performed, and the amplicon was sequenced as described above (see Amplicons pool library construction and sequencing).…”

“…The copyright holder for this preprint this version posted December 2, 2020. ; https://doi.org/10.1101/2020.11.30.20234971 doi: medRxiv preprint previously that NGS of a LR PCR over this region provides good coverage and we have developed a secondary data analysis pipeline to improve sensitivity and specificity. 27,29 This strategy permitted the identification of a novel 20 bp insertion (NM_001034853.1:c.2819_2838dup, Table 2, and Table S4) that was not detected by standard analysis pipelines.…”