PacMAGI: A pipeline including accurate indel detection for the analysis of PacBio sequencing data applied to RPE65.

• An automated pipeline for PacBio sequencing data was developed (PacMAGI) which includes QC, alignment, variant calling, phasing, annotation, variant interpretation. • A validated custom algorithm for the detection of indels was developed as the chosen variant caller, Longshot, was not able to call them. • PacMAGI, applied to 13 patients affected by Leber congenital amaurosis or Retinitis Pigmentosa, found all variants detected with NGS in the diagnostic setting plus a set of variants in intron and promoter regions. • In addition, in a patient in which a pathogenic variant was detected with NGS, PacMAGI discovered a second pathogenic variant giving to the patient the possibility to be a candidate for gene therapy. Third generation sequencing methods, like PacBio, provide information about structural variants, introns, enhancers and promoters. We developed an automated pipeline, called PacMAGI, including quality control, alignment, SNV, INDELs, structural variant calling, phasing, annotation and variant interpretation, for the analysis of PacBio data for any target region. Bi-allelic mutations in the RPE65 gene are associated with different inherited retinal dystrophies, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Diagnostic panel-based NGS analysis is performed on coding regions and intron/exon junctions of genes. To obtain a more conclusive diagnosis, we applied PacMAGI to obtain a second hit on RPE65 in LCA or RP patients who showed a single heterozygous variant by NGS. We used PacBio to sequence the full gene and identify putative second-hits in intronic, problematic and promoter regions. All variants identified in the diagnostic setting with NGS were correctly detected by the pipeline, and thanks to our custom algorithm for INDELs, a previously undetected 'Pathogenic' frameshift variant was found in a RP patient already identified to carry a 'Likely Pathogenic' variant. [ABSTRACT FROM AUTHOR]