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

Sorrentino, Elisa; Albion, Elena; Modena, Chiara; Daja, Muharrem; Cecchin, Stefano; Paolacci, Stefano; Miertuš, Ján; Bertelli, Matteo; Maltese, Paolo Enrico; Chiurazzi, Pietro; Stuppia, Liborio; Colombo, Leonardo; Marceddu, Giuseppe

doi:10.1016/j.gene.2022.146554

Cited by 4 publications

(4 citation statements)

References 23 publications

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“…The incorporation of NGS into clinical diagnostics has uncovered imminent needs in terms of clarifying the huge amount of uncertainty that arises from large-scale sequencing studies. Aside from disease-specific strategies, which allow more detailed and complete designs to reach a molecular diagnosis [14][15][16], generic solutions when confronting any kind of rare disease are difficult to achieve. Predicting the clinical significance of a change at the DNA level is a challenging task due to the myriad of structures/processes that could be affected.…”

Section: Discussionmentioning

confidence: 99%

Molecular Modelling Hurdle in the Next-Generation Sequencing Era

Fernàndez

Yubero

Palau

et al. 2022

IJMS

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There are challenges in the genetic diagnosis of rare diseases, and pursuing an optimal strategy to identify the cause of the disease is one of the main objectives of any clinical genomics unit. A range of techniques are currently used to characterize the genomic variability within the human genome to detect causative variants of specific disorders. With the introduction of next-generation sequencing (NGS) in the clinical setting, geneticists can study single-nucleotide variants (SNVs) throughout the entire exome/genome. In turn, the number of variants to be evaluated per patient has increased significantly, and more information has to be processed and analyzed to determine a proper diagnosis. Roughly 50% of patients with a Mendelian genetic disorder are diagnosed using NGS, but a fair number of patients still suffer a diagnostic odyssey. Due to the inherent diversity of the human population, as more exomes or genomes are sequenced, variants of uncertain significance (VUSs) will increase exponentially. Thus, assigning relevance to a VUS (non-synonymous as well as synonymous) in an undiagnosed patient becomes crucial to assess the proper diagnosis. Multiple algorithms have been used to predict how a specific mutation might affect the protein’s function, but they are far from accurate enough to be conclusive. In this work, we highlight the difficulties of genomic variability determined by NGS that have arisen in diagnosing rare genetic diseases, and how molecular modelling has to be a key component to elucidate the relevance of a specific mutation in the protein’s loss of function or malfunction. We suggest that the creation of a multi-omics data model should improve the classification of pathogenicity for a significant amount of the detected genomic variability. Moreover, we argue how it should be incorporated systematically in the process of variant evaluation to be useful in the clinical setting and the diagnostic pipeline.

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Section: Discussionmentioning

confidence: 99%

Molecular Modelling Hurdle in the Next-Generation Sequencing Era

Fernàndez

Yubero

Palau

et al. 2022

IJMS

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“…Minor allele frequencies (MAF) were retrieved from the Genome Aggregation Database [33]. Long-read sequencing data were analyzed using an in-house bioinformatics pipeline [37].…”

Section: Bioinformatics and Genetic Variants Classificationmentioning

confidence: 99%

“…The process was repeated for two other peptides, namely EEGGEEE734GDREE and EGEGEEE870GEEGE. Docking was performed using Autodock Vina 1.2 [38] and MAGI-Dock [39], a PyMol plugin that generates the docking boxes [37] around the active site crevice. Two separate docking runs were performed, one for each of the upstream and downstream segments of Glu737.…”

Section: Molecular Modeling Of Ttll5-rpgr Orf15 Interactionsmentioning

confidence: 99%

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Bonetti,

Cozza,

Bernini

et al. 2023

IJMS

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Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases.

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“…With regard to the bioinformatic analysis of the sequencing data obtained, we referred to our internally developed bioinformatic pipeline “PacMAGI” [ 24 ]. The workflow of such bioinformatic pipeline presents a quality control performed by using FastQC and MultiQC, an alignment phase with reference genotype, through the use of Pbmm2, and the generation of mapping statistics through Picard and Qualimap tool.…”

Section: Introductionmentioning

confidence: 99%

Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing

Mareso¹,

Albion²,

Cozza³

et al. 2023

Mol Biol Rep

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Background The filaggrin (FLG) protein, encoded by the FLG gene, is an intermediate filament-associated protein that plays a crucial role in the terminal stages of human epidermal differentiation. Loss-of-function mutations in the FLG exon 3 have been associated with skin diseases. The identification of causative mutations is challenging, due to the high sequence homology within its exon 3 (12,753 bp), which includes 10 to 12 filaggrin tandem repeats. With this study we aimed to obtain the whole FLG exon 3 sequence through PacBio technology, once 13-kb amplicons have been generated. Methods and results For the preparation of SMRTbell libraries to be sequenced using PacBio technology, we focused on optimizing a 2-step long-range PCR protocol to generate 13-kb amplicons covering the whole FLG exon 3 sequence. The performance of three long-range DNA polymerases was assessed in an attempt to improve the PCR conditions required for the enzymes to function properly. We focused on optimization of the input template DNA concentration and thermocycling parameters to correctly amplify the entire FLG exon 3 sequence, minimizing non-specific amplification. Conclusions Taken together, our findings suggested that the PrimeSTAR protocol is suitable for producing the amplicons of the 13-kb FLG whole exon 3 to prepare SMRTbell libraries. We suggest that sequencing the generated amplicons may be useful for identifying LoF variants that are causative of the patients’ disorders.

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

Cited by 4 publications

References 23 publications

Molecular Modelling Hurdle in the Next-Generation Sequencing Era

Molecular Modelling Hurdle in the Next-Generation Sequencing Era

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing

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