Search

Background: Liquid biopsy is considered a complementary and recently also an alternative method to surgical biopsy. It allows for the acquisition of valuable information regarding the potential presence of tumors, particularly through the analysis of circulating tumor DNA (ctDNA). CtDNA is a fraction of circulating free DNA (cfDNA) that can be extracted from various tissues, with blood being the most readily available., Results: To maximize the yield of plasma separation, specific Streck tubes are recommended for blood collection. The MagPurix CFC DNA Extraction Kit can be used for cfDNA extraction, and the TWIST Library Preparation protocol can be optimized for further analysis. Next-generation sequencing (NGS) can be employed to compare somatic and germline lineages, enabling the identification of somatic variants with a Variant Allele Frequency (VAF) of 5 % or higher, which are absent in the germline lineage., Conclusion: This analysis helps in the assessment of recurrence, analysis, and monitoring of cancer tissue., Competing Interests: Declaration of competing interest The authors of this scientific article often publish together, being part of the same group and/or scientific society. The scientific journal designated to publish this article has a specific policy to ensure the optimal handling of these situations. The journal’s policy ensures that the peer review process is kept independent by selecting reviewers independent of the authors of the article. All affiliations of the authors with private companies have been declared to make clear the position regarding the interests of these companies. The authors are affiliated with private companies for which there could be a possible conflict of interest. The authors state that the research was conducted in the absence of any commercial relationship that could be construed as a possible direct conflict of interest avoiding mention of any products or services sold by private companies related to the authors. The authors of this article are reported to be patents inventors., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)