Author(s): Silva, Catarina ; Ferrão, José ; Marques, Bárbara ; Pedro, Sónia ; Correia, Hildeberto ; Valente, Ana ; Rodrigues, António Sebastião ; Vieira, Luís
Date: 2025
Persistent ID: http://hdl.handle.net/10400.18/10573
Origin: Repositório Científico do Instituto Nacional de Saúde
Subject(s): Copy Number Variation; Hybrid-SNP Microarray; Nanopore Sequencing; Structural Variation; Third-Generation Sequencing; Variant Calling; Whole Genome Sequencing; Tecnologias de Análise de DNA
Background: Nanopore sequencing is a technology that holds great promise for identifying all types of human genome variations, particularly structural variations. In this work, we used nanopore sequencing technology to sequence 2 human cell lines at low depth of coverage to call copy number variations (CNV), and compared the results variant by variant with chromosomal microarray (CMA) results. Results: We analysed sequencing data using CuteSV and Sniffles2 variant callers, compared breakpoints based on hybrid-SNP microarray, nanopore sequencing and Sanger sequencing, and analysed CNV coverage. From a total of 48 high confidence variants (truth set), variant calling detected 79% of the truth set variants, increasing to 86% for interstitial CNV. Simultaneous use of the 2 callers slightly increased variant calling. Both callers performed better when calling CNV losses than gains. Variant sizes from CMA and nanopore sequencing showed an excellent correlation, with breakpoints determined by nanopore sequencing differing by only 20 base pairs on average from Sanger sequencing. Nanopore sequencing also revealed that four variants concealed genomic inversions undetectable by CMA. In the 10 CNV not called in nanopore sequencing, 8 showed coverage evidence of genomic loss or gain, highlighting the need to improve SV calling algorithms performance. Conclusions: Nanopore sequencing offers advantages over CMA for structural variant detection, including the identification of multiple variant types and their breakpoints with increased precision. However, further improvements in variant calling algorithms are still needed for nanopore sequencing to become a highly robust and standardized approach for a comprehensive analysis of genomic structural variation.
