Advances in forensic genetics: Exploring the potential of long read sequencing

• Published in: Forensic science international : genetics Vol. 74; p. 103156
• Main Authors: Ferreira, Marcel Rodrigues, Carratto, Thássia Mayra Telles, Frontanilla, Tamara Soledad, Bonadio, Raphael Severino, Jain, Miten, de Oliveira, Silviene Fabiana, Castelli, Erick C., Mendes-Junior, Celso Teixeira
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-01-2025
• Subjects: Bioinformatics; Forensic Genetics; Massively Parallel Sequencing; Next-generation sequencing; Oxford Nanopore; Third-generation sequencing; Forensic Genetics; Third-generation sequencing; Oxford Nanopore; Next-generation sequencing; Bioinformatics; Massively Parallel Sequencing
• ISSN: 1872-4973; 1878-0326; 1878-0326
• DOI: 10.1016/j.fsigen.2024.103156

DNA-based technologies have been used in forensic practice since the mid-1980s. While PCR-based STR genotyping using Capillary Electrophoresis remains the gold standard for generating DNA profiles in routine casework worldwide, the research community is continually seeking alternative methods capable of providing additional information to enhance discrimination power or contribute with new investigative leads. Oxford Nanopore Technologies (ONT) and PacBio third-generation sequencing have revolutionized the field, offering real-time capabilities, single-molecule resolution, and long-read sequencing (LRS). ONT, the pioneer of nanopore sequencing, uses biological nanopores to analyze nucleic acids in real-time. Its devices have revolutionized sequencing and may represent an interesting alternative for forensic research and routine casework, given that it offers unparalleled flexibility in a portable size: it enables sequencing approaches that range widely from PCR-amplified short target regions (e.g., CODIS STRs) to PCR-free whole transcriptome or even ultra-long whole genome sequencing. Despite its higher error rate compared to Illumina sequencing, it can significantly improve accuracy in read alignment against a reference genome or de novo genome assembly. This is achieved by generating long contiguous sequences that correctly assemble repetitive sections and regions with structural variation. Moreover, it allows real-time determination of DNA methylation status from native DNA without the need for bisulfite conversion. LRS enables the analysis of thousands of markers at once, providing phasing information and eliminating the need for multiple assays. This maximizes the information retrieved from a single invaluable sample. In this review, we explore the potential use of LRS in different forensic genetics approaches.
•Sequencing methods have evolved considerably over the last two decades•Third-generation sequencing is an alternative for forensic research and casework•Various applications rely on PCR-amplified short targets to PCR-free whole genomes•DNA and RNA methylation as direct byproducts contribute with investigative leads•Flexibility, portability and real-time capabilities are useful for on-site analysis