Biology subjects

Tytgat, O.

Publications and source records attributed to Tytgat, O..

3 recordsLinked to original sources

bioRxiv2025

Recombinase Polymerase Amplification of Forensic Short Tandem Repeat Loci

Short tandem repeats (STRs) are highly polymorphic repetitive DNA sequences extensively used in forensic science for identification of individuals. STR genotyping is usually performed by capillary electrophoresis (CE) or next-generation sequencing (NGS) in centralized laboratories. However, there is an increasing need for a low cost, portable and rapid STR genotyping method. Multiple methods for miniaturization have been explored, all relying on polymerase chain reaction (PCR) for generating amplicons. PCR requires precise thermal cycling, which complicates the design of the STR genotyping microfluidic device. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification method that operates between 37{degrees}C and 42{degrees}C and completes within 40 minutes. This, along with the robustness of reagents and reduced stutter rate compared to PCR makes RPA a suitable candidate for implementation in an STR genotyping microfluidics device as well as a part of the established STR genotyping work flows. In this proof-of-concept study, we evaluate RPA assay for amplification of forensically relevant STR loci. Thirteen core STR loci of the Combined DNA Index System (CODIS) were amplified using RPA in both singleplex and multiplex formats. The amplicons were then analyzed using three different methods: CE, Illumina and Oxford Nanopore Technologies (ONT). A subset of 5 loci was used for CE analysis. CE, Illumina and ONT sequencing of singleplex RPA each resulted in complete and correct STR profiles across all samples. Sensitivity assessment demonstrated that complete and correct genotypes were achieved with DNA inputs of 62 pg and above for all but locus D8S1179. Attempts at multiplex RPA amplification resulted in incomplete or incorrect STR profiles. This outcome highlights a challenge in adapting RPA for simultaneous amplification of multiple STR loci, which is a standard requirement in forensic DNA profiling.

genomics↗

bioRxiv2022

Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping

CYP2D6 is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants. Author SummaryDuring the last decades, the usefulness of personalized medicine has become increasingly apparent. Directly linked to that is the need for accurate genotyping assays to determine the pharmacogenetic profile of patients. Continuing research has led to the development of genotyping assays that perform quite robustly. However, complex genes remain an issue when it comes to determining the complete sequence correctly. An example of such a complex but very important pharmacogene is CYP2D6. Therefore, we developed a genotyping assay in an attempt to generate complete allele-specific consensus sequences of CYP2D6, by optimizing a targeted amplification-free long-read sequencing method and developing a new analysis pipeline. In reference samples, we showed that our genotyping assay performed accurately and confirmed the presence of variants that go undetected by most current assays. However, the implementation of this assay in practice is still hampered as the selected enrichment strategies inherently lead to a low percentage of on-target reads, resulting in low on-target sequencing depths. Further optimization and validation of the assay is thus needed, but definitely worth considering for follow-up research as we already demonstrated the added value for generating more complete genotypes, which on its turn will result in more accurate gene function predictions.

genetics↗

bioRxiv2018

Forensic STR profiling using Oxford Nanopore Technologies’ MinION sequencer

Forensic STR profiling using massively parallel sequencing (MPS) has gained much attention as an alternative for the traditional capillary electrophoresis (CE) methods. Oxford Nanopore Technologies recently developed the MinION, a pocket-sized nanopore sequencer with promising features that could be useful in the field of forensic genetics. We applied this technology for forensic sequencing in a pilot study. Using standard STR primers, originally designed for multiplex PCR and CE, we developed a library preparation method suited for nanopore sequencing. Several analysis approaches were evaluated to explore the usefulness of the generated data: we developed and applied both a sequence-based and an amplicon length-based analysis on data originating from a 14-loci multiplex PCR on a single contributor DNA sample. Despite the high sequencing error rate, the analyses yielded partial forensic profiles with some useful evidential value.

genomics↗