Biology subjects

Deserranno, K.

Publications and source records attributed to Deserranno, K..

3 recordsLinked to original sources

bioRxiv2026

Recombinase polymerase amplification: characterization and mitigation of undescribed multimeric artefacts

Recombinase polymerase amplification (RPA) enables rapid nucleic acid testing in low-resource environments, but poorly characterized byproducts can compromise assay specificity and cause false-positive results. Here, we amplified the thirteen original CODIS core loci and Amelogenin to characterize recurrent RPA artefacts and establish conditions that reduce their formation. First, RPA products were analyzed for two reference samples by Oxford Nanopore Technologies sequencing. This revealed two distinct classes of multimeric products: primer multimers and amplicon multimers, consisting of repeated primer or amplicon sequences, respectively. Individual artefacts contained up to 281 primer copies or 22 amplicon copies, demonstrating the extensive range of these products. Next, we performed an optimization study to evaluate the effects of reaction temperature and reagent concentrations at two representative loci, D3S1358 and D5S818. Among the conditions tested, temperature had the most pronounced effect. Reducing the temperature from 42{degrees}C to 34{degrees}C increased the relative target amplicon fraction from 15% to 83% for D3S1358 and from 84% to 98% for D5S818, while maintaining or increasing absolute target concentration. Lower primer concentrations and higher T4 UvsX concentrations also reduced multimer formation, although lower primer concentrations reduced target yield and caused allelic dropout. Finally, amplification at 34{degrees}C was evaluated across all fourteen loci by sequencing. Relative to 42{degrees}C, the target read fraction increased by more than 5 percentage points for 7/14 loci in one reference sample and 9/14 loci in the other, with the largest improvements at multimer-prone loci. These findings identify multimers as an important class of RPA artefacts and establish reaction temperature and T4 UvsX concentration as promising conditions to improve RPA specificity.

biochemistry↗

bioRxiv2025

A laser capture microdissection-based method for high-sensitivity transcriptomics from archived FFPE tissue slides with single-cell resolution using LCM-FFPEseq

Understanding gene expression within its spatial context is essential for unravelling biological processes. Laser Capture Microdissection (LCM) has emerged as a transformative technology, enabling targeted isolation of individual cells or regions from tissue sections while preserving spatial context. However, its application to formalin-fixed, paraffin-embedded (FFPE) tissues has been limited by RNA degradation, leaving the vast repository of clinical FFPE samples underutilized. To address this, we introduce LCM-FFPEseq, a novel method combining LCM with the advanced Smart-seq3xpress protocol and FFPE-specific adaptations for spatial transcriptomics of FFPE sections. Unlike traditional protocols requiring thousands of cells to generate high-quality libraries, LCM-FFPEseq achieves high sensitivity, reproducibility, and transcript coverage. With as few as 30 FFPE-embedded K562 cells and Sertoli cells, we detected over 14,000 protein-coding genes per sample, with no substantial gains when a higher number of cells were isolated. Even individual LCM-isolated cells yielded an average of 7,353 or 6,490 protein-coding genes per K562 or Sertoli single cell, respectively. To demonstrate its clinical utility, we applied LCM-FFPEseq to archived testicular FFPE samples from transgender females receiving gender-affirming hormone therapy. Transcriptomic profiling of isolated seminiferous tubules revealed tubular hyalinization to be associated with greater upregulation of extracellular matrix remodelling and inflammatory pathways, alongside stronger downregulation of spermatogenesis-associated pathways. These findings suggest that testicular fibrosis and/or tubular hyalinization may contribute to germ cell loss following inappropriate hormonal exposure. By enabling high-resolution transcriptomics in archived FFPE samples, LCM-FFPEseq unlocks new possibilities for investigating rare cell types, spatial heterogeneity, and therapy-induced tissue remodelling in vast FFPE repositories.

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↗