Biology subjects

Waldvogel, D.

Publications and source records attributed to Waldvogel, D..

2 recordsLinked to original sources

bioRxiv2023

Detection of genetically modified organisms using highly multiplexed amplicon sequencing

The circulation of products based on genetically modified (GM) organisms is highly regulated by some governments having implemented strict rules on the breeding, planting, marketing, labelling, and trading of such products. To ensure compliance, accurate detection methods for GM events are necessary, along with assurance that GM material falls within relevant threshold levels. The increasing complexity and potential of undocumented GM are a growing challenge for genetic screening. In this study, we developed and assessed a highly multiplexed amplicon sequencing assay for the detection of GM events based on a microfluidics platform and next-generation sequencing (NGS). To probe GM events comprehensively, we designed a total of 230 new amplicons to cover flanking, promoter, junction and coding sequences of GM sequences. In addition, we designed and implemented parallel amplification of ribosomal and chloroplast markers to define crop species identity from potentially mixed samples. Using reference GM material of 11 crop species and multiple amplicons, we successfully detected the presence of 10 known modifications per GM event. We also find that reported flanking sequences of GM events may not be all useful for diagnostic. We assessed the assays potential to detect GM events in mixed samples as well as in highly diluted DNA. Finally, we performed a prospective search of potentially undocumented GM events in plant material. Our microfluidics-based amplicon GM detection approach fills important gaps in detecting potentially undocumented and complex GM events by recovering a wide range of specific amplicon sequences for evaluation. Integrating highly parallel amplicon assays in GM screening efforts should be an effective complement to aid post-market monitoring and regulatory compliance efforts.

genomics↗

bioRxiv2020

A robust sequencing assay of a thousand amplicons for the high-throughput population monitoring of Alpine ibex immunogenetics

Genetic variation is a major factor determining susceptibility to diseases. Polymorphism at the major histocompatibility complex (MHC) and other immune function loci can underlie health and reproductive success of individuals. Endangered species of low population size could be severely compromised to evolve disease resistance due to reduced adaptive variation. A major impediment to screen adaptive genetic variation in wild species is the difficulty to comprehensively genotype immune-related loci based on low input material. Here, we design and validate a targeted amplicon sequencing assay to parallelize the analysis of a thousand loci of the MHC, other immunity-related genes, and genome-wide markers for the assessment of population structure. We apply the approach to Alpine ibex, one of the most successful examples of restoration of a large mammal in Europe. We used 51 whole genome sequenced individuals to select representative target SNPs. We integrated SNP call data from four related species for amplification robustness and genotyped 158 Alpine ibex individuals for validation. We show that the genome-wide markers perform equally well at resolving population structure as RAD-seq or low-coverage genome sequencing datasets with orders of magnitude more markers. The targeted amplicon sequencing assay is robust to >100-fold variation in input DNA quantity and generates useful genotype information from fecal samples. The amplicon marker set also identified recent species hybridization events with domestic goats. The immune loci show unexpectedly high degrees of differentiation within the species. Our assay strategy can realistically be implemented into population genetic surveys of a large range of species.

genomics↗