S.J.J. Brouns
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Comparing Outbreak Reconstruction Tools: The Role of Within-Host Variation
Comparing Outbreak Reconstruction Tools: The Role of Within-Host Variation
Reconstructing transmission dynamics is a key challenge in outbreak analysis, as identifying who infected whom enables targeted interventions and improved understanding of disease spread. While many reconstruction methods rely on consensus genomes, this approach ignores within-host variation, which may contain additional transmission information. This study evaluates to what extent incorporating within-host variation improves outbreak reconstruction and under which conditions. To address this, simulated outbreaks were generated using the seedy framework under varying mutation rates, bottleneck sizes, and noise conditions. Three reconstruction tools were benchmarked: SeqTrack (consensus-based), BaDTrIP (iSNV-based), and Phyloscanner (haplotype-based). Performance was evaluated by comparing inferred transmission links to the ground truth. Results show that within-host variation provides an advantage in reconstruction accuracy, but mainly when sufficient genetic diversity is present. The mutation rate of the pathogen has a strong effect on performance, while bottleneck size has limited impact. Higher mutation rates improved performance across all three tools, but BaDTrIP and Phyloscanner substantially outperform SeqTrack. BaDTrIP achieved higher accuracy overall, but was more sensitive to noise, whereas Phyloscanner showed greater robustness to sequencing noise. Overall, the value of within-host variation depends on outbreak conditions. These findings highlight the importance of selecting reconstruction methods based on outbreak characteristics.
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Reconstructing transmission dynamics is a key challenge in outbreak analysis, as identifying who infected whom enables targeted interventions and improved understanding of disease spread. While many reconstruction methods rely on consensus genomes, this approach ignores within-host variation, which may contain additional transmission information. This study evaluates to what extent incorporating within-host variation improves outbreak reconstruction and under which conditions. To address this, simulated outbreaks were generated using the seedy framework under varying mutation rates, bottleneck sizes, and noise conditions. Three reconstruction tools were benchmarked: SeqTrack (consensus-based), BaDTrIP (iSNV-based), and Phyloscanner (haplotype-based). Performance was evaluated by comparing inferred transmission links to the ground truth. Results show that within-host variation provides an advantage in reconstruction accuracy, but mainly when sufficient genetic diversity is present. The mutation rate of the pathogen has a strong effect on performance, while bottleneck size has limited impact. Higher mutation rates improved performance across all three tools, but BaDTrIP and Phyloscanner substantially outperform SeqTrack. BaDTrIP achieved higher accuracy overall, but was more sensitive to noise, whereas Phyloscanner showed greater robustness to sequencing noise. Overall, the value of within-host variation depends on outbreak conditions. These findings highlight the importance of selecting reconstruction methods based on outbreak characteristics.
Master thesis
(2020)
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Rodrigo Gonzalez Linares, A.R. Martins Costa, C. Almendros Romero, S.J.J. Brouns
CRISPR-Cas effectors (e.g. Cas9) have been widely used to perform genetic knock-outs. Performing knock-ins however, remains challenging due to the inefficiency of the endogenous pathway cells use to integrate a donor genetic cargo into its genome (homology directed repair) when compared to other repair pathways like non-homologous end joining. CRISPR-associated transposases are complexes formed by a catalysis-deficient effector and a transposase. These complexes are able to sequester a transposon, localize a genomic target specified by a CRISPR RNA (crRNA), and integrate the transposon near the targeted site; thereby bypassing homology directed repair. In this study we aimed at developing a screening method using a CRISPR- associated transposase known as CAST, to detect integration events based on the disruption of lacZ. During the development, we found that CAST is unable to integrate a cargo in this highly active gene, most likely due to RNA polymerase-mediated dislodgment of the complex, and physical impediment for transposition proteins to reach the target DNA.
...
CRISPR-Cas effectors (e.g. Cas9) have been widely used to perform genetic knock-outs. Performing knock-ins however, remains challenging due to the inefficiency of the endogenous pathway cells use to integrate a donor genetic cargo into its genome (homology directed repair) when compared to other repair pathways like non-homologous end joining. CRISPR-associated transposases are complexes formed by a catalysis-deficient effector and a transposase. These complexes are able to sequester a transposon, localize a genomic target specified by a CRISPR RNA (crRNA), and integrate the transposon near the targeted site; thereby bypassing homology directed repair. In this study we aimed at developing a screening method using a CRISPR- associated transposase known as CAST, to detect integration events based on the disruption of lacZ. During the development, we found that CAST is unable to integrate a cargo in this highly active gene, most likely due to RNA polymerase-mediated dislodgment of the complex, and physical impediment for transposition proteins to reach the target DNA.