Speaker
Description
Detecting extremely rare DNA variants is key in multiple clinical applications, such as circulating tumour DNA detection or transplant monitoring. A serious limitation of detection ability comes from polymerase‑induced sequencing errors. Unique molecular identifiers (UMIs) help suppress these errors, but their efficiency depends on how many amplified copies each barcoded molecule yields - an outcome shaped by protocol design.
Here we present a stochastic branching process-based model that captures the key steps of ultrasensitive UMI‑tagged sequencing: barcoding PCR, dilution, amplification PCR with saturation, and final sequencing. We quantitatively fit the model to data generated with the SiMSen‑Seq protocol \cite{Stahlberg:2017aa} using Approximate Bayesian Computation. We demonstrate that the model accurately reproduces the experimentally observed linear relationship between sequencing depth and average UMI cluster size, and suggests that even UMI-affecting polymerase error rates are fragment dependent. We then utilise the model to explore the effect of mid‑protocol dilution on final UMI yields, and find negligible impact, indicating experimental flexibility without loss of sensitivity.
In summary, our model provides a computationally efficient framework for evaluating protocol modifications and understanding sources of variability in UMI‑based ultrasensitive sequencing.
Bibliography
@article{Stahlberg:2017aa,
abstract = {St{\aa}hlberg et al. describe SiMSen-seq, a barcoding NGS library preparation approach to enable detection of rare variant alleles from as little as 5 ng of input DNA. The method also works for fragmented templates such as cell-free DNA.},
author = {St{\aa}hlberg, Anders and Krzyzanowski, Paul M and Egyud, Matthew and Filges, Stefan and Stein, Lincoln and Godfrey, Tony E},
date = {2017/04/01},
date-added = {2025-05-19 15:02:44 +0200},
date-modified = {2025-05-19 15:02:44 +0200},
doi = {10.1038/nprot.2017.006},
id = {St{\aa}hlberg2017},
isbn = {1750-2799},
journal = {Nature Protocols},
number = {4},
pages = {664--682},
title = {Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing},
url = {https://doi.org/10.1038/nprot.2017.006},
volume = {12},
year = {2017},
bdsk-url-1 = {https://doi.org/10.1038/nprot.2017.006}}
