Presentation description

Unique Molecular Identifiers (UMIs) are short sequences of degenerate nucleotides used to uniquely ""barcode"" individual molecules in a heterogenous mixture so that those of interest can be specifically isolated and analyzed independently of other molecules. Adding these UMI labels through PCR is quick, effective, and precise-these barcoded molecules can be used in combination with deep and high throughput sequencing offered by Oxford Nanopore to accurately distinguish between PCR duplicates and variants for a multitude of bioinformatic approaches. Although this combination has been described previously, it is an unpolished system with minimally tested software. Here, we show an array of mutational assays to validate the functionality of our streamlined approach to individual molecular labeling and deep mutational sequencing through our own pipeline. As part of a wide array of experiments to test this system, here we describe a series of mutagenesis-based experiments: an ongoing multi-week culturing of two E. coli strains transfected with pcDNA3.1 derivative plasmids to observe compensatory mutations that promote cell survivability, inducing random mutagenesis within PCR-amplified material by a deoxynucleotide triphosphate analog mutagen, and controlling a string of hundreds of successive cycles of PCR amplification to quantify the rate of ultra rare base misincorporation by DNA polymerase. We explore the extent to which UMI-labeling followed by Oxford Nanopore sequencing can be applied through these, especially in cases such as these where target DNA to be analyzed is long (> 1 kb) and exists in a non-homogenous mixture of minutely distinct molecules. Using the UMIs as primers, we have been able to isolate individual molecules from a mixed pool. This partnership is necessary for many molecular biology and bioengineering approaches for cell signaling and gene expression understanding, such as in directed evolution-both mutation detection with high sensitivity and long read sequencing of a collection of diverse molecules are required. |