Amplified DNA heterogeneity assessment with Oxford Nanopore sequencing applied to cell free expression templates.

ABSTRACT: In this work, Oxford Nanopore sequencing is tested as an accessible method for quantifying heterogeneity of amplified DNA. This method enables rapid quantification of deletions, insertions, and substitutions, the probability of each mutation error, and their locations in the replicated sequences. Amplification techniques tested were conventional polymerase chain reaction (PCR) with varying levels of polymerase fidelity (OneTaq, Phusion, and Q5) as well as rolling circle amplification (RCA) with Phi29 polymerase. Plasmid amplification using bacteria was also assessed. By analyzing the distribution of errors in a large set of sequences for each sample, we examined the heterogeneity and mode of errors in each sample. This analysis revealed that Q5 and Phusion polymerases exhibited the lowest

SUBMITTER: Hejazi S 

PROVIDER: S-EPMC11614277 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature