Evaluating the benefits and limits of multiple displacement amplification with whole-genome Oxford Nanopore sequencing

Authors: AGYABENG-DADZIE, FIIFI - University Of Georgia; BEAUDRY, MEAGAN - University Of Georgia; DEYANOV, ALEX - Houston Methodist Research Institute; SLANIS, HALEY - Houston Methodist Research Institute; DUONG, MINH - Houston Methodist Research Institute; TURNER, RANDI - University Of Georgia; Khan, Asis; ARIAS, CESAR - Houston Methodist Research Institute; KISSINGER, JESSICA - University Of Georgia; GLENN, TRAVIS - University Of Georgia; BAPTISTA, RODRIGO - Houston Methodist Research Institute

Submitted to: Molecular Ecology Resources
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/18/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Long-read sequencing using Oxford Nanopore Sequencing (ONT)typically still requires higher quality and higher quantities of DNA inputs than may be available for many projects. Multiple Displacement Amplification (MDA) is a better alternative to conventional PCR for whole genome amplification when combined with long-read sequencing for low-input samples. Here, we successfully generated almost complete genome sequences for different organisms including Cryptosporidium meleagridis, Staphylococcus aureus, Enterococcus faecium, and Escherichia coli with only 0.025 ng of DNA. We also developed CADECT, a pipeline to remove concatemeric sequences to improve de novo alignment significantly. These results are significant for studying organisms that are difficult to culture and for clinical and produce industry settings with limited DNA from foodborne pathogens.

Technical Abstract: Multiple Displacement Amplification (MDA) outperforms conventional PCR in long fragment and whole genome amplification which makes it attractive to couple with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for genome sequence assembly using Oxford Nanopore Technologies (ONT) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Cryptosporidium meleagridis, Staphylococcus aureus, Enterococcus faecium, and Escherichia coli, with the ability to generate high-quality data from samples starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size-increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.