RT Journal Article
SR Electronic
T1 MAUI-seq: Multiplexed, high-throughput amplicon diversity profiling using unique molecular identifiers
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 538587
DO 10.1101/538587
A1 Bryden Fields
A1 Sara Moeskjær
A1 Ville-Petri Friman
A1 Stig U. Andersen
A1 J. Peter W. Young
YR 2019
UL http://biorxiv.org/content/early/2019/02/03/538587.abstract
AB Correcting for sequencing and PCR errors is a major challenge when characterising genetic diversity using high-throughput amplicon sequencing (HTAS). Clustering amplicons by sequence similarity is a robust and frequently used approach, but it reduces sensitivity and makes it more difficult to detect differences between closely related strains. We have developed a multiplexed HTAS method, MAUI-seq, that incorporates unique molecular identifiers (UMIs) to improve correction. We profile Rhizobium leguminosarum biovar trifolii (Rlt) synthetic DNA mixes and Rlt diversity in white clover (Trifolium repens) root nodules by multiplexed sequencing of two plasmid-borne nodulation genes and two chromosomal genes. We show that the two main advantages of UMIs are efficient elimination of chimeric reads and the ability to confidently distinguish alleles that differ at only a single position. In addition, multi-amplicon profiling of genes from different replicons enables increased diversity profiling resolution, allowing us to demonstrate limited strain overlap between geographically distinct locations. The method does not rely on commercial library preparation kits and provides cost-effective, sensitive and flexible profiling of intra-species diversity.