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UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution.

Accepted version
Peer-reviewed

Type

Article

Change log

Authors

Knyphausen, Philipp 
Neufeld, Katharina 
Pushpanath, Ahir 
Hollfelder, Florian  ORCID logo  https://orcid.org/0000-0002-1367-6312

Abstract

The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein's amino acids ('intra-gene epistasis'). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non-additive interactions between mutations within a full gene showing sign epistasis. Access to deep and accurate long reads will facilitate prediction of key beneficial mutations and adaptive potential based on in silico analysis of large sequence datasets.

Description

Keywords

Biocatalysis, Cloning, Molecular, Computational Biology, Consensus Sequence, Datasets as Topic, Directed Molecular Evolution, Enzyme Assays, Epistasis, Genetic, Gene Library, High-Throughput Nucleotide Sequencing, High-Throughput Screening Assays, Mutagenesis, Mutation, Oxidoreductases Acting on CH-NH Group Donors, Phylogeny, Protein Engineering, Recombinant Proteins, Software

Journal Title

Nat Commun

Conference Name

Journal ISSN

2041-1723
2041-1723

Volume Title

11

Publisher

Springer Science and Business Media LLC

Rights

All rights reserved
Sponsorship
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (722610)
European Commission Horizon 2020 (H2020) ERC (695664)
Eu H2020