UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution

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.

Freshwater monitoring by nanopore sequencing

“…Using Minimap2, 16.2% of freshwater-derived sequencing reads were assigned to a bacterial species on average, thereby primarily encouraging automated analyses on the genus (65.6% assigned) or family level (76.6% assigned). As nanopore sequencing quality continues to increase through refined pore chemistries, basecalling algorithms and consensus sequencing workflows ( Calus et al, 2018 ; Karst et al, 2021 ; Latorre-Pérez et al, 2020 ; Rang et al, 2018 ; Santos et al, 2020 ; Zurek et al, 2020 ), future bacterial taxonomic classifications are likely to improve and advance opportunities for species discovery.…”

Recent trends in biocatalysis

“…An increased absorbance signal triggers an electric pulse that moves the droplet into the sorting channel. 44,45 (D) Droplets can be split, evenly or unevenly, into smaller droplets. This is useful for example if a destructive detection method like mass spectrometry is used, so the first droplet is sacrificed, and the second droplet sorted.…”

“…Just recently, the Hollfelder group published the first follow up AADS papers, albeit for the same type of reaction and detection system. 45,57 As far as we know, no other group has published the use of AADS for directed evolution. There might be several reasons for this.…”

(R)evolution-on-a-chip

“…The utility of the approach was demonstrated by evolving a dehydrogenase variant with improved activity after two rounds of directed evolution [74]. AADS was again applied for the evolution of an amine dehydrogenase [75]. This time best hit droplets were isolated by AADS and their content was sequenced by nanopore sequencing to generate…”