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Reconstruction of evolving gene variants and fitness from short sequencing reads

Nature Chemical Biology volume 17, pages 1188–1198 (2021)Cite this article

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Abstract

Directed evolution can generate proteins with tailor-made activities. However, full-length genotypes, their frequencies and fitnesses are difficult to measure for evolving gene-length biomolecules using most high-throughput DNA sequencing methods, as short read lengths can lose mutation linkages in haplotypes. Here we present Evoracle, a machine learning method that accurately reconstructs full-length genotypes (R2 = 0.94) and fitness using short-read data from directed evolution experiments, with substantial improvements over related methods. We validate Evoracle on phage-assisted continuous evolution (PACE) and phage-assisted non-continuous evolution (PANCE) of adenine base editors and OrthoRep evolution of drug-resistant enzymes. Evoracle retains strong performance (R2 = 0.86) on data with complete linkage loss between neighboring nucleotides and large measurement noise, such as pooled Sanger sequencing data (~US$10 per timepoint), and broadens the accessibility of training machine learning models on gene variant fitnesses. Evoracle can also identify high-fitness variants, including low-frequency ‘rising stars’, well before they are identifiable from consensus mutations.

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Fig. 1: Genotype reconstruction during evolution from short-read sequencing data with incomplete physical linkage.
Fig. 2: Evolutionary fitness reconstruction from pooled Sanger sequencing of OrthoRep campaigns.
Fig. 3: Evolutionary time-series frequency reconstruction from non-continuous directed evolution data.
Fig. 4: Robustness to shorter read lengths.
Fig. 5: Robustness to measurement noise.
Fig. 6: Model-guided fitness optimization.

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Data availability

The sequencing data generated during this study are available at the NCBI Sequence Read Archive database under accession code PRJNA625117. Processed data have been deposited at https://doi.org/10.6084/m9.figshare.12121359.

Code availability

The code used for data processing and analysis are available at https://github.com/maxwshen/evoracle-dataprocessinganalysis. The Evoracle model is available at https://github.com/maxwshen/evoracle.

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Acknowledgements

This work was supported by NIH R01 EB031172, R01 EB027793 and R35 GM118062, and the HHMI. We acknowledge an NSF Graduate Research Fellowship to M.W.S. We thank A. Vieira for assistance editing the manuscript.

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Authors and Affiliations

  1. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Max W. Shen, Kevin T. Zhao & David R. Liu

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Max W. Shen, Kevin T. Zhao & David R. Liu

  3. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Max W. Shen, Kevin T. Zhao & David R. Liu

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Contributions

Conceptualization, investigation, and computational and statistical analyses were performed by M.W.S. Data curation and formal analysis were conducted by M.W.S. Software and methodology were provided by M.W.S. and resources by K.T.Z. Validation was performed by M.W.S. Project administration was carried out by M.W.S. and D.R.L. The manuscript was written by M.W.S. and D.R.L. Visualization was provided by M.W.S. Supervision and funding acquisition were performed by D.R.L. TadA next-generation sequencing was performed by K.T.Z.

Corresponding author

Correspondence to David R. Liu.

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Competing interests

D.R.L. is a co-founder of Beam Therapeutics, Prime Medicine, Editas Medicine and Pairwise Plants, companies that use genome editing technologies.

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Extended data

Extended Data Fig. 1 Evoracle model properties.

a, Regularization strategies. Comparison of loss incurred by L2 norm, variance, normalized statistical skew, and unnormalized statistical skew (our skew) regularizers for distributions of three variables. b, Synthetic data to demonstrate the utility of the skew regularizer. The top left graph shows a ground-truth simulated population containing only a wild-type genotype and a double mutant. Observed single-mutation frequencies from the ground-truth simulation were used by Evoracle to infer full-length genotype trajectories of the wild-type genotype, both single mutants, and the double mutant. Evoracle was performed with varying values of beta (top right, bottom left, and bottom right). When beta is higher, Evoracle more correctly infers the ground-truth trajectories. Inferred genotype frequencies are plotted with a small jitter to show overlapping lines clearly. c, Robustness to hyperparameters. Performance while varying hyperparameters alpha and beta for Cry1Ac data. Reported statistics summarize performance across ten replicates with random parameter initializations.

Extended Data Fig. 2 Evaluating Evoracle’s genotype proposal strategy.

a-b, Sequence proposal strategies. Performance with varying full-length genotype proposal strategies for (a) Cry1Ac data, and (b) TadA data. N = 40 replicates. Box plot depicts median and interquartile range. Default strategy is described in the Methods; x2 to x100 represent adding full-length genotypes comprising combinations of mutations to increase the total number of reconstructed full-length genotypes by the stated multiplicative factor of the default number. See Methods for more details.

Extended Data Fig. 3 Evolutionary fitness reconstruction from pooled Sanger sequencing of OrthoRep campaigns.

a, Comparison of ground-truth and inferred fitness, indicating a negative epistatic interaction between A-76V and D384Y, S404C in Cry1Ac. b-d, Comparison of MIC values and inferred fitness for evolved PfDHFR variants.

Extended Data Fig. 4 Evoracle performance on ABE8e evolution replicates.

Model evaluation on replicate PACE experiments 1 and 3 of the ABE8e directed evolution campaign. Samples 1-20 are from low-stringency PANCE, samples 21-29 are from high- stringency PANCE, and samples 30-36 are from PACE. a, Observed frequencies of 34 mutations. Colors represent amino acid mutations, using the same coloring scheme as in Fig. 2a-b. b, Observed full-length genotype trajectories. Colors represent full-length genotypes, using the same coloring scheme as in Fig. 2c-d. c, Inferred full-length genotype trajectories. Colors represent full-length genotypes, using the same coloring scheme as in Fig. 2c-d. d, Consistency between observed and predicted full-length genotype frequencies; scatter plot and swarm plot with kernel density estimate.

Extended Data Fig. 5 Evaluation of fitness inference.

a-b, Comparison of inferred fitness to fitness calculated from full-length reads for (a) Cry1Ac and (b) TadA.

Extended Data Fig. 6 Evoracle performance with varying sequencing read depth.

a-b, Full-length genotype reconstruction performance across timepoints with varying simulated read depths using binomial samples for (a) Cry1Ac and (b) TadA. Box plot depicts median and interquartile range. N = 50 independent replicates with random seeds.

Extended Data Fig. 7 Comparison to related methods.

a, Observed Cry1Ac (2,138 nt) genotypes from 34 timepoints (spanning 528 h) of PACE from PacBio long-read sequencing data. Colors represent distinct genotypes. Figure is the same as Fig. 1c and reproduced for convenience. b, Cry1Ac genotype frequencies reconstructed by SGML. Gray lines indicate genotypes that are not present in PacBio data. c, Comparison of performance by clonal Sanger sequencing depth compared to pooled Sanger sequencing. Box plots indicate median and interquartile range, and whiskers indicate extrema. N = 50 random seed replicates. d, Comparison of rising star performance by clonal Sanger sequencing depth vs pooled Sanger sequencing on 12 h interpolated Cry1Ac data.

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Shen, M.W., Zhao, K.T. & Liu, D.R. Reconstruction of evolving gene variants and fitness from short sequencing reads. Nat Chem Biol 17, 1188–1198 (2021). https://doi.org/10.1038/s41589-021-00876-6

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