Millstone: Software for Multiplex Microbial Genome Analysis and Engineering

Goodman, Daniel B.; Kuznetsov, Gleb; Lajoie, Marc J.; Ahern, Brian W.; Napolitano, Michael G.; Chen, Kevin Y.; Chen, Changping; Church, George M.

doi:10.1101/086868

Cited by 2 publications

(2 citation statements)

References 17 publications

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“…CP006698.1) and the sequence of the plasmid encoding nsAA incorporation machinery. The Millstone software suite was used to identify variants, provide measures of sequencing confidence, and predict their likelihood of altering gene function (49). Genomic variants of low confidence, low sequence coverage, or presence in the ancestral strain were discarded, prioritizing variants observed in three nonessential genes that encode membrane proteins: acrB, emrD, and trkH.…”

Section: Genome Resequencing and Analysismentioning

confidence: 99%

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells

et al. 2021

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Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.

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Section: Genome Resequencing and Analysismentioning

confidence: 99%

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells

et al. 2021

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“…To determine the abundance of retron donors conferring SNPs, reads from amplicons obtained after selection were aligned to the MG1655 reference genome and SNPs inferred using Millstone 10 . Depth of SNP coverage in the resulting output reflects abundance in the pool, and was used for visualization and analysis.…”

Section: Methodsmentioning

confidence: 99%

High throughput functional variant screens via in-vivo production of single-stranded DNA

Schubert

Goodman

Wannier

et al. 2020

Preprint

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5Tremendous genetic variation exists in nature, but our ability to create and characterize 6 individual genetic variants remains far more limited in scale. Likewise, engineering proteins and 7 phenotypes requires the introduction of synthetic variants, but design of variants outpaces 8 experimental measurement of variant effect. Here, we optimize efficient and continuous 9 generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded 10 DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing 11 efficiency can be obtained using this method, enabling multiplexed applications. We introduce 12 Retron Library Recombineering (RLR), a system for high-throughput screens of variants, 13 wherein the association of introduced edits with their retron elements enables a targeted deep 14 sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized 15 variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared 16 genomic DNA of an evolved bacterium, experimentally querying millions of sequences for 17 antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize 18 non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus 19 present new avenues for exploring variation, both designed and not, across the entire genome. 20 21 Introduction: 22Constructing genotypes of interest and observing their effect on phenotype critically aids 23 our understanding of genetics and genome function. As methods for editing genomes have 24 progressed, this "reverse genetics" approach has expanded in breadth and scale, from knockout 25 libraries 1 to refactored genomes 2,3 . These experiments can now be performed within multiplexed 26 pools, which allow an ever greater number of mutations to be explored across varied conditions. 27Critically, both creating genotypes and observing phenotype within pools has necessitated 28 2 development of new techniques. Transposon insertions 4 , marked integrations 5 and CRISPR-29 inhibition 6,7 can create thousands of variants simultaneously within pooled experiments, and 30 targeted sequencing of these elements enables pooled measurement of variant phenotypes. These 31 advancements in experimental scale have fundamentally transformed our understanding of 32 genome function 8 . 33However, these current high-throughput genetic techniques remain limited, in that they 34 typically introduce, ablate, or regulate kilobases of DNA to create variation. This contrasts with 35 point mutations, which are ubiquitous in natural variation 9 , and are indispensable for engineering 36 proteins 10 and metabolic pathways 11 . While modifying kilobases of DNA can add and subtract 37 functional elements such as genes and regulatory sequences from the genome, point mutations 38 can alter the function of these elements, accessing a larger phenotypic landscape. 39Point mutations and other precision edits can be performed by oligonucleo...

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Millstone: Software for Multiplex Microbial Genome Analysis and Engineering

Abstract: Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform which automates genotype comparison and

Cited by 2 publications

References 17 publications

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells

High throughput functional variant screens via in-vivo production of single-stranded DNA

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