Rapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

Rapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with C q values between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

The expanding knowledge of the variety of synthetic genetic elements has enabled the construction of new and more efficient genetic circuits and yielded novel insights into molecular mechanisms. However, context dependence, in which interactions between cis‐ or trans‐genetic elements affect the behavior of these elements, can reduce their general applicability or predictability. Genetic insulators, which mitigate unintended context‐dependent cis‐interactions, have been used to address this issue. One of the most commonly used genetic insulators is a self‐splicing ribozyme called RiboJ, which can be used to decouple upstream 5′ UTR in mRNA from downstream sequences (e.g., open reading frames). Despite its general use as an insulator, there has been no systematic study quantifying the efficiency of RiboJ splicing or whether this autocatalytic activity is robust to trans‐ and cis‐genetic context. Here, we determine the robustness of RiboJ splicing in the genetic context of six widely divergent E. coli strains. We also check for possible cis‐effects by assessing two SNP versions close to the catalytic site of RiboJ. We show that mRNA molecules containing RiboJ are rapidly spliced even during rapid exponential growth and high levels of gene expression, with a mean efficiency of 98%. We also show that neither the cis‐ nor trans‐genetic context has a significant impact on RiboJ activity, suggesting this element is robust to both cis‐ and trans‐genetic changes.

Bacteria often respond to dynamically changing environments by regulating gene expression. Despite this regulation being critically important for growth and survival, little is known about how selection shapes gene regulation in natural populations. To better understand the role natural selection plays in shaping bacterial gene regulation, here we compare differences in the regulatory behaviour of naturally segregating promoter variants from Escherichia coli (which have been subject to natural selection) to randomly mutated promoter variants (which have never been exposed to natural selection). We quantify gene expression phenotypes (expression level, plasticity, and noise) for hundreds of promoter variants across multiple environments, and show that segregating promoter variants are enriched for mutations with minimal effects on expression level. In many promoters, we infer that there is strong selection to maintain high levels of plasticity, and direct selection to decrease or increase cell-to-cell variability in expression. Finally, taking an integrated view, we show that across all phenotypes combined, segregating promoter variants are far more phenotypically similar than would be expected given their genetic divergence. This is the consequence of both stabilizing and directional selection acting on individual phenotypes to minimize differences among segregating variants. Taken together, these results expand our knowledge of how gene regulation is affected by natural selection and highlight the power of comparing naturally segregating polymorphisms to de novo random mutations to quantify the action of selection.

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The expanding knowledge of the variety of synthetic genetic elements has enabled the construction of new and more efficient genetic circuits and yielded novel insights into molecular mechanisms. However, context dependence, in which interactions between proximal (cis) or distal (trans) elements affect the behaviour of these elements, can reduce their general applicability or predictability. Genetic insulators, which mitigate unintended context-dependent cis-interactions, have been used to address this issue. One of the most commonly used genetic insulators is a self-splicing ribozyme called RiboJ, which can be used to decouple upstream 5’ UTR in mRNA from downstream sequences (e.g., open reading frames). Despite its general use as an insulator, there has been no systematic study quantifying the efficiency of RiboJ splicing or whether this autocatalytic activity is robust to trans- and cis-genetic context. Here, we determine the robustness of RiboJ splicing in the genetic context of six widely divergent E. coli strains. We also check for possible cis-effects by assessing two SNP versions close to the catalytic site of RiboJ. We show that mRNA molecules containing RiboJ are rapidly spliced even during rapid exponential growth and high levels of gene expression, with a mean efficiency of 98%. We also show that neither the cis- nor trans-genetic context has a significant impact on RiboJ activity, suggesting this element is robust to both cis- and trans-genetic changes.

Bacterial cells often respond to changes in the environment by modifying protein expression. This can be achieved through changes in transcriptional or translational activity, or both. Recent research has shed some light on how natural selection shapes overall protein expression. Still, little is known about how selection acts on transcription or translation individually. To address part of this question, we implement an experimental system which allows us to measure how genetic changes affect transcription only, excluding the effects on translation. We use this system to quantify changes in three regulatory phenotypes of the lacZ promoter: transcriptional activity, plasticity, and cell-to-cell variability. We compare these phenotypes from segregating variants that have been subject to natural selection, and random variants that have never been subjected to natural selection. We show that natural selection filters out mutations causing large changes in transcriptional levels from the lacZ promoter. Further, we detect directional selection acting on transcriptional plasticity in combinations of glucose, galactose and lactose environments. Focusing on cell-to-cell variability in transcription, we describe both directional and diversifying selection acting on this phenotype depending on the environment used. We also observe a link between the phylogeny of the environmental E. coli strains and high and low transcriptional noise levels in glucose which are mediated by just one or two SNPs. Our results thus provide new insight into how one of the most well-characterized bacterial promoters is shaped in nature by selection.