Mutational and fitness landscapes of an RNA virus revealed through population sequencing

Acevedo, Ashley; Brodsky, Leonid; Andino, Raul

doi:10.1038/nature12861

Cited by 362 publications

(481 citation statements)

References 39 publications

(33 reference statements)

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“…In such cases, care should be taken to minimize amplification cycles upstream of the circle-sequencing pipeline. Alternately, circle sequencing can also be applied directly to RNA templates (11).…”

Section: Resultsmentioning

confidence: 99%

High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing

Lou¹,

Hussmann²,

McBee³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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234

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A major limitation of high-throughput DNA sequencing is the high rate of erroneous base calls produced. For instance, Illumina sequencing machines produce errors at a rate of ∼0.1-1 × 10 −2 per base sequenced. These technologies typically produce billions of base calls per experiment, translating to millions of errors. We have developed a unique library preparation strategy, "circle sequencing," which allows for robust downstream computational correction of these errors. In this strategy, DNA templates are circularized, copied multiple times in tandem with a rolling circle polymerase, and then sequenced on any high-throughput sequencing machine. Each read produced is computationally processed to obtain a consensus sequence of all linked copies of the original molecule. Physically linking the copies ensures that each copy is independently derived from the original molecule and allows for efficient formation of consensus sequences. The circlesequencing protocol precedes standard library preparations and is therefore suitable for a broad range of sequencing applications. We tested our method using the Illumina MiSeq platform and obtained errors in our processed sequencing reads at a rate as low as 7.6 × 10 −6 per base sequenced, dramatically improving the error rate of Illumina sequencing and putting error on par with low-throughput, but highly accurate, Sanger sequencing. Circle sequencing also had substantially higher efficiency and lower cost than existing barcode-based schemes for correcting sequencing errors.next-generation sequencing | barcoding | rare variants

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Section: Resultsmentioning

confidence: 99%

High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing

Lou¹,

Hussmann²,

McBee³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

250

234

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show abstract

“…This is one of a growing number of approaches that enable and promote cross-disciplinary biological research (Harms and Thornton 2013). Pioneering approaches to sequence virus populations with extreme depth have recently provided valuable views of the landscape of mutation effects across a genome (Acevedo et al 2014). This genome-wide study of poliovirus showed a bimodal distribution of fitness effects with a large cluster of mutations that were of minimal impact and a second cluster that were null.…”

Section: Concluding Thoughtsmentioning

confidence: 99%

Viewing Protein Fitness Landscapes Through a Next-Gen Lens

Boucher¹,

Cote²,

Flynn³

et al. 2014

Genetics

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High-throughput sequencing has enabled many powerful approaches in biological research. Here, we review sequencing approaches to measure frequency changes within engineered mutational libraries subject to selection. These analyses can provide direct estimates of biochemical and fitness effects for all individual mutations across entire genes (and likely compact genomes in the near future) in genetically tractable systems such as microbes, viruses, and mammalian cells. The effects of mutations on experimental fitness can be assessed using sequencing to monitor time-dependent changes in mutant frequency during bulk competitions. The impact of mutations on biochemical functions can be determined using reporters or other means of separating variants based on individual activities (e.g., binding affinity for a partner molecule can be interrogated using surface display of libraries of mutant proteins and isolation of bound and unbound populations). The comprehensive investigation of mutant effects on both biochemical function and experimental fitness provide promising new avenues to investigate the connections between biochemistry, cell physiology, and evolution. We summarize recent findings from systematic mutational analyses; describe how they relate to a field rich in both theory and experimentation; and highlight how they may contribute to ongoing and future research into protein structure-function relationships, systems-level descriptions of cell physiology, and population-genetic inferences on the relative contributions of selection and drift.

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“…Circle sequencing (cirseq) deep-sequencing methods can be used in the future to more thoroughly characterize the manner in which 3D pol mutations and RNA recombination impact the fidelity of viral RNA replication (52,53). Cirseq methods would be especially helpful when 45,000 bases of TOPO-TA cDNA reveal limited numbers of mutations per genome (Table 4).…”

Section: Dmentioning

confidence: 99%

Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

Kempf

Peersen

Barton

2016

J Virol

107

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RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3D pol , as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3D pol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3D pol -RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3D pol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3D pol increased the frequency of recombination. The 3D pol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G¡A and C¡U mutations in viral RNA. We conclude that 3D pol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs).The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a polymerase mutation that disables recombination renders the virus more susceptible to the antiviral drug ribavirin, suggesting that recombination contributes to ribavirin resistance. Elucidating the molecular mechanisms of RNA replication and recombination may help mankind achieve and maintain poliovirus eradication. Picornaviruses are widespread in nature. Based on their ancient origins (1) and shared molecular features (2), picornaviruses are taxonomically organized by order, family, genus, species, and virus. Viruses in the family Picornaviridae are classified within 29 genera and 50 species groups. The genus Enterovirus, with 12 species groups (enterovirus species A to J and rhinovirus species A to C), contains the vast majority of medically important picornaviruses, including the polioviruses (PV), coxsackieviruses, enteroviruses, echoviruses, and rhinoviruses. These viruses, which possess single-stranded positive-sense RNA genomes, replicate via RNA intermediates in the cytoplasm of infected cells. A virus-encoded RNA-dependent RNA polymerase, 3D pol...

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Mutational and fitness landscapes of an RNA virus revealed through population sequencing

Cited by 362 publications

References 39 publications

High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing

High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing

Viewing Protein Fitness Landscapes Through a Next-Gen Lens

Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

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