(A) Following 20 serial infections of the ΔE3L strain (MOI 0.1 for 48 hr) in HeLa cells (see Materials and methods for further details), replication was measured in triplicate in HeLa cells for …
Data used to generate Figure 1A.
Data used to generate Figure 1D.
Statistics for Figure 1D, One-way ANOVA followed by Dunnett’s multiple comparison test.
Replication was measured for plaque purified clones (as in Figure 1D) in BHK cells by 48 hr infection (MOI 0.1) in triplicate. All titers were measured multiple times in BHK cells by plaque assay, …
Data used to generate Figure 1—figure supplement 1.
Statistics for Figure 1—figure supplement 1, One-way ANOVA followed by Dunnett’s multiple comparison test.
Population-level K3LHis47Arg and E9LGlu495Gly allele frequencies were estimated using freebayes on Illumina MiSeq reads from different passages.
Data used to generate Figure 1—figure supplement 2.
A virus clone containing the E9LGlu495Gly variant as the only genetic change relative to ΔE3L was isolated following four rounds of plaque purification in BHK cells (clone a in Figure 1C). …
Data used to generate Figure 1—figure supplement 3.
Statistics for Figure 1—figure supplement 3, unpaired 2-tailed t test with Welch’s correction.
(A) Representative structure of the K3L locus in the VC-2 reference genome is shown on top, with representative Illumina MiSeq and ONT MinION reads shown to scale below. The K3LHis47Arg variant …
Single nucleotide variants in virus populations from Illumina or ONT datasets, used to generate Figure 2B.
Population-level E9LGlu495Gly allele frequencies were estimated using freebayes and nanopolish on Illumina or ONT reads, respectively, from different passages as in Figure 2B.
Data used to generate Figure 2—figure supplement 1.
The proportions of non-reference bases aligned to the 5-mers containing the K3LWT (A), K3LHis47Arg (B), E9LWT (C), or E9LGlu495Gly (D) sequences were calculated from alignments of ONT reads from the …
K3L copy number was assessed in ONT reads from P10, P15, and P20 as described in Figure 2C. Stacked bar plots indicate overall proportions of sequencing reads that contain between 6 and 16 copies of …
Stacked bar plots representing the proportions of mixed and homogeneous K3L arrays were generated from ONT reads for the indicated virus populations (passages are listed above each plot). The …
The K3LHis47Arg allele was uniformly distributed in simulated vaccinia populations with copy number distributions identical to passages P10, P15, and P20 (see Materials and methods for further …
The P15 population was sequenced using R7.3, R9, and R9.4 ONT flowcell chemistries, and stacked bar plots representing the proportions of mixed and homogeneous K3L arrays were generated as in Figure …
(A) Using reads from passage 15 populations sequenced with R7.3, R9, and R9.4 flowcell chemistries, all mixed arrays were converted into homogeneous arrays (see Materials and methods for further …
(A) The proportions of 3-copy K3L arrays containing each possible combination of K3LWT and K3LHis47Arg alleles at P10, P15, and P20 were counted. Dotted red lines separate mixed and homogeneous …
(A) ONT reads from every 5th passage were grouped by K3L copy number, and each K3L copy was assessed for the presence or absence of the K3LHis47Arg SNV. Reads containing 1–5 K3L copies are shown. (B)…
The P10 population was serially passaged in HeLa cells at different MOIs (listed above each plot), and each of the resulting P15 populations was sequenced with ONT. Stacked bar plots representing …
The P10 population was serially passaged five times in BHK cells (MOI = 0.1, 48 hr; P15-BHK). P10 and P15 data are included from previous figures for comparison with P15-BHK. (A) K3L copy number was …
Data used to generate Figure 6D.
Single nucleotide variants in all sequenced virus populations from Illumina or ONT datasets.
Data used to generate Figure 6B.
We selected a random set of 20 vaccinia genomes of the specified copy number from the P15 sequencing data (a passage with a large proportion of mixed genomes), and plotted the distribution of K3LHis4…
(A) Kernel density plots representing the distribution of error rates for T>C, T>A, T>G, and T>deletion error across all TATGC 5-mers in data from each flowcell chemistry. (B) Kernel density plots …
The P15 vaccinia population was sequenced with R7.3, R9, and R9.4 chemistry ONT flowcells. (A) Stacked bar plots representing the diversity of allele combinations within single-copy and multicopy …
Population* | Total sequenced reads | Mean read length (bp) | Read length N50 (bp) | Total sequenced bases (Gbp) | Reads containing K3L |
---|---|---|---|---|---|
P5 | 239,737 | 2168 | 5932 | 0.52 | 1190 |
P10 | 91,815 | 3523 | 7693 | 0.32 | 912 |
P15 | 388,502 | 4493 | 6908 | 1.75 | 4317 |
P20 | 94,050 | 2893 | 7702 | 0.27 | 789 |
*ONT sequencing datasets for all populations are available in Table 1-source data 1
Complete summary of ONT sequencing datasets
Mutation and context (amino acid change) | R7.3 | R9 | R9.4 |
---|---|---|---|
TA[T > C]GC (His47Arg) | 0.023 | 0.023 | 0.005 |
TA[C > T]GC (Arg47His) | 0.015 | 0.024 | 0.026 |
AT[T > C]CG (Glu495Gly) | 0.014 | 0.018 | 0.009 |
AT[C > T]CG (Gly495Glu) | 0.025 | 0.009 | 0.009 |
Breakpoint frequency* | ||||||
---|---|---|---|---|---|---|
Breakpoint | K2L break | K4L break | P5 | P10 | P15 | P20 |
1 | 30,284 | - | 0.76 | 0.69 | 0.76 | 0.66 |
1 | - | 30,837 | 0.76 | 0.63 | 0.72 | 0.62 |
2 | 30,287 | - | 0.14 | 0.06 | 0.10 | 0.08 |
2 | - | 30,840 | 0.12 | 0.04 | 0.09 | 0.05 |
*Due to sequencing errors, a proportion of reads do not match either breakpoint
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Vaccinia virus) | K3L | NA | NCBI_Gene ID:3707649 | |
Strain, strain background (Vaccinia virus) | VC-2, Copenhagen | (Goebel et al., 1990) PMID: 2219722 | NCBI_txid:10249; NCBI_GenBank:M35027.1 | |
Strain, strain background (Vaccinia virus) | ΔE3L, Copenhagen | (Beattie et al., 1995) PMID: 7527085 | ||
Cell line (Homo sapiens) | HeLa | Other | Obtained from Geballelab, University of Washington | |
Cell line (Mesocricetus auratus) | BHK | Other | Obtained from Geballelab, University of Washington | |
Commercial assay or kit | Covaris g-TUBE | Covaris, Inc. | Catalog no: 520079 | |
Commercial assay or kit | DIG High-Prime DNA Labeling and Detection Starter Kit II | Roche | Catalog no: 11585614910 | |
Commercial assay or kit | Nextera XT DNA library preparation kit | Illumina | Catalog no: FC-131–1024 | |
Commercial assay or kit | SQK-NSK007; SQK-LSK208; SQK-LSK308; SQK-RAD002 | Oxford Nanopore Technologies | Catalog no: SQK-NSK007; SQK-LSK208; SQK-LSK308; SQK-RAD002 | |
Commercial assay or kit | FLO-MIN104; FLO-MIN106; FLO-MIN107 | Oxford Nanopore Technologies | Catalog no: FLO-MIN104; FLO-MIN106; FLO-MIN107 | |
Chemical compound, drug | DMEM | HyClone, VWR | Catalog no: 16777–129 | |
Chemical compound, drug | FBS | HyClone, VWR | Catalog no: 26-140-079 | |
Chemical compound, drug | Penicillin-streptomycin | GE Life Sciences, VWR | Catalog no: 16777–164 | |
Chemical compound, drug | SG-2000 | GE Life Sciences, VWR | Catalog no: 82024–258 | |
Software, algorithm | GraphPad Prism | GraphPad Software | ||
Software, algorithm | BWA-MEM | (Li, 2013) | v0.7.15 | arxiv.org/abs/1303.3997 |
Software, algorithm | samblaster | (Faust and Hall, 2014) PMID: 24812344 | v0.1.24 | https://github.com/GregoryFaust/samblaster |
Software, algorithm | freebayes | (Garrison and Marth, 2012) | v1.0.2–14 | arxiv.org/abs/1207.3907 |
Software, algorithm | Metrichor | Oxford Nanopore Technologies | v2.40 | |
Software, algorithm | Albacore | Oxford Nanopore Technologies | v1.2.4 | |
Software, algorithm | poretools | (Loman and Quinlan, 2014) PMID: 25143291 | v0.6.0 | https://github.com/arq5x/poretools |
Software, algorithm | Porechop | Other | v0.2.3 | https://github.com/rrwick/Porechop |
Software, algorithm | nanopolish | (Loman et al., 2015) PMID: 26076426 | v0.8.4 | https://github.com/jts/nanopolish |
Software, algorithm | source code | this paper | See Materials andmethods, https://github.com/tomsasani/vacv-ont-manuscript; copy archived at https://github.com/elifesciences-publications/vacv-ont-manuscript) | |
Software, algorithm | raw sequencing data | this paper | SRP128569; SRP128573; DOI: 10.5281/zenodo.1319732 | See Materials and methods |
Software, algorithm | raw sequencing data | (Elde et al., 2012) PMID: 22901812 | SRP013146 |