Percentage of errors by type of acquired diversity determined during sample preparation.

<p>(A) Percentage of error attributed to synonymous vs. non-synonymous variants. (B) Percentage of errors attributed to transitions vs. transversions. (C) Percentage of errors attributed to insertions or deletions vs. Intra-host single nucleotide variants (iSNV).</p

Study sample diagram.

<p>Study sample diagram.</p

Sources of error per sample preparation method.

<p>(A) Intra-host single nucleotide variants (iSNVs) that were present in multiple samples at greater than 1% of population are shown in a heat map format to visualize patterned diversity acquired during sample preparation. The total number of samples containing iSNVs in greater than 1% of population are summarized in the column “#> 1% of Pop” in green. “Codon” column (second column from right) provides both the nucleotide and protein translation of the site. (B) The detected mean error rates of iSNVs greater than 0. 2% of population (error/site/copy) are stratified by presence in the plasmid (Origin), detection after transcription/reverse transcription (Transc) or preparation, and preparation/sequencer error (Prep). (C) Error profiles are expressed as the number of iSNVs per percent of population obtained from each of the 5 sample preparation methods.</p

Total error by sample preparation method.

<p>(A) The mean read depth per position from two DNA controls (PLASMID and P_AMP) and five RNA sample preparation methods (n = 2 of replicate experiments). (B) Errors calculated as error/site/copy (plasmid or transcript) are presented as the average of duplicate experiments as in A. Intra-host single nucleotide variants present in the original plasmid were removed from the calculation of the error (reference positions: 4,697 and 4,725). Student t-tests were performed to demonstrate differences between the mean error per site per copy in the control plasmid (* = <i>p</i><0.05) and each sample preparation method. (C) The error calculated in B is converted to fold change over the control (“PLASMID”). Error bars in all panels represent standard deviation of the mean.</p

Intrahost single nucleotide variants (iSNVs) of EBOV/Kik-9510621 “R4415” (passage 3) comprising ≥2% of total population as compared to “134” (GenBank #AY354458).

<p>Intrahost single nucleotide variants (iSNVs) of EBOV/Kik-9510621 “R4415” (passage 3) comprising ≥2% of total population as compared to “134” (GenBank #AY354458).</p

Intrahost single nucleotide variants (iSNVs) of EBOV/Kik-9510621 “R4368” (passage 4) comprising ≥2% of total population as compared to “134” (GenBank #AY354458).

<p>Intrahost single nucleotide variants (iSNVs) of EBOV/Kik-9510621 “R4368” (passage 4) comprising ≥2% of total population as compared to “134” (GenBank #AY354458).</p

<i>GP</i> gene editing site composition in EBOV/Kik-9510621 NHP challenge stocks.

<p><i>GP</i> gene editing site composition in EBOV/Kik-9510621 NHP challenge stocks.</p

History of Ebola virus variant Kikwit (isolate 9510621) challenge stocks used at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID), CDC, Centers for Disease Control and Prevention; NHP, nonhuman primate; SPBLOG, Special Pathogens Branch Log; UTMB, University of Texas Medical Branch; VSP, virus seed pool.

<p>History of Ebola virus variant Kikwit (isolate 9510621) challenge stocks used at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID), CDC, Centers for Disease Control and Prevention; NHP, nonhuman primate; SPBLOG, Special Pathogens Branch Log; UTMB, University of Texas Medical Branch; VSP, virus seed pool.</p

Stock identifier alias table.

*<p>Stock ID is used to identify the stock in the text.</p

Comparison of EBOV-Kik <i>in vitro</i> passage with <i>in vivo</i> infection.

<p>EBOV-Kik samples from passage 2, 3, and 4 in Vero E6 cells (represent 1, 4 and 3 isolates respectively) were compared to six in vivo samples from lethally challenged crab-eating macaques collected after day 4. A passage 3 viral stock from this study (16502) was used as the challenge material for the <i>in vivo</i> samples. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050316#pone-0050316-g001" target="_blank">Figure 1</a> for passage and seed stock information. Numbers are reported here as percent of population for sub-clonal variants. Error bars represent variance between multiple independently propagated lineages or infected animals (i.e. The data is summarized based on passage number or infection day rather than experimental or individual bounds). a) GP Poly-U transition. Here, we compare two variants of the 8U form that expressed predominantly the full length GP_1, and the 7U variant that predominantly expressed sGP. <i>In vitro</i>, we observed a dramatic and rapid shift between the 7U variant and the 8U variant at positions 6,925 and 6,926. By passage 3, there is an inversion in variant levels and there is an equally rapid reversion observed by day 8 <i>in vivo</i>. b) Stop Codon detection. There is a twofold increase in the amount of the sub-clonal variant encoding for a truncated form of GP_1,2 at position 6,677.* Note: the scale is changed to 10% for better visualization. c) and d). Marker increase <i>in vivo</i>. We identify two changes 6,179, and 10,833, which result in amino acid changes ₂ protein in GP_1,2 and VP24 respectively. As with the 7U variant, the subclonal variants at these positions decrease and revert rapidly when switching between cell culture passage and infection.</p