Validation of PBJelly

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047768#s2" target="_blank"><b>Results</b></a><b>.</b> Using Sanger sequencing of Dpse we validated 7 negative gap closures (A) and 45 closed gaps (B). We also compared PBJelly's gap closing sequence with the original Dmel reference (C).</p

Gap filling Improvements and categories produced by PBJelly.

<p>Histograms showing gap-size distribution in the original and upgraded (A) D .mel, (B) Dpse, (C) Mund, and (D) Caty references as well as a summary of the upgrade categories for gaps.</p

A schematic of PBJelly's workflow and decision-making.

<p>(A) A flow chart of PBJelly's steps. (B) A schematic describing two hypothetical gaps supported by reads and the classifications used during the Support step. (C) A detailed flow chart for local assembly of PacBio reads in a gap region used during the assembly step.</p

Distribution of amount of sequence placed in closed gaps compared to overfilled gaps.

<p>Frequency plots of the absolute value of sequence placed into gaps subtracted from the predicted gap size in closed gaps versus overfilled gaps in (A) Dpse (B) Mund (C) Caty. Data for Dmel is not shown because synthetically inserted gaps' predicted gap sizes matched the amount of sequence that should have been placed into the gaps.</p

Sanger Validation Results Per Gap.

<p>Negative gaps are marked with an asterisk.</p

Description of sequencing data sets used.

<p>Histograms of read lengths in (A) Dmel, (B) Dpse, (C) Mund, (D) Caty. Panel (E) contains detailed metrics of each dataset.</p

Generation and Characterization of Antibodies against Asian Elephant (<i>Elephas maximus</i>) IgG, IgM, and IgA

<div><p>Asian elephant (<i>Elephas maximus</i>) immunity is poorly characterized and understood. This gap in knowledge is particularly concerning as Asian elephants are an endangered species threatened by a newly discovered herpesvirus known as elephant endotheliotropic herpesvirus (EEHV), which is the leading cause of death for captive Asian elephants born after 1980 in North America. While reliable diagnostic assays have been developed to detect EEHV DNA, serological assays to evaluate elephant anti-EEHV antibody responses are lacking and will be needed for surveillance and epidemiological studies and also for evaluating potential treatments or vaccines against lethal EEHV infection. Previous studies have shown that Asian elephants produce IgG in serum, but they failed to detect IgM and IgA, further hampering development of informative serological assays for this species. To begin to address this issue, we determined the constant region genomic sequence of Asian elephant IgM and obtained some limited protein sequence information for putative serum IgA. The information was used to generate or identify specific commercial antisera reactive against IgM and IgA isotypes. In addition, we generated a monoclonal antibody against Asian elephant IgG. These three reagents were used to demonstrate that all three immunoglobulin isotypes are found in Asian elephant serum and milk and to detect antibody responses following tetanus toxoid booster vaccination or antibodies against a putative EEHV structural protein. The results indicate that these new reagents will be useful for developing sensitive and specific assays to detect and characterize elephant antibody responses for any pathogen or vaccine, including EEHV.</p></div

Anti-tetanus toxoid IgG titers in milk and serum following booster vaccination with tetanus toxoid.

<p>The graphs show anti-tetanus IgG antibody titers (y-axis) for milk and serum from an adult female Asian elephant. Samples were tested on days indicated below the x-axis. The results are shown as the mean plus standard deviations from three independent experiments.</p

Immunoblot analysis of Asian elephant serum following fractionation by gel filtration.

<p>Liquid fractions eluting off from gel filtration of Asian elephant serum were resolved and analyzed by immunoblotting. Fraction numbers are labeled at the top and the approximate sizes of marker proteins are indicated. Three identical gels were then probed with isotype specific antisera or monoclonal antibody (anti-IgG) as indicated to the left of each blot. The bottom panel is a coomassie stained gel identical to the gels used for the Westerns above. Molecular weight markers for the blots are shown on the right of each panel.</p

Anti-EEHV1 gL serum titers in two elephants with documented EEHV viremia and shedding.

<p>A) Recombinant EEHV gL expressed in e. coli and purified using metal affinity chromatography. The panel on the left is a coomassie stained gel of purified gL protein and the panel on the right is the same protein detected in an immunoblot using an anti-histidine antibody. Molecular weight markers are indicated on the right. B) The graphs show anti-gL IgG antibody titers (y-axis) for serum from an adult female Asian elephant (elephant 1) and a juvenile male elephant (elephant 2). Samples were tested on days indicated below the x-axis. The results are shown as the mean plus standard deviations from three independent experiments in which each independent experiment had four replicates for each dilution.</p