High-throughput NAb Support
Britt Piehler
LabKey Software
July 20, 2010
Background
The Atlas scientific data portal includes a widely-adopted tool for uploading and processing Neutralizing
Antibody (NAb) assay run data. Users provide this tool with a raw instrument file produced by their
luminometer, as well as metadata about the batch, run, and samples being processed. This information
is combined and analyzed automatically by the system to estimate the titers at which a specified level of
neutralization is seen for each sample/virus combination.
This tool has been successfully adopted by many labs performing "standard" NAb assay runs. A single
run for these labs utilizes a 96-well plate, typically prepared with five specimens in eight replicated
dilutions. The current Atlas NAb tool has been optimized for this scenario.
Revisions/extensions to this tool have been requested by NVITAL, which seeks to utilize
automated/robotic systems capable of high throughput NAb data processing. This system differs from
low-throughput NAb in the following ways:



The system uses 384-well plates, allowing for more samples per run.
A run may consist of up to eight plates, further multiplying the possible throughput.
Each sample is diluted across plates, rather than within a single plate.
Assumptions and limitations
Several assumptions have been made while preparing this plan.
Analysis
We assume that the existing analysis techniques employed for low-throughput runs are sufficient for
high-throughput runs. Specifically, we assume that current curve fitting needs are met by the 5
parameter, 4 parameter, and polynomial approaches, and that curve-based titers, point-based titers,
areas under the curves, positive areas under the curves, and curve fit error estimates are sufficient
analysis results.
Required plate layouts
We assume that it will be sufficient to support two types of NAb runs:

High-throughput 384-well NAb runs with dilutions across plates , where all dilutions for a given
sample exist in the same location on each plate.
1

Low-throughput 96-well NAb runs, with dilutions within a single plate.
[QUESTION: Is it necessary to also support dilution across multiple 96-well plates?]
As a result, support for many theoretical plate layouts is not planned. Two examples are:


The use of a 96-well plate layout multiplied four times on a single 384-well plate.
Hybrid dilution schemes, where the first four dilutions of a given sample are on one plate, while
the second four dilutions are on a second plate.
Visualization
We assume that it is not necessary to present a graphical "run details" view for a high-throughput run,
since it is not practical to present a graph containing up to 140 samples. Instead, run results will be
presented in the current results grid view, which is a sortable, filterable grid of data with one row per
sample. It should be possible to generate dilution/neutralization graphs for specific samples by selecting
them in the grid view.
[QUESTION: Some information on the low-throughput graphical run details view is not available in the
current results grid views. This information includes control range, deviation of replicates, and the
raw plate data. Will it be necessary to make any of this data available in the results grid view?]
Planned support for 10.3
384-well plates
Development estimate: 4 days
Support for 384 well plates will require modifications to the Atlas plate designer and the NAb data
processing algorithms. With substantially more wells per plate, the plate designer will need to support
bulk selection of wells, either through a click-and-drag mechanism, or via simple selection of entire rows
and columns. Keyboard shortcuts for creating new well groups may be required. Additional
modifications will be required to generalize the NAb data processing code to eliminate any assumptions
that plates consist of 96 wells.
[QUESTION: What plate designer enhancements will be most valuable for SCHARP's LDO group?]
Multi-plate data upload and processing
Development estimate: 10 days
Because a single high-throughput NAb run may consist of up to 8 plates of data, it will be necessary to
collect multiple plates of data for each run. More information is needed from the lab regarding how this
data will be generated in the file system by the luminometer. Three possible data formats have been
identified:
2



Each plate of data may be stored in a separate TSV or Excel data file. In this case, the uploading
user will be asked to select each of the data files individually, specifying dilution order.
Each plate of data may be saved in a separate worksheet in a single Excel data file. In this case,
the uploading user will select the single file, and dilution order will be assumed based on sheet
order.
Data for all plates may be found in a single Excel sheet or TSV file, with the plates listed
sequentially (either horizontally or vertically) within the file. In this case, the uploading user will
select the single file, and dilution order will be assumed based on location in the file.
Only one of these three formats will be supported in the final tool, based on what is most convenient for
NVITAL's staff and equipment. Once a file format is decided, internal data processing will be modified to
support sample dilution across plates.
[QUESTION: When NVITAL know which data file format is produced?]
File-based sample metadata
Development estimate: 3 days
Because it is not practical for a data uploader to enter metadata for up to 140 samples through a
browser-based form on the Atlas website, an additional metadata file will be required. This file will be a
simple Excel or TSV file, with one line per sample. The user will be prompted to provide this file along
with the plate data files. The file must contain the following metadata for each sample:





Unique sample identifier. May consist of a vial ID, participant/visit ID combination, or
participant/date combination.
Dilution/concentration of sample on the first plate
Dilution/concentration factor for each successive plate
Whether the sample is being diluted or concentrated
Plate location identifier. This may be an absolute plate location (e.g., "A1"), or a reference to an
identifier from the plate template. [QUESTION: What is the best way to identify a sample's
location?]
Additional metadata may be configured, collected, and saved with the sample results, but this
information will not be used as part of the analysis.
[QUESTION: What metadata file format does NVITAL prefer?]
3