I N N O V A T I O N S F O R U M

High-throughput genotyping
N. Stover
Amersham Biosciences Inc., Sunnyvale, CA

MegaBACE™ 1000 Genotyping System is a high-
G throughput, fluorescence-based DNA genotyping
E system utilizing capillary electrophoresis with 96
N capillaries operating in parallel. The system automates
O sample injection, gel matrix replacement, DNA
T separation, detection, and data analysis.
Y
P Introduction
I Genotyping is used to help determine which genes are
N responsible for specific inherited traits. Identifying
G these genetic markers can help researchers understand
the mechanisms behind certain, inherited medical
disorders. The knowledge gained through genotyping
can then be used to improve early detection and
treatment of disease.
MegaBACE 1000 Genotyping System
The current method for production-scale genotyping
is a time-consuming process because researchers must
manually analyse data in order to locate the informa-
tion of interest. MegaBACE 1000 Genotyping System
streamlines this process by automating all aspects of
genotyping. Combined with MegaBACE Genetic
Profiler analysis software, MegaBACE 1000 Geno-
typing System automatically processes 96 samples per
run. The total turnaround time per run is less than two
hours from injection to injection, corresponding to
more than 20 000 genotypes per system in 24 hours
of operation.
MegaBACE Genetic Profiler
MegaBACE Genetic Profiler analysis software is the
first genotyping program to fully integrate fragment
sizing with allele calling in a Windows NT™ format.
MegaBACE Genetic Profiler greatly reduces manual
review of data by assigning quality scores to every
marker and sorting the samples based on how well a
marker fits within a specified pattern. Genotypes with
high quality scores do not require further review,
enabling researchers to focus on genotypes with
lower quality markers.
The software further streamlines data management by
enabling multiple runs to be viewed and assembled
together in one display as an entire project. The runs
added to a project share the same analysis parameters,
such as sizing, dye set and marker panel set.

Instrument Control Manager MegaBACE Genetic Profiler

MegaBACE Data Collection Automated Genotyping
Run n
Trace Processing
Run ... ■ Background subtraction
Run 2 ■ Spectral separation
■ Peak smoothing
MegaBACE Genetic Profiler
Run 1 Manual Review
and Editing
Fragment Sizing
■ Size standard peak finding
■ Label all marker peaks
MegaBACE Genetic Profiler
MegaBACE Allele Calling Project Summary Reports
Genetic Profiler ■ Peak selection ■ Allele calls of all runs
Project Setup ■ Allele histogram or selected runs for
■ Dye set each marker
■ Marker panels ■ Run-by-run comparisons
■ Sizing parameters Genotype Calling for each marker
■ Genotype quality scores ■ Allele tables

Fig 1. MegaBACE Genetic Profiler workflow.

Life Science News 4, 2000 Amersham Biosciences

1
I N N O V A T I
Automated genotyping
MegaBACE Genetic Profiler automates genotyping
through a progression of steps, including trace pro-
cessing, fragment sizing, allele calling, and genotype
calling (Fig 1).
Trace processing consists of baseline subtraction,
spectral separation, and peak smoothing. Baseline
subtraction places the baseline signal intensity of all
four traces of the raw electropherogram at the same
level to provide a better comparison of relative signal
intensity. Spectral separation uses a spectral cross-talk
matrix that removes the unwanted signals from other
colours in each data channel.
MegaBACE Genetic Profiler then calculates the
length of the DNA fragments and identifies the
peaks in the “unknown” sample data using an internal
DNA standard of known fragment size. The ET dye-
labelled size standard (ET400-R) is recommended for
MegaBACE 1000 Genotyping System because its high
intensity assures that every band will be detected,
making it ideal for reproducible sizing.
Allele calling
Allele calling is performed on the microsatellite PCR
products by selecting the allele peaks from the sized
peak list for each trace. Peak selections are based on
the size range of the marker and the expected peak
pattern for the marker type. The software then assigns
the best fit of the peaks to allele bins. Peak selections
that fall within a given allele bin are called as the same
allele. Alleles for a given marker can then be quickly
viewed together in a histogram format where the dark
vertical bars indicate the frequency of alleles within a
given bin (Fig 2).
Fig 2. The main window displaying data for a selected marker. The
histogram of the marker is in top right window; the trace area in lower
right and the allele tables in the lower left window.
Life Science News 4, 2000 Amersham Biosciences
O N S F O R U M
MegaBACE Genetic Profiler gives the sample identifi-
cation and the two alleles for each marker genotype in
tabular format. In addition, it assigns a quality value
for each genotype based on how well the designated
allele peaks fit the expected peak pattern for the
marker. With high quality markers, analysis time
amounts to scanning the quality scores—less than
one minute. With markers of varying quality, manual
review and editing can be focused on those giving
lower quality scores. Both sized peaks and alleles
can be manually reviewed and edited within
MegaBACE Genetic Profiler.
MegaBACE Genetic Profiler also has the flexibility to
create several Project Summary Reports that enable
the researcher to view the data in different ways and
easily export in HTML or text format to downstream
software programs.
The Allele Summary of Runs Report summarizes allele
data for all runs or selected runs for each marker in a
project. It gives statistical information including
expected and observed frequency of heterozygosity
and Chi-square values for each marker. The Marker
Comparison Report compares the summarized allele
data for each run of the selected marker and gives the
minimum and maximum allele sizes and frequency of
the allele. The Allele Summary Report lists the allele
calls for all runs of each marker in the project with
their sizes and quality scores.
Conclusions
MegaBACE Genetic Profiler provides the automation
that is needed for high throughput microsatellite
analysis. Combined with the flexibility and auto-
mation of the MegaBACE 1000 system, it is faster
and easier to use than gel electrophoresis platforms.
MegaBACE 1000 Genotyping System provides
cleaner data and quality scores that will accelerate
the genotyping process, enabling more rapid
scientific discovery.
ORDERING INFORMATION
MegaBACE 1000 Genotyping 1 63-0027-77
System with Genetic Profiler Software
MegaBACE ET400-R Size Standard 500 µl 25-0205-01
MegaBACE ET400-R Size Standard 2.5 ml 25-0205-02