Software Programs

The three software programs used in conjunction with the FMBIO scanner to perform STR genotyping include (1) Read Image, (2) FMBIO® Analysis Software, and (3) STaR Call™ Genotyping Software. Read Image controls the scan area, scan resolution, and photomultiplier sensitivity as the FMBIO II scanning unit generates the digital image of the gel. The user can indicate which fluorescent emission filters to use and add comments to the scanned image. The scanned image for each fluorescent wavelength of the experimental data is converted into a 16-bit digital TIFF file and stored for future data analysis.

The generated gel data images are next examined by the FMBIO® Analysis Software, which performs functions such as DNA fragment sizing and quantitation of peak height and area. DNA bands are sized through logarithmic comparison to size standards. Data can be displayed as either full gel images or electropherograms that are a virtual slice through one of the gel lanes. The gel images can also be examined one color at a time following application of the color separation matrix.

The analysis software includes a DNA band finding program. Because of fluorescence intensity variation between gels and even within samples on a gel, this step requires some user review and editing of the data. An analyst manually evaluates each called DNA band. Stutter bands can be highlighted and edited out of the processed data or removed based on user-defined criteria in the genotyping software described below.

Once the DNA bands have been sized, the STR alleles are genotyped using STaR Call™ (Genotyping Software. Band sizes from STR alleles are compared to sized alleles from allelic ladders run in adjacent lanes and converted to the appropriate genotype. Band sizes calculated by FMBIO® Analysis Software are imported into STaR Call™ and compared to values for each STR locus in a multiplex set. STR 'lookup tables' are exported to a Microsoft® Excel worksheet for evaluation of genotypes and manual confirmation that the expected size ranges are obtained for each allele. A 'lookup table' typically includes the DNA band size, STR allele call, and band quantitation in the form of optical density (OD) units.

Based on comparison of DNA fragment sizes with allele ranges and allelic ladders, each band is assigned a locus name and repeat number. The program looks for bands with weaker intensities, assigns them as stutter products if they are one repeat unit less than a 'normal' allele, and appropriately excludes them from the final data output. All of these genotyping steps can be performed automatically by the software. However, the final genotype information is typically reviewed carefully in a manual fashion to insure that correct calls were made by the genotyping software.

SAMPLE PROCESSING ON THE FMBIO II

One of the downsides of performing sample processing with the FMBIO II is that the process involves the use of gel electrophoresis, which is more labor intensive than capillary electrophoresis. However, the FMBIO system can lead to higher sample throughputs per instrument. Unlike the ABI 310 capillary system that performs online detection during electrophoresis, a gel's image is captured after electrophoretic separation with the FMBIO system. In other words, gels are run separately from the detection portion of the analysis with the FMBIO approach (Figure 14.7). Therefore, with staggered start times, multiple gel electrophoresis systems can feed into a single FMBIO II/III Fluorescence Analysis System, leading to higher throughput for the cost of a single instrument. For example, in the late 1990s, the Bode Technology Group, a private contract DNA typing laboratory in Springfield, Virginia, ran on average 20 gels per day with each gel containing 25 samples plus allelic ladders and controls. Thus, in this case a single scanner processed 450 new samples every day.

Figure 14.7 Sample processing steps using the Hitachi FMBIO II or III. Multiple gels can be prepared simultaneously and then quickly scanned after electrophoresis to improve sample throughput.

Figure 14.7 Sample processing steps using the Hitachi FMBIO II or III. Multiple gels can be prepared simultaneously and then quickly scanned after electrophoresis to improve sample throughput.

Electrophoresis and Detection steps are separate

Of course this kind of throughput has been superseded with newer multi-capillary electrophoresis systems such as the ABI 3700 described earlier.

Was this article helpful?

0 0
Stammering Its Cause and Its Cure

Stammering Its Cause and Its Cure

This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.

Get My Free Ebook


Post a comment