It was not possible to address issues of reproducibility or precision with this study. Most participating laboratories ran each sample only once (14 of 23).
With regard to the accuracy of allele size calling, it appears that overall, slab gel sequencers produce more accurate results than capillary sequencers.
When analyzing accuracy in terms of specific instruments or parameters, it is sometimes difficult to determine whether trends revealed by the data are significant. In some instances, when the results are broken down into parameters or instruments, the number of responses are small. Also, when parameters are specific to an instrument or model, as was the case particularly with the capillary instruments, it is impossible to discern whether observed effects are the direct result of specific parameters (polymer temperature, capillary length), or result from the instrument model.
SLAB GEL INSTRUMENTS (373, 377, LI-COR)
Analysis of the following parameters revealed a possible correlation to the accuracy of allele size calling:
- Instrument model (377/373)
- Well to Read length (34/36 cm)
- Percentage of acrylamide used in gel solution (5%)
- Amount of time that the gel is allowed to polymerize (3-6 hours).
- Electrophoresis conditions (for 34/36 cm plates: 3000 V, 60 Amp, 200 Watts
for 12 cm plates: 750 V, 50 Amp, 150 Watts).
- Temperature (51°C).
- Lane Standard (High density- around 20 fragments).
- NOTE: having any of the fragment sizes of the lane standard improperly defined or not defined will affect the accuracy of size calling.
Analysis of the following parameters did not reveal a correlation to the accuracy of allele size calling:
- The use of beveled plates for loading.
- Upgrades to the PE Bio instruments.
- Number of Lanes on the comb used for loading.
- Type of comb used for loading.
- Brand of Gel Solution.
- Type of running buffer.
- Age of APS.
- Deionizing, Degassing, and Filtering the gel solution prior to polymerization.
- Run time.
- Size calling software.
- Analysis software.
- Size calling method.
CAPILLARY INSTRUMENTS (310, 3700, MEGABASE, CEQ2000)
Analysis of the following parameters revealed a possible correlation to the accuracy of allele size calling:
- Instrument model (Beckman CEQ2000)
- Type of polymer (PE POP6, Beckman)
- Capillary Length (30 cm- CEQ2000))
- Temperature (50°C).
- Lane Standard (High density- around 20 fragments).
- NOTE: having any of the fragment sizes of the lane standard improperly defined or not defined will affect the accuracy of size calling.
Analysis of the following parameters did not reveal a correlation to the accuracy of allele size calling:
- Run time.
- Size calling software.
- Analysis software.
- Size calling method.
BACK
CONTENTS