A general approach to the analysis of errors and failure modes in the base-calling function in automated fluorescent DNA sequencing.
We describe the analysis of errors and failure modes in the base-calling function in automated DNA sequencing, on instruments in which fluorescently-labeled Sanger dideoxy-sequencing ladders are detected via their times of migration past a fixed detector. A general approach entails the joint use of: (i) well-defined control samples such as M13mp18, and (ii) mathematical simulation of sequencing electropherograms, with the deliberate introduction of different types of distortion and noise. An algorithm, the electrophoretic trace simulator (ETS), is used to calculate electrophoresis traces corresponding to the output data stream of an automated fluorescent DNA sequencer. The ETS accepts a user-defined sequence of nucleotide bases (A, C, G, T) as input, and employs user-adjustable functions to compute the following critical parameters of an electropherogram: peak intensity, peak spacing, peak shape as a function of base number; background, noise, and spectral cross-talk correction (for a sequencer using multiple dyes). We use a combination of M13mp18 controls and simulated electropherograms to analyze two problems of considerable practical importance: (i) variation in electrophoretic migration rates between different lanes of a gel, and (ii) variation in signal intensity due to user-dependent loading artifacts. The issue of base-calling errors and failure modes, for electropherograms that contain noise and distortion, is addressed.
Izmailov
Alexandre
A
Visible Genetics Inc., 700 Bay Street, Suite 1000, Toronto, Ontario M5G 1Z6, Canada. izmailov@visgen.com
Goloubentzev
Dmitri
D
Jin
Caroline
C
Sunay
Susan
S
Wisco
Valiente
V
Yager
Thomas D
TD
D000465:Algorithms; D001331:Automation; D004591:Electrophoresis, Polyacrylamide Gel / Q000592:standards*; D019544:Equipment Failure Analysis; D005456:Fluorescent Dyes; D009711:Nucleotides; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000592:standards
D005456:Fluorescent Dyes; D009711:Nucleotides