Data acquisition and processing using single photon counting in DNA sequencing

In this dissertation, a system for acquisition and processing of DNA sequencing data was developed. The system may perform all necessary procedures from fluorescence intensity measurement, data digitization to placement of base-calls and preparation of data for further processing by standard software package PHRED/PHRAP/CONSED.; The system is using single photon counting devices (SPC) for acquisition of fluorescence intensity. Using SPC ensures superiority of the system over existing instruments in terms of sensitivity, dynamic range and quality of obtained data. Properties of SPC data are discussed in detail. A method for measurement of SPC non-linearity is offered and its performance is estimated. Direct counting approach to SPC data digitization is offered and implemented. Multicolor fluorescence detection schemes with one-laser and multi-laser excitation are discussed and analyzed. Application of spectral analysis and synchronous detection for channel separation in multi-laser excitation based system is discussed.; A complete system for processing of DNA sequencing data was developed and implemented. The system includes tools for data preparation, baseline removal, cross-talk filtering, smoothing, mobility shift correction and placement of base-calls. The system also has ability of converting the results of processing into .SCF format which is accepted by standard base-caller PHRED.; The project included design and implementation of hardware prototypes of fluorescence detection devices based on the discussed processing schemes. A set of software tools for data recording and processing was developed. The discussed principles were utilized as part of the design of SBS-2000 (Stony Brook, NY) sequencing instrument.