Electrophoresis

Referring to Figure 1, a CE device 100 in one embodiment comprises a voltage bias source 102, a capillary 104, a body 114, a detector 106, a sample injection port 108, a heater 110, and a separation media 112. A sample is injected into the sample injection port 108, which is maintained at an above-ambient temperature by the heater 110. Once injected the sample engages the separation media 112 and is split into component molecules. The components migrate through the capillary 104 under the influence of an electric field established by the voltage bias source 102, until they reach the detector 106.

Referring to Figure 2, a CE device 200, in one embodiment,  comprises a voltage bias source 202, a capillary 204, a body 214, a detector 206, a sample injection port 208, a heater 210, and a separation media 212. A sample is injected into the sample injection port 208, which is maintained at an above-ambient temperature by the heater 210. Once injected the sample engages the separation media 212 and is split into component molecules. The components migrate through the capillary 204 under the influence of an electric field established by the voltage bias source 202, until they reach the detector 206. The CE device 200 may be a component of an instrument 216 that include a computational device to collect and process image signals from the detector. The instrument 216 may be a capillary electrophoresis genetic analyzer providing many similar features found in exemplary commercial CE instruments

Referencing Figure 3, a CE system 300 in one embodiment comprises a source buffer 316 initially comprising the fluorescently labeled sample 318, a capilary 320, a destination buffer 324, a power supply 326, a computing device 302 comprising a processor 308, memory 306 comprising basecaller algorithm 304, and a controller 310. The source buffer 316 is in fluid communication with the destination buffer 324 by way of the capilary 320. The power supply 326 applies voltage to the source buffer 316 and the destination buffer 324 generating a voltage bias through an anode 328 in the source buffer 316 and a cathode 330 in the destination buffer 324. The voltage applied by the power supply 326 is configured by a controller 310 operated by the computing device 302. The fluorescently labeled sample 318 near the source buffer 316 is pulled through the capilary 320 by the voltage gradient and optically labeled nucleotides of the DNA fragments within the sample are detected as they pass through an optical sensor 322. Differently sized DNA fragments within the fluorescently labeled sample 318 are pulled through the capilary at different times due to their size. The optical sensor 322 detects the fluorescent labels on the nucleotides as an image signal and communicates the image signal to the to the computing device 302. The computing device 302  aggregates the image signal as sample data and utilizes a basecaller algorithm 304 stored in memory 306 to transform the sample data into processed data and generate an electropherogram 314 to be shown in a display device 312.

Referencing Figure 4, a CE process 400 involves a computing device 412 communicating a configuration control 416 to a controller 408 to control the voltage applied by a power supply 406 to the buffers 402. After the prepared flourscently labled sample has been added to the source buffer, the controller 408 communicates an operation control 418 to the power supply 406 to apply a voltage 420 to the buffers creating a voltage bias/electrical gradient. The applied voltage cause the  fluorescently labled sample 422 to move through capilary 404 between the buffers 402 and pass by the optical sensor 410. The optical sensor 410 detects fluorescent labels on the nucleotides of the DNA fragments that pass through the capilary and communicates the image signal 424 to the computing device 412. The computing device 412 aggregates the image signal 424 to generate the sample data for further processing. A basecaller algorithm processes the sample data (e.g., signal values) to generate processed data. The computing device 412 then generates a display control 426 to display an electropherogram of the processed data on a display device 414. 

Referencing Figure 5, a CE process 500 involves configuring a capillary electrophoresis instrument operating parameters to sequence at least one fluorescently labeled sample (block 502). The configuration of the insturment may include creating or importing a plate setting for running a series of samples and assigning labels to the plate samples to assist in the processing of the collected imaging data. The process may also include communicating configuration controls to a controller to start applying voltage at a predetermined time. In block 504, the CE process 500 loads the fluorescently labled sample into the instrument. After the sample is loaded into the insturment, the insturment may transfer the sample from a plate well into the capilary tube and then position the capilary tube into the starting buffer at the beginning of the capilary electrophoresis process. In block 506, the CE process 500 begins the insturment run after the sample has been loaded into the capilary by applying a voltage to the buffer solutions postioned at opposite ends of the capilary, forming an electrical gradient to transport DNA fragments of the fluorescently labeled sample fromt the starting buffer to a destination buffer and traversing an optical sensor. In block 508, the CE process 500 detects the individual  fluorescent signals on the nucleotides of the DNA fragments as they move towards the destination buffer through the optical sensor and communicates the image signal to the computing device. In block 510, the CE process 500 aggregates the image signal at the computing device from the optical sensor and generates sample data that corresponds to the fluorescent intensity of the nucleotides DNA fragments. In block 512, the CE process 500 processes the sample data to identify the bases called in the DNA fragments at the particular time point. In block 514, the CE process 500 displays processed data through an electrpherogram through a display device. 

Referencing Figure 6,  sequencing data 600 shows Sanger sequencing data 602 in a electropherogram. Capillary electrophoresis technologies (CE) can examine several varieties of biopolymer; for example, DNA, methylated DNA, mRNA, proteins tagged with variable length oligos.  The resulting data appears as a series of peaks.  Minor variations in a biopolymer appear as smaller peaks, possibly overlapped with the peaks corresponding to the dominant form of the biopolymer.  The smaller peaks can be confused with peaks that arise from biochemical noise associated with the chemical reactions used to process the biological sample. With the Sanger sequencing data 602, a number of peaks 604 are seen at 116 nucleotide position corresponding to a cytosine. Thorough exisitng analysis techniques that involve examining the characteristics of the peaks such as peak height and width and data maninpulation 608,  the Sanger sequencing data 602 may be presented as adjusted data 610 showing the peaks 604 as a detected variant 606 for the 116 nucleotide position. 

Current solutions, such as commerically available variant detection software (e.g., Thermo Fisher Minor variant Finder, Multilocus variant Analysis, variant Reporter Software v1.1, variant analysis and identitifcation modules in MicrobeBridge Software, etc.,) to recognize the smaller peaks that are associated with the minor biopolymeric forms present in a sample involve examining the characteristics of the peaks such as peak height and width. 

The Sanger sequencing data 602 and the adjusted data 610, as well as the analysis process utilized to identify the peak characteristics, may be stored within sequencing data storage to be used in the statistical analysis of similar results or applied as inputs to machine learning algorithms to distinguish dominant from minor variants of biomolecules.