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ExperimentDNASequencing

ExperimentDNASequencing[Samples]⟹Protocol

creates a Protocol object for running a Sanger DNA sequencing experiment, which uses fluorescently labeled DNA Samples, with a genetic analyzer instrument containing a capillary electrophoresis array to determine the nucleotide sequences from the provided Samples.

ExperimentDNASequencing[Samples,Primers]⟹Protocol

creates a Protocol object for running a Sanger DNA sequencing experiment, which uses fluorescent dideoxynucleotide chain-terminating PCR to label provided Samples using provided Primers and then a genetic analyzer instrument with a capillary electrophoresis array to determine the nucleotide sequences from the provided Samples.

    
DNA sequencing is the process by which the order of the bases, or nucleotides, in a strand of DNA is determined. Sanger sequencing is a method of DNA sequencing where a DNA sample is reacted with fluorescently labeled chain terminating nucleotides that are incorporated into the DNA strands at different strand lengths. Each fluorophore corresponds to one of the four DNA bases (C, T, G, or A). Once these labeled bases are incorporated, the DNA samples are analyzed using capillary gel electrophoresis (CGE). In CGE, the negatively charged DNA strands are drawn into the polymer-filled capillary via an electrokinetic phenomenon, and the strands separated based on size as they travel through the gel in the capillary. The separated DNA fragments pass a laser that excites the dye molecules. A fluorescence detector measures the fluorescence emission of the dye molecules on the strands. From this emission wavelength, the identity of the base is determined. Sanger sequencing is useful for many applications, including identification of species, confirmation or identification of mutagenesis, or verifying results using next generation sequencing techniques.
    

Experimental Principles

    Figure 1.1: Experimental workflow for a DNA sequencing experiment. 1. Prepare samples by loading DNA samples of up to 800 base pairs onto a plate and performing chain termination PCR with fluorescently labeled nucleotides. 2. Purify samples to remove unreacted materials if necessary. 3. Load samples (10-100 Microliter/sample) into autosampler holder of the instrument. The holder can accommodate up to 96 samples in a well plate. Samples must be in groups of 4, as there are 4 capillaries that inject samples from adjacent wells simultaneously; extra wells are filled with buffer. Load the sequencing cartridge containing the capillary array and the buffer cartridge containing the cathode buffer onto the instrument. Start the run. 4. Once the run begins, samples are electrokinetically drawn into the capillaries. Negatively charged DNA fragments travel through the polymer in the capillaries and separate based on size. Longer DNA fragments carry more negative charge compared to shorter fragments. 5. Separated DNA fragments are excited by a laser and fluorescence is detected. 6. Fluorescence data is analyzed using AnalyzeDNASequencing according to calibration to the specific dye set used and the identity of the base in the sequence is determined.

Instrumentation

    SeqStudio

    Figure 2.1.1: Instrument schematic of Applied Biosystems SeqStudio Genetic Analyzer, an instrument used for determining the base pair sequence of DNA samples using Sanger sequencing techniques. The SeqStudio comprises a removeable cartridge housing the polymer injection system and capillary array of 4 capillaries, an autosampler, and a fluorescence detector. Once fluorescently labeled samples are loaded and the run method is set up, the instrument electrokinetically draws up samples into the capillaries, and employs capillary gel electrophoresis techniques to separate DNA fragments. The fluorescence detector measures spectral data of emissions from the fluorophores, and further analysis determines the base pairs based on calibration to the fluorophores in the dye set used to label the template. The instrument can analyze up to 96 samples in one run. DNA samples of up to 800 base pairs can be analyzed. Analysis of short DNA strands (<300 base pairs) takes 30 min for 4 samples, processing 96 samples in 12 hours. Analysis of longer strands (>600 base pairs) takes up to 2 hours for 4 samples, and 48 samples can be analyzed in a 24 hour period.

Experiment Options

    General

    Instrument

    The cartridge-based capillary electrophoresis instrument for running the DNA sequencing experiment using fluorescence detection to determine nucleotide sequence.
    Default Value: Model[Instrument, GeneticAnalyzer, SeqStudio]
    Pattern Description: An object of type or subtype Model[Instrument, GeneticAnalyzer] or Object[Instrument, GeneticAnalyzer]
    Programmatic Pattern: ObjectP[{Model[Instrument, GeneticAnalyzer], Object[Instrument, GeneticAnalyzer]}]

    SequencingCartridge

    The cartridge containing the polymer, capillary array, and anode buffer that fits into the instrument for running the DNA sequencing experiment.
    Default Value: Model[Item, Cartridge, DNASequencing, SeqStudio Sequencing Cartridge v2]
    Pattern Description: An object of type or subtype Object[Item, Cartridge, DNASequencing] or Model[Item, Cartridge, DNASequencing]
    Programmatic Pattern: ObjectP[{Object[Item, Cartridge, DNASequencing], Model[Item, Cartridge, DNASequencing]}]

    BufferCartridge

    The cartridge containing the cathode buffer and waste container that fits into the instrument for running the DNA sequencing experiment.
    Default Value: Model[Container, Vessel, BufferCartridge, SeqStudio Buffer Cartridge]
    Pattern Description: An object of type or subtype Object[Container, Vessel, BufferCartridge] or Model[Container, Vessel, BufferCartridge]
    Programmatic Pattern: ObjectP[{Object[Container, Vessel, BufferCartridge], Model[Container, Vessel, BufferCartridge]}]

    Temperature

    The target temperature of the capillary array throughout the experiment.
    Default Value: 60 degrees Celsius
    Pattern Description: Greater than or equal to 40 degrees Celsius and less than or equal to 60 degrees Celsius.
    Programmatic Pattern: RangeP[40*Celsius, 60*Celsius]

    PreparedPlate

    Indicates if the plate containing the samples for the DNA sequencing experiment has been previously prepared with primers and Master Mix and does not need to run sample preparation steps.
    Default Value: Automatic
    Default Calculation: Automatically resolves to False if 'primers' are specified as an input, and True if 'primers' are not an input.
    Pattern Description: True or False.
    Programmatic Pattern: BooleanP | Automatic

    NumberOfInjections

    The number of times each amplified sample is injected for a capillary electrophoresis run using identical experimental parameters. All samples are injected for each NumberOfInjections, and a separate Data object is created for each sample for every injection.
    Default Value: 1
    Pattern Description: Greater than or equal to 1 in increments of 1.
    Programmatic Pattern: GreaterEqualP[1, 1]

    Sample Preparation

    ReadLength

    The estimated number of base pairs in the template samples to be sequenced. Template sample length informs default capillary electrophoresis experiment parameters as described in Figure 3.1.
    Figure 3.1: Capillary gel electrophoresis separation parameters are set based on the number of base pairs in the sample being analyzed according to the table.
    Default Value: Automatic
    Default Calculation: Automatically resolves based on SequenceLength[Object[Sample][Composition]], or to 500 base pairs if Composition is not known, unless otherwise specified.
    Pattern Description: Greater than or equal to 1 and less than or equal to 2000 in increments of 1.
    Programmatic Pattern: RangeP[1, 2000, 1] | Automatic
    Index Matches to: experiment samples

    SampleVolume

    The volume of each template sample to add to the polymerase reaction.
    Default Value: 2 microliters
    Pattern Description: Greater than or equal to 1 microliter and less than or equal to 100 microliters.
    Programmatic Pattern: RangeP[1*Microliter, 100*Microliter]
    Index Matches to: experiment samples

    ReactionVolume

    The total volume of the polymerase reaction including the template, primer, master mix, and diluent.
    Default Value: 20 microliters
    Pattern Description: Greater than or equal to 10 microliters and less than or equal to 100 microliters.
    Programmatic Pattern: RangeP[10*Microliter, 100*Microliter]
    Index Matches to: experiment samples

    PrimerConcentration

    The desired final concentration of the primer in the polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically resolves to 0.1 Micromolar, or Null if PreparedPlate is set to True.
    Pattern Description: Greater than or equal to 0.1 picomolar and less than or equal to 10 micromolar or Null.
    Programmatic Pattern: (RangeP[0.1*Picomolar, 10*Micromolar] | Automatic) | Null
    Index Matches to: experiment samples

    PrimerVolume

    The volume of the primer to add to each polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically set according to the equation PrimerVolume=(PrimerConcentration)*(ReactionVolume)/(inital primer concentration), or 1 Microliter if the calculated volume is too small to be pipetted accurately, or Null otherwise.
    Pattern Description: Greater than or equal to 1 microliter and less than or equal to 100 microliters or Null.
    Programmatic Pattern: (RangeP[1*Microliter, 100*Microliter] | Automatic) | Null
    Index Matches to: experiment samples

    MasterMix

    The stock solution composed of the polymerase, nucleotides, fluorescent dideoxynucleotides, and buffer for the polymerase reaction.
    Default Value: Model[Sample, BigDye Terminator v3.1 Ready Reaction Mix]
    Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
    Programmatic Pattern: (ObjectP[{Model[Sample], Object[Sample]}] | _String) | Null
    Index Matches to: experiment samples

    MasterMixVolume

    The volume of master mix to add to the reaction.
    Default Value: Automatic
    Default Calculation: Automatically set to 0.5*ReactionVolume if MasterMix is not set to Null, or Null otherwise.
    Pattern Description: Greater than or equal to 1 microliter and less than or equal to 100 microliters or Null.
    Programmatic Pattern: (RangeP[1*Microliter, 100*Microliter] | Automatic) | Null
    Index Matches to: experiment samples

    AdenosineTriphosphateTerminator

    The dye molecule (dideoxynucelotide triphosphate) used to terminate DNA chains at locations where the complementary base on the template strand is thymine.
    Default Value: Automatic
    Default Calculation: Automatically resolves by finding the corresponding dideoxynucleotide dye in the MasterMix Object[Sample][Composition].
    Pattern Description: An object of type or subtype Model[Molecule] or Model[ProprietaryFormulation] or Null.
    Programmatic Pattern: (ObjectP[{Model[Molecule], Model[ProprietaryFormulation]}] | Automatic) | Null
    Index Matches to: experiment samples

    ThymidineTriphosphateTerminator

    The dye molecule (dideoxynucelotide triphosphate) used to terminate DNA chains at locations where the complementary base on the template strand is adenine.
    Default Value: Automatic
    Default Calculation: Automatically resolves by finding the corresponding dideoxynucleotide dye in the MasterMix Object[Sample][Composition].
    Pattern Description: An object of type or subtype Model[Molecule] or Model[ProprietaryFormulation] or Null.
    Programmatic Pattern: (ObjectP[{Model[Molecule], Model[ProprietaryFormulation]}] | Automatic) | Null
    Index Matches to: experiment samples

    GuanosineTriphosphateTerminator

    The dye molecule (dideoxynucelotide triphosphate) used to terminate DNA chains at locations where the complementary base on the template strand is cytosine.
    Default Value: Automatic
    Default Calculation: Automatically resolves by finding the corresponding dideoxynucleotide dye in the MasterMix Object[Sample][Composition].
    Pattern Description: An object of type or subtype Model[Molecule] or Model[ProprietaryFormulation] or Null.
    Programmatic Pattern: (ObjectP[{Model[Molecule], Model[ProprietaryFormulation]}] | Automatic) | Null
    Index Matches to: experiment samples

    CytosineTriphosphateTerminator

    The dye molecule (dideoxynucelotide triphosphate) used to terminate DNA chains at locations where the complementary base on the template strand is guanine.
    Default Value: Automatic
    Default Calculation: Automatically resolves by finding the corresponding dideoxynucleotide dye in the MasterMix Object[Sample][Composition].
    Pattern Description: An object of type or subtype Model[Molecule] or Model[ProprietaryFormulation] or Null.
    Programmatic Pattern: (ObjectP[{Model[Molecule], Model[ProprietaryFormulation]}] | Automatic) | Null
    Index Matches to: experiment samples

    Diluent

    The solution for bringing each reaction to ReactionVolume once all the reaction components (template, primers, and master mix) are added.
    Default Value: Automatic
    Default Calculation: Automatically resolves to Model[Sample,"Nuclease-free Water"], or Null if PreparedPlate is set to True.
    Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
    Programmatic Pattern: ((ObjectP[{Model[Sample], Object[Sample]}] | _String) | Automatic) | Null
    Index Matches to: experiment samples

    DiluentVolume

    The volume of buffer to add to bring each reaction to ReactionVolume.
    Default Value: Automatic
    Default Calculation: Automatically set according to the equation DiluentVolume=ReactionVolume-(SampleVolume+MasterMixVolume+PrimerVolume) if Diluent is not set to Null, or Null otherwise.
    Pattern Description: Greater than or equal to 1 microliter and less than or equal to 100 microliters or Null.
    Programmatic Pattern: (RangeP[1*Microliter, 100*Microliter] | Automatic) | Null
    Index Matches to: experiment samples

    Post Experiment

    PrimerStorageCondition

    For each sample, the non-default conditions under which the primers of this experiment should be stored after the protocol is completed. If left unset, the primers will be stored according to their current StorageCondition.
    Default Value: Null
    Pattern Description: {AmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, BacteriaIncubation, MammalianIncubation, TissueCultureCellsIncubation, MicrobialCellsIncubation, MicrobialCellsShakingIncubation, YeastCellsIncubation, YeastCellsShakingIncubation, ViralIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
    Programmatic Pattern: (SampleStorageTypeP | Disposal) | Null
    Index Matches to: experiment samples

    MasterMixStorageCondition

    For each sample, the non-default condition under which MasterMix of this experiment should be stored after the protocol is completed. If left unset, MasterMix will be stored according to their current StorageCondition.
    Default Value: Null
    Pattern Description: {AmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, BacteriaIncubation, MammalianIncubation, TissueCultureCellsIncubation, MicrobialCellsIncubation, MicrobialCellsShakingIncubation, YeastCellsIncubation, YeastCellsShakingIncubation, ViralIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
    Programmatic Pattern: (SampleStorageTypeP | Disposal) | Null
    Index Matches to: experiment samples

    SamplesInStorageCondition

    The non-default conditions under which the SamplesIn of this experiment should be stored after the protocol is completed. If left unset, SamplesIn will be stored according to their current StorageCondition.
    Default Value: Null
    Pattern Description: {AmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, BacteriaIncubation, MammalianIncubation, TissueCultureCellsIncubation, MicrobialCellsIncubation, MicrobialCellsShakingIncubation, YeastCellsIncubation, YeastCellsShakingIncubation, ViralIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
    Programmatic Pattern: (Alternatives[SampleStorageTypeP | Disposal]) | Null
    Index Matches to: experiment samples

    Polymerase Activation

    Activation

    Indicates if hot start activation should be performed in the polymerase reaction. In order to reduce non-specific amplification, enzymes can be made room temperature stable by inhibiting their activity via thermolabile conjugates. Once an experiment is ready to be run, this inhibition is disabled by heating the reaction to ActivationTemperature.
    Default Value: Automatic
    Default Calculation: Automatically set to True if other Activation options are set, or False otherwise.
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null

    ActivationTime

    The length of time for which the sample is held at ActivationTemperature to remove the thermolabile conjugates inhibiting polymerase activity in the polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically set to 60 seconds if Activation is set to True, or Null otherwise.
    Pattern Description: Greater than or equal to 1 second and less than or equal to 1 hour or Null.
    Programmatic Pattern: (RangeP[1*Second, 1*Hour] | Automatic) | Null

    ActivationTemperature

    The temperature to which the sample is heated to remove the thermolabile conjugates inhibiting polymerase activity in the polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically set to 95 degrees Celsius if Activation is set to True, or Null otherwise.
    Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 105 degrees Celsius or Null.
    Programmatic Pattern: (RangeP[4*Celsius, 105*Celsius] | Automatic) | Null

    ActivationRampRate

    The rate at which the sample is heated to reach ActivationTemperature in the polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically set to 3.5 degrees Celsius per second if Activation is set to True, or Null otherwise.
    Pattern Description: Greater than or equal to 0.1 degrees Celsius per second and less than or equal to 3.5 degrees Celsius per second or Null.
    Programmatic Pattern: (RangeP[0.1*(Celsius/Second), 3.5*(Celsius/Second)] | Automatic) | Null

    Denaturation

    DenaturationTime

    The length of time for which the sample is held at DenaturationTemperature to allow the dissociation of the double stranded template into single strands in the polymerase reaction.
    Default Value: 10 seconds
    Pattern Description: Greater than or equal to 1 second and less than or equal to 1 hour or Null.
    Programmatic Pattern: RangeP[1*Second, 1*Hour] | Null

    DenaturationTemperature

    The temperature to which the sample is heated to allow the dissociation of the double stranded template into single strands in the polymerase reaction.
    Default Value: 96 degrees Celsius
    Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 105 degrees Celsius or Null.
    Programmatic Pattern: RangeP[4*Celsius, 105*Celsius] | Null

    DenaturationRampRate

    The rate at which the sample is heated to reach DenaturationTemperature in the polymerase reaction.
    Default Value: 1 degree Celsius per second
    Pattern Description: Greater than or equal to 0.1 degrees Celsius per second and less than or equal to 3.5 degrees Celsius per second or Null.
    Programmatic Pattern: RangeP[0.1*(Celsius/Second), 3.5*(Celsius/Second)] | Null

    Primer Annealing

    PrimerAnnealing

    Indicates if annealing should be performed as a separate step instead of as part of extension in the polymerase reaction. Lowering the temperature during annealing allows primers to bind to the template and serve as anchor points for the polymerase in the subsequent extension.
    Default Value: Automatic
    Default Calculation: Automatically set to True if other PrimerAnnealing options are set, or False otherwise.
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null

    PrimerAnnealingTime

    The length of time for which the sample is held at PrimerAnnealingTemperature to allow primers to bind to the template in the polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically set to 5 seconds if PrimerAnnealing is set to True, or Null otherwise.
    Pattern Description: Greater than or equal to 1 second and less than or equal to 1 hour or Null.
    Programmatic Pattern: (RangeP[1*Second, 1*Hour] | Automatic) | Null

    PrimerAnnealingTemperature

    The temperature to which the sample is cooled to allow primers to bind to the template in the polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically set to 50 degrees Celsius if PrimerAnnealing is set to True, or Null otherwise.
    Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 105 degrees Celsius or Null.
    Programmatic Pattern: (RangeP[4*Celsius, 105*Celsius] | Automatic) | Null

    PrimerAnnealingRampRate

    The rate at which the sample is cooled to reach PrimerAnnealingTemperature in the polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically set to 1 degrees Celsius per second if PrimerAnnealing is set to True, or Null otherwise.
    Pattern Description: Greater than or equal to 0.1 degrees Celsius per second and less than or equal to 3.5 degrees Celsius per second or Null.
    Programmatic Pattern: (RangeP[0.1*(Celsius/Second), 3.5*(Celsius/Second)] | Automatic) | Null

    Strand Extension

    ExtensionTime

    The length of time for which sample is held at ExtensionTemperature to allow the polymerase to synthesize a new strand using the template and primers in the polymerase reaction.
    Default Value: 240 seconds
    Pattern Description: Greater than or equal to 1 second and less than or equal to 1 hour or Null.
    Programmatic Pattern: RangeP[1*Second, 1*Hour] | Null

    ExtensionTemperature

    The temperature to which the sample is heated/cooled to allow the polymerase to synthesize a new strand using the template and primers in the polymerase reaction.
    Default Value: 60 degrees Celsius
    Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 105 degrees Celsius or Null.
    Programmatic Pattern: RangeP[4*Celsius, 105*Celsius] | Null

    ExtensionRampRate

    The rate at which the sample is heated/cooled to reach ExtensionTemperature in the polymerase reaction.
    Default Value: 1 degree Celsius per second
    Pattern Description: Greater than or equal to 0.1 degrees Celsius per second and less than or equal to 3.5 degrees Celsius per second or Null.
    Programmatic Pattern: RangeP[0.1*(Celsius/Second), 3.5*(Celsius/Second)] | Null

    Thermocycling

    NumberOfCycles

    The number of times the polymerase reaction will undergo repeated cycles of denaturation, primer annealing (optional), and strand extension.
    Default Value: 25
    Pattern Description: Greater than or equal to 1 and less than or equal to 60 in increments of 1 or Null.
    Programmatic Pattern: RangeP[1, 60, 1] | Null

    HoldTemperature

    The temperature to which the sample is cooled and held after the polymerase reaction thermocycling procedure.
    Default Value: 4 degrees Celsius
    Pattern Description: Greater than or equal to 4 degrees Celsius and less than or equal to 105 degrees Celsius or Null.
    Programmatic Pattern: RangeP[4*Celsius, 105*Celsius] | Null

    Post-PCR Sample Preparation

    PurificationType

    The method of purification of the DNA template samples after undergoing chain termination PCR. Ethanol precipitation selectively precipitates DNA out of solution by adding solutions of ethanol and EDTA to the sample, mixing, and centrifuging. Then the supernatant containing reaction reagents is discarded and the pure DNA pellet is resuspended. BigDye XTerminator purification removes unincorporated BigDye terminators and salts by adding BigDye XTerminator solution to the reactions, then mixing and centrifuging.
    Default Value: Automatic
    Default Calculation: Automatically set to Null if PreparedPlate->True, or BigDye XTerminator if a BigDye master mix is used, or Ethanol precipitation otherwise.
    Pattern Description: Ethanol precipitation or BigDye XTerminator or Null.
    Programmatic Pattern: (("Ethanol precipitation" | "BigDye XTerminator") | Automatic) | Null

    QuenchingReagents

    The reagents used to quench the polymerase chain reaction and remove unreacted materials.
    Default Value: Automatic
    Default Calculation: Automatically set to Model[Sample,"Absolute Ethanol, Anhydrous"] and Model[Sample, StockSolution, "125 mM EDTA"] if PurificationType->Ethanol precipitation, or Model[Sample,"SAM Solution"] and Model[Sample,"BigDye XTerminator Solution"] if PurificationType->BigDye XTerminator, or Null otherwise.
    Pattern Description: List of one or more an object of type or subtype Model[Sample] or Object[Sample] or a prepared sample entries or Null.
    Programmatic Pattern: ({(ObjectP[{Model[Sample], Object[Sample]}] | _String)..} | Automatic) | Null

    QuenchingReagentVolumes

    The volumes of the reagents added to quench the polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically set to {30 Microliter, 1 Microliter} if PurificationType->Ethanol precipitation, or {4.5*ReactionVolume, ReactionVolume} of the ReactionVolume of the first sample if PurificationType->BigDye XTerminator, or Null otherwise.
    Pattern Description: List of one or more greater than or equal to 1 microliter and less than or equal to 100 microliters entries or Null.
    Programmatic Pattern: ({RangeP[1*Microliter, 100*Microliter]..} | Automatic) | Null

    SequencingBuffer

    The buffer to be used to resuspend DNA samples prior to loading in the genetic analyzer instrument. This buffer will also be used to fill empty wells that are needed to complete injection groups as a blank.
    Default Value: Automatic
    Default Calculation: Automatically set to Model[Sample,"Tris EDTA 0.1 buffer solution"] if PurificationType->Ethanol precipitation, or Null if PurificationType->BigDye XTerminator and otherwise.
    Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
    Programmatic Pattern: ((ObjectP[{Model[Sample], Object[Sample]}] | _String) | Automatic) | Null

    SequencingBufferVolume

    The volume of the buffer to be used to resuspend DNA samples prior to loading in the genetic analyzer instrument.
    Default Value: Automatic
    Default Calculation: Automatically set to 40 microliters if PurificationType->Ethanol precipitation, or Null if PurificationType->BigDye XTerminator and otherwise.
    Pattern Description: Greater than or equal to 1 microliter and less than or equal to 100 microliters or Null.
    Programmatic Pattern: (RangeP[1*Microliter, 100*Microliter] | Automatic) | Null

    Separation

    DyeSet

    The set of dye terminator molecules used to terminate DNA chains, with different emission spectra corresponding to the different nucleotides. For optimal performance use the dye set that most closely matches the Master Mix and dyes used in the polymerase reaction.
    Default Value: Automatic
    Default Calculation: Automatically resolves based on the name of the Master Mix used.
    Pattern Description: E_BigDye Terminator v1.1, Z_BigDye Terminator v3.1, or Z_BigDye Direct.
    Programmatic Pattern: ("E_BigDye Terminator v1.1" | "Z_BigDye Terminator v3.1" | "Z_BigDye Direct") | Automatic
    Index Matches to: experiment samples

    PrimeTime

    The length of time for which cathode buffer is drawn into the capillary array in order to prime the capillaries prior to the samples being injected.
    Default Value: 180 seconds
    Pattern Description: Greater than or equal to 0 seconds and less than or equal to 1000 seconds.
    Programmatic Pattern: RangeP[0*Second, 1000*Second]
    Index Matches to: experiment samples

    PrimeVoltage

    The voltage applied to the capillary array in order to prime the capillaries prior to the samples being injected.
    Default Value: 13000 volts
    Pattern Description: Greater than or equal to 0 volts and less than or equal to 13000 volts.
    Programmatic Pattern: RangeP[0*Volt, 13000*Volt]
    Index Matches to: experiment samples

    InjectionTime

    The length of time for which sample is drawn into the capillary array. A longer injection time will lead to an increase in the signal, as more DNA molecules will be electrokinetically drawn into the capillary.
    Default Value: 10 seconds
    Pattern Description: Greater than or equal to 1 second and less than or equal to 600 seconds.
    Programmatic Pattern: RangeP[1*Second, 600*Second]
    Index Matches to: experiment samples

    InjectionVoltage

    The voltage applied to the capillary array while the samples are being drawn into the capillaries.
    Default Value: Automatic
    Default Calculation: Automatically set according to the ReadLength option specifying the length of the sequence to be read.
    Pattern Description: Greater than or equal to 1 volt and less than or equal to 13000 volts.
    Programmatic Pattern: RangeP[1*Volt, 13000*Volt] | Automatic
    Index Matches to: experiment samples

    RampTime

    The length of time for which the voltage will ramp from the injection voltage to the run voltage.
    Default Value: 300 seconds
    Pattern Description: Greater than or equal to 1 second and less than or equal to 1800 seconds.
    Programmatic Pattern: RangeP[1*Second, 1800*Second]
    Index Matches to: experiment samples

    RunVoltage

    The voltage applied to the capillary array while the template samples move through the capillary and the fragments separate.
    Default Value: Automatic
    Default Calculation: Automatically set according to the ReadLength option specifying the length of the sequence to be read.
    Pattern Description: Greater than or equal to 1 volt and less than or equal to 13000 volts.
    Programmatic Pattern: RangeP[1*Volt, 13000*Volt] | Automatic
    Index Matches to: experiment samples

    RunTime

    The length of time for which the separation of the template fragments in the capillaries will occur.
    Default Value: Automatic
    Default Calculation: Automatically set according to the ReadLength option specifying the length of the sequence to be read.
    Pattern Description: Greater than or equal to 300 seconds and less than or equal to 14000 seconds.
    Programmatic Pattern: RangeP[300*Second, 14000*Second] | Automatic
    Index Matches to: experiment samples

    Storage

    SequencingCartridgeStorageCondition

    The storage condition for the SequencingCartridge once the experiment has concluded.
    Default Value: Model[StorageCondition, Refrigerator]
    Pattern Description: Storage Object or Storage Type.
    Programmatic Pattern: (SampleStorageTypeP | Disposal) | ObjectP[Model[StorageCondition]]

Sample Prep Options

    Sample Preparation

    PreparatoryUnitOperations

    Specifies a sequence of transferring, aliquoting, consolidating, or mixing of new or existing samples before the main experiment. These prepared samples can be used in the main experiment by referencing their defined name. For more information, please reference the documentation for ExperimentSampleManipulation.
    Default Value: Null
    Pattern Description: List of one or more unit Operation ManualSamplePreparation or RoboticSamplePreparation or unit Operation must match SamplePreparationP entries or Null.
    Programmatic Pattern: {((ManualSamplePreparationMethodP | RoboticSamplePreparationMethodP) | SamplePreparationP)..} | Null

    PreparatoryPrimitives

    Specifies a sequence of transferring, aliquoting, consolidating, or mixing of new or existing samples before the main experiment. These prepared samples can be used in the main experiment by referencing their defined name. For more information, please reference the documentation for ExperimentSampleManipulation.
    Default Value: Null
    Pattern Description: List of one or more a primitive with head Define, Transfer, Mix, Aliquot, Consolidation, FillToVolume, Incubate, Filter, Wait, Centrifuge, or Resuspend entries or Null.
    Programmatic Pattern: {SampleManipulationP..} | Null

    Preparatory Incubation

    Incubate

    Indicates if the SamplesIn should be incubated at a fixed temperature prior to starting the experiment or any aliquoting. Sample Preparation occurs in the order of Incubation, Centrifugation, Filtration, and then Aliquoting (if specified).
    Default Value: Automatic
    Default Calculation: Resolves to True if any of the corresponding Incubation options are set. Otherwise, resolves to False.
    Pattern Description: True or False.
    Programmatic Pattern: BooleanP | Automatic
    Index Matches to: experiment samples

    IncubationTemperature

    Temperature at which the SamplesIn should be incubated for the duration of the IncubationTime prior to starting the experiment.
    Default Value: Automatic
    Pattern Description: Ambient or greater than or equal to -20 degrees Celsius and less than or equal to 500 degrees Celsius or Null.
    Programmatic Pattern: ((Ambient | RangeP[$MinIncubationTemperature, $MaxIncubationTemperature]) | Automatic) | Null
    Index Matches to: experiment samples

    IncubationTime

    Duration for which SamplesIn should be incubated at the IncubationTemperature, prior to starting the experiment.
    Default Value: Automatic
    Pattern Description: Greater than or equal to 1 minute and less than or equal to 72 hours or Null.
    Programmatic Pattern: (RangeP[1*Minute, $MaxExperimentTime] | Automatic) | Null
    Index Matches to: experiment samples

    Mix

    Indicates if this sample should be mixed while incubated, prior to starting the experiment.
    Default Value: Automatic
    Default Calculation: Automatically resolves to True if any Mix related options are set. Otherwise, resolves to False.
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null
    Index Matches to: experiment samples

    MixType

    Indicates the style of motion used to mix the sample, prior to starting the experiment.
    Default Value: Automatic
    Default Calculation: Automatically resolves based on the container of the sample and the Mix option.
    Pattern Description: Roll, Vortex, Sonicate, Pipette, Invert, Stir, Shake, Homogenize, Swirl, Disrupt, or Nutate or Null.
    Programmatic Pattern: (MixTypeP | Automatic) | Null
    Index Matches to: experiment samples

    MixUntilDissolved

    Indicates if the mix should be continued up to the MaxIncubationTime or MaxNumberOfMixes (chosen according to the mix Type), in an attempt dissolve any solute. Any mixing/incubation will occur prior to starting the experiment.
    Default Value: Automatic
    Default Calculation: Automatically resolves to True if MaxIncubationTime or MaxNumberOfMixes is set.
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null
    Index Matches to: experiment samples

    MaxIncubationTime

    Maximum duration of time for which the samples will be mixed while incubated in an attempt to dissolve any solute, if the MixUntilDissolved option is chosen. This occurs prior to starting the experiment.
    Default Value: Automatic
    Default Calculation: Automatically resolves based on MixType, MixUntilDissolved, and the container of the given sample.
    Pattern Description: Greater than or equal to 1 minute and less than or equal to 72 hours or Null.
    Programmatic Pattern: (RangeP[1*Minute, $MaxExperimentTime] | Automatic) | Null
    Index Matches to: experiment samples

    IncubationInstrument

    The instrument used to perform the Mix and/or Incubation, prior to starting the experiment.
    Default Value: Automatic
    Default Calculation: Automatically resolves based on the options Mix, Temperature, MixType and container of the sample.
    Pattern Description: An object of type or subtype Model[Instrument, Roller], Model[Instrument, OverheadStirrer], Model[Instrument, Vortex], Model[Instrument, Shaker], Model[Instrument, BottleRoller], Model[Instrument, Roller], Model[Instrument, Sonicator], Model[Instrument, HeatBlock], Model[Instrument, Homogenizer], Model[Instrument, Disruptor], Model[Instrument, Nutator], Model[Instrument, Thermocycler], Model[Instrument, EnvironmentalChamber], Model[Instrument, Pipette], Object[Instrument, Roller], Object[Instrument, OverheadStirrer], Object[Instrument, Vortex], Object[Instrument, Shaker], Object[Instrument, BottleRoller], Object[Instrument, Roller], Object[Instrument, Sonicator], Object[Instrument, HeatBlock], Object[Instrument, Homogenizer], Object[Instrument, Disruptor], Object[Instrument, Nutator], Object[Instrument, Thermocycler], Object[Instrument, EnvironmentalChamber], or Object[Instrument, Pipette] or Null.
    Programmatic Pattern: (ObjectP[Join[MixInstrumentModels, MixInstrumentObjects]] | Automatic) | Null
    Index Matches to: experiment samples

    AnnealingTime

    Minimum duration for which the SamplesIn should remain in the incubator allowing the system to settle to room temperature after the IncubationTime has passed but prior to starting the experiment.
    Default Value: Automatic
    Pattern Description: Greater than or equal to 0 minutes and less than or equal to 72 hours or Null.
    Programmatic Pattern: (RangeP[0*Minute, $MaxExperimentTime] | Automatic) | Null
    Index Matches to: experiment samples

    IncubateAliquotContainer

    The desired type of container that should be used to prepare and house the incubation samples which should be used in lieu of the SamplesIn for the experiment.
    Default Value: Automatic
    Pattern Description: An object of type or subtype Model[Container] or {Index, Container} or Null.
    Programmatic Pattern: ((ObjectP[Model[Container]] | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
    Index Matches to: experiment samples

    IncubateAliquotDestinationWell

    The desired position in the corresponding IncubateAliquotContainer in which the aliquot samples will be placed.
    Default Value: Automatic
    Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
    Pattern Description: Any well from A1 to H12 or Null.
    Programmatic Pattern: (WellPositionP | Automatic) | Null
    Index Matches to: experiment samples

    IncubateAliquot

    The amount of each sample that should be transferred from the SamplesIn into the IncubateAliquotContainer when performing an aliquot before incubation.
    Default Value: Automatic
    Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container.
    Pattern Description: All or greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
    Programmatic Pattern: ((RangeP[1*Microliter, 20*Liter] | All) | Automatic) | Null
    Index Matches to: experiment samples

    Preparatory Centrifugation

    Centrifuge

    Indicates if the SamplesIn should be centrifuged prior to starting the experiment or any aliquoting. Sample Preparation occurs in the order of Incubation, Centrifugation, Filtration, and then Aliquoting (if specified).
    Default Value: Automatic
    Default Calculation: Resolves to True if any of the corresponding Centrifuge options are set. Otherwise, resolves to False.
    Pattern Description: True or False.
    Programmatic Pattern: BooleanP | Automatic
    Index Matches to: experiment samples

    CentrifugeInstrument

    The centrifuge that will be used to spin the provided samples prior to starting the experiment.
    Default Value: Automatic
    Pattern Description: An object of type or subtype Model[Instrument, Centrifuge] or Object[Instrument, Centrifuge] or Null.
    Programmatic Pattern: (ObjectP[{Model[Instrument, Centrifuge], Object[Instrument, Centrifuge]}] | Automatic) | Null
    Index Matches to: experiment samples

    CentrifugeIntensity

    The rotational speed or the force that will be applied to the samples by centrifugation prior to starting the experiment.
    Default Value: Automatic
    Pattern Description: Greater than 0 revolutions per minute or greater than 0 standard accelerations due to gravity on the surface of the earth or Null.
    Programmatic Pattern: ((GreaterP[0*RPM] | GreaterP[0*GravitationalAcceleration]) | Automatic) | Null
    Index Matches to: experiment samples

    CentrifugeTime

    The amount of time for which the SamplesIn should be centrifuged prior to starting the experiment.
    Default Value: Automatic
    Pattern Description: Greater than 0 minutes or Null.
    Programmatic Pattern: (GreaterP[0*Minute] | Automatic) | Null
    Index Matches to: experiment samples

    CentrifugeTemperature

    The temperature at which the centrifuge chamber should be held while the samples are being centrifuged prior to starting the experiment.
    Default Value: Automatic
    Pattern Description: Ambient or greater than or equal to -10 degrees Celsius and less than or equal to 40 degrees Celsius or Null.
    Programmatic Pattern: ((Ambient | RangeP[-10*Celsius, 40*Celsius]) | Automatic) | Null
    Index Matches to: experiment samples

    CentrifugeAliquotContainer

    The desired type of container that should be used to prepare and house the centrifuge samples which should be used in lieu of the SamplesIn for the experiment.
    Default Value: Automatic
    Pattern Description: An object of type or subtype Model[Container] or {Index, Container} or Null.
    Programmatic Pattern: ((ObjectP[Model[Container]] | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
    Index Matches to: experiment samples

    CentrifugeAliquotDestinationWell

    The desired position in the corresponding AliquotContainer in which the aliquot samples will be placed.
    Default Value: Automatic
    Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
    Pattern Description: Any well from A1 to H12 or Null.
    Programmatic Pattern: (WellPositionP | Automatic) | Null
    Index Matches to: experiment samples

    CentrifugeAliquot

    The amount of each sample that should be transferred from the SamplesIn into the CentrifugeAliquotContainer when performing an aliquot before centrifugation.
    Default Value: Automatic
    Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container.
    Pattern Description: All or greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
    Programmatic Pattern: ((RangeP[1*Microliter, 20*Liter] | All) | Automatic) | Null
    Index Matches to: experiment samples

    Preparatory Filtering

    Filtration

    Indicates if the SamplesIn should be filter prior to starting the experiment or any aliquoting. Sample Preparation occurs in the order of Incubation, Centrifugation, Filtration, and then Aliquoting (if specified).
    Default Value: Automatic
    Default Calculation: Resolves to True if any of the corresponding Filter options are set. Otherwise, resolves to False.
    Pattern Description: True or False.
    Programmatic Pattern: BooleanP | Automatic
    Index Matches to: experiment samples

    FiltrationType

    The type of filtration method that should be used to perform the filtration.
    Default Value: Automatic
    Default Calculation: Will automatically resolve to a filtration type appropriate for the volume of sample being filtered.
    Pattern Description: PeristalticPump, Centrifuge, Vacuum, Syringe, or AirPressure or Null.
    Programmatic Pattern: (FiltrationTypeP | Automatic) | Null
    Index Matches to: experiment samples

    FilterInstrument

    The instrument that should be used to perform the filtration.
    Default Value: Automatic
    Default Calculation: Will automatically resolved to an instrument appropriate for the filtration type.
    Pattern Description: An object of type or subtype Model[Instrument, FilterBlock], Object[Instrument, FilterBlock], Model[Instrument, PeristalticPump], Object[Instrument, PeristalticPump], Model[Instrument, VacuumPump], Object[Instrument, VacuumPump], Model[Instrument, Centrifuge], Object[Instrument, Centrifuge], Model[Instrument, SyringePump], or Object[Instrument, SyringePump] or Null.
    Programmatic Pattern: (ObjectP[{Model[Instrument, FilterBlock], Object[Instrument, FilterBlock], Model[Instrument, PeristalticPump], Object[Instrument, PeristalticPump], Model[Instrument, VacuumPump], Object[Instrument, VacuumPump], Model[Instrument, Centrifuge], Object[Instrument, Centrifuge], Model[Instrument, SyringePump], Object[Instrument, SyringePump]}] | Automatic) | Null
    Index Matches to: experiment samples

    Filter

    The filter that should be used to remove impurities from the SamplesIn prior to starting the experiment.
    Default Value: Automatic
    Default Calculation: Will automatically resolve to a filter appropriate for the filtration type and instrument.
    Pattern Description: An object of type or subtype Model[Container, Plate, Filter], Model[Container, Vessel, Filter], or Model[Item, Filter] or Null.
    Programmatic Pattern: (ObjectP[{Model[Container, Plate, Filter], Model[Container, Vessel, Filter], Model[Item, Filter]}] | Automatic) | Null
    Index Matches to: experiment samples

    FilterMaterial

    The membrane material of the filter that should be used to remove impurities from the SamplesIn prior to starting the experiment.
    Default Value: Automatic
    Default Calculation: Resolves to an appropriate filter material for the given sample is Filtration is set to True.
    Pattern Description: Cellulose, Cotton, Polyethylene, PTFE, Nylon, PES, PLUS, PVDF, GlassFiber, GHP, UHMWPE, EPDM, DuraporePVDF, GxF, ZebaDesaltingResin, NickelResin, Silica, or HLB or Null.
    Programmatic Pattern: (FilterMembraneMaterialP | Automatic) | Null
    Index Matches to: experiment samples

    PrefilterMaterial

    The material from which the prefilter filtration membrane should be made of to remove impurities from the SamplesIn prior to starting the experiment.
    Default Value: Automatic
    Default Calculation: By default, no prefiltration is performed on samples, even when Filter->True.
    Pattern Description: Cellulose, Cotton, Polyethylene, PTFE, Nylon, PES, PLUS, PVDF, GlassFiber, GHP, UHMWPE, EPDM, DuraporePVDF, GxF, ZebaDesaltingResin, NickelResin, Silica, or HLB or Null.
    Programmatic Pattern: (FilterMembraneMaterialP | Automatic) | Null
    Index Matches to: experiment samples

    FilterPoreSize

    The pore size of the filter that should be used when removing impurities from the SamplesIn prior to starting the experiment.
    Default Value: Automatic
    Default Calculation: Resolves to an appropriate filter pore size for the given sample is Filtration is set to True.
    Pattern Description: 0.008 micrometers, 0.1 micrometers, 0.22 micrometers, 0.45 micrometers, 1. micrometer, 1.1 micrometers, 2.5 micrometers, 6. micrometers, 20. micrometers, 30. micrometers, or 100. micrometers or Null.
    Programmatic Pattern: (FilterSizeP | Automatic) | Null
    Index Matches to: experiment samples

    PrefilterPoreSize

    The pore size of the filter; all particles larger than this should be removed during the filtration.
    Default Value: Automatic
    Default Calculation: By default, no prefiltration is performed on samples, even when Filter->True.
    Pattern Description: 0.008 micrometers, 0.1 micrometers, 0.22 micrometers, 0.45 micrometers, 1. micrometer, 1.1 micrometers, 2.5 micrometers, 6. micrometers, 20. micrometers, 30. micrometers, or 100. micrometers or Null.
    Programmatic Pattern: (FilterSizeP | Automatic) | Null
    Index Matches to: experiment samples

    FilterSyringe

    The syringe used to force that sample through a filter.
    Default Value: Automatic
    Default Calculation: Resolves to an syringe appropriate to the volume of sample being filtered, if Filtration is set to True.
    Pattern Description: An object of type or subtype Model[Container, Syringe] or Object[Container, Syringe] or a prepared sample or Null.
    Programmatic Pattern: ((ObjectP[{Model[Container, Syringe], Object[Container, Syringe]}] | _String) | Automatic) | Null
    Index Matches to: experiment samples

    FilterHousing

    The filter housing that should be used to hold the filter membrane when filtration is performed using a standalone filter membrane.
    Default Value: Automatic
    Default Calculation: Resolve to an housing capable of holding the size of the membrane being used, if filter with Membrane FilterType is being used and Filtration is set to True.
    Pattern Description: An object of type or subtype Model[Instrument, FilterHousing], Object[Instrument, FilterHousing], Model[Instrument, FilterBlock], or Object[Instrument, FilterBlock] or Null.
    Programmatic Pattern: (ObjectP[{Model[Instrument, FilterHousing], Object[Instrument, FilterHousing], Model[Instrument, FilterBlock], Object[Instrument, FilterBlock]}] | Automatic) | Null
    Index Matches to: experiment samples

    FilterIntensity

    The rotational speed or force at which the samples will be centrifuged during filtration.
    Default Value: Automatic
    Default Calculation: Will automatically resolve to 2000 GravitationalAcceleration if FiltrationType is Centrifuge and Filtration is True.
    Pattern Description: Greater than 0 revolutions per minute or greater than 0 standard accelerations due to gravity on the surface of the earth or Null.
    Programmatic Pattern: ((GreaterP[0*RPM] | GreaterP[0*GravitationalAcceleration]) | Automatic) | Null
    Index Matches to: experiment samples

    FilterTime

    The amount of time for which the samples will be centrifuged during filtration.
    Default Value: Automatic
    Default Calculation: Will automatically resolve to 5 Minute if FiltrationType is Centrifuge and Filtration is True.
    Pattern Description: Greater than 0 minutes or Null.
    Programmatic Pattern: (GreaterP[0*Minute] | Automatic) | Null
    Index Matches to: experiment samples

    FilterTemperature

    The temperature at which the centrifuge chamber will be held while the samples are being centrifuged during filtration.
    Default Value: Automatic
    Default Calculation: Will automatically resolve to 22 Celsius if FiltrationType is Centrifuge and Filtration is True.
    Pattern Description: Greater than or equal to 4 degrees Celsius or Null.
    Programmatic Pattern: ((Alternatives[GreaterEqualP[4*Celsius]]) | Automatic) | Null
    Index Matches to: experiment samples

    FilterContainerOut

    The desired container filtered samples should be produced in or transferred into by the end of filtration, with indices indicating grouping of samples in the same plates, if desired.
    Default Value: Automatic
    Default Calculation: Automatically set as the PreferredContainer for the Volume of the sample. For plates, attempts to fill all wells of a single plate with the same model before using another one.
    Pattern Description: An object of type or subtype Model[Container] or Object[Container] or a prepared sample or {Index, Container} or Null.
    Programmatic Pattern: (((ObjectP[{Model[Container], Object[Container]}] | _String) | {GreaterEqualP[1, 1] | Automatic, (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
    Index Matches to: experiment samples

    FilterAliquotDestinationWell

    The desired position in the corresponding AliquotContainer in which the aliquot samples will be placed.
    Default Value: Automatic
    Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
    Pattern Description: Any well from A1 to H12 or Null.
    Programmatic Pattern: (WellPositionP | Automatic) | Null
    Index Matches to: experiment samples

    FilterAliquotContainer

    The desired type of container that should be used to prepare and house the filter samples which should be used in lieu of the SamplesIn for the experiment.
    Default Value: Automatic
    Pattern Description: An object of type or subtype Model[Container] or {Index, Container} or Null.
    Programmatic Pattern: ((ObjectP[Model[Container]] | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
    Index Matches to: experiment samples

    FilterAliquot

    The amount of each sample that should be transferred from the SamplesIn into the FilterAliquotContainer when performing an aliquot before filtration.
    Default Value: Automatic
    Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container.
    Pattern Description: All or greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
    Programmatic Pattern: ((RangeP[1*Microliter, 20*Liter] | All) | Automatic) | Null
    Index Matches to: experiment samples

    FilterSterile

    Indicates if the filtration of the samples should be done in a sterile environment.
    Default Value: Automatic
    Default Calculation: Resolve to False if Filtration is indicated. If sterile filtration is desired, this option must manually be set to True.
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null
    Index Matches to: experiment samples

    Aliquoting

    Aliquot

    Indicates if aliquots should be taken from the SamplesIn and transferred into new AliquotSamples used in lieu of the SamplesIn for the experiment. Note that if NumberOfReplicates is specified this indicates that the input samples will also be aliquoted that number of times. Note that Aliquoting (if specified) occurs after any Sample Preparation (if specified).
    Default Value: Automatic
    Pattern Description: True or False.
    Programmatic Pattern: BooleanP | Automatic
    Index Matches to: experiment samples

    AliquotAmount

    The amount of a sample that should be transferred from the input samples into aliquots.
    Default Value: Automatic
    Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container if a liquid, or the current Mass or Count if a solid or counted item, respectively.
    Pattern Description: All or Count or Count or Mass or Volume or Null.
    Programmatic Pattern: ((RangeP[1*Microliter, 20*Liter] | RangeP[1*Milligram, 20*Kilogram] | GreaterP[0*Unit, 1*Unit] | GreaterP[0., 1.] | All) | Automatic) | Null
    Index Matches to: experiment samples

    TargetConcentration

    The desired final concentration of analyte in the AliquotSamples after dilution of aliquots of SamplesIn with the ConcentratedBuffer and BufferDiluent which should be used in lieu of the SamplesIn for the experiment.
    Default Value: Automatic
    Default Calculation: Automatically calculated based on aliquot and buffer volumes.
    Pattern Description: Greater than 0 molar or greater than 0 grams per liter or Null.
    Programmatic Pattern: ((GreaterP[0*Molar] | GreaterP[0*(Gram/Liter)]) | Automatic) | Null
    Index Matches to: experiment samples

    TargetConcentrationAnalyte

    The substance whose final concentration is attained with the TargetConcentration option.
    Default Value: Automatic
    Default Calculation: Automatically set to the first value in the Analytes field of the input sample, or, if not populated, to the first analyte in the Composition field of the input sample, or if none exist, the first identity model of any kind in the Composition field.
    Pattern Description: An object of type or subtype Model[Molecule], Model[Molecule, cDNA], Model[Molecule, Oligomer], Model[Molecule, Transcript], Model[Molecule, Protein], Model[Molecule, Protein, Antibody], Model[Molecule, Carbohydrate], Model[Molecule, Polymer], Model[Resin], Model[Resin, SolidPhaseSupport], Model[Lysate], Model[ProprietaryFormulation], Model[Virus], Model[Cell], Model[Cell, Mammalian], Model[Cell, Bacteria], Model[Cell, Yeast], Model[Tissue], Model[Material], or Model[Species] or Null.
    Programmatic Pattern: (ObjectP[IdentityModelTypes] | Automatic) | Null
    Index Matches to: experiment samples

    AssayVolume

    The desired total volume of the aliquoted sample plus dilution buffer.
    Default Value: Automatic
    Default Calculation: Automatically determined based on Volume and TargetConcentration option values.
    Pattern Description: Greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
    Programmatic Pattern: (RangeP[1*Microliter, 20*Liter] | Automatic) | Null
    Index Matches to: experiment samples

    ConcentratedBuffer

    The concentrated buffer which should be diluted by the BufferDilutionFactor in the final solution (i.e., the combination of the sample, ConcentratedBuffer, and BufferDiluent). The ConcentratedBuffer and BufferDiluent will be combined and then mixed with the sample, where the combined volume of these buffers is the difference between the AliquotAmount and the total AssayVolume.
    Default Value: Automatic
    Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
    Programmatic Pattern: ((ObjectP[{Model[Sample], Object[Sample]}] | _String) | Automatic) | Null
    Index Matches to: experiment samples

    BufferDilutionFactor

    The dilution factor by which the concentrated buffer should be diluted in the final solution (i.e., the combination of the sample, ConcentratedBuffer, and BufferDiluent). The ConcentratedBuffer and BufferDiluent will be combined and then mixed with the sample, where the combined volume of these buffers is the difference between the AliquotAmount and the total AssayVolume.
    Default Value: Automatic
    Default Calculation: If ConcentratedBuffer is specified, automatically set to the ConcentrationFactor of that sample; otherwise, set to Null.
    Pattern Description: Greater than or equal to 1 or Null.
    Programmatic Pattern: (GreaterEqualP[1] | Automatic) | Null
    Index Matches to: experiment samples

    BufferDiluent

    The buffer used to dilute the aliquot sample such that ConcentratedBuffer is diluted by BufferDilutionFactor in the final solution. The ConcentratedBuffer and BufferDiluent will be combined and then mixed with the sample, where the combined volume of these buffers is the difference between the AliquotAmount and the total AssayVolume.
    Default Value: Automatic
    Default Calculation: Automatically resolves to Model[Sample, "Milli-Q water"] if ConcentratedBuffer is specified; otherwise, resolves to Null.
    Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
    Programmatic Pattern: ((ObjectP[{Model[Sample], Object[Sample]}] | _String) | Automatic) | Null
    Index Matches to: experiment samples

    AssayBuffer

    The buffer that should be added to any aliquots requiring dilution, where the volume of this buffer added is the difference between the AliquotAmount and the total AssayVolume.
    Default Value: Automatic
    Default Calculation: Automatically resolves to Model[Sample, "Milli-Q water"] if ConcentratedBuffer is not specified; otherwise, resolves to Null.
    Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
    Programmatic Pattern: ((ObjectP[{Model[Sample], Object[Sample]}] | _String) | Automatic) | Null
    Index Matches to: experiment samples

    AliquotSampleStorageCondition

    The non-default conditions under which any aliquot samples generated by this experiment should be stored after the protocol is completed.
    Default Value: Automatic
    Pattern Description: {AmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, BacteriaIncubation, MammalianIncubation, TissueCultureCellsIncubation, MicrobialCellsIncubation, MicrobialCellsShakingIncubation, YeastCellsIncubation, YeastCellsShakingIncubation, ViralIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
    Programmatic Pattern: ((SampleStorageTypeP | Disposal) | Automatic) | Null
    Index Matches to: experiment samples

    DestinationWell

    The desired position in the corresponding AliquotContainer in which the aliquot samples will be placed.
    Default Value: Automatic
    Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
    Pattern Description: Any well from A1 to H12 or list of one or more any well from A1 to H12 or any well from A1 to H12 entries or Null.
    Programmatic Pattern: ((WellPositionP | {((Automatic | Null) | WellPositionP)..}) | Automatic) | Null

    AliquotContainer

    The desired type of container that should be used to prepare and house the aliquot samples, with indices indicating grouping of samples in the same plates, if desired. This option will resolve to be the length of the SamplesIn * NumberOfReplicates.
    Default Value: Automatic
    Default Calculation: Automatically set as the PreferredContainer for the AssayVolume of the sample. For plates, attempts to fill all wells of a single plate with the same model before aliquoting into the next.
    Pattern Description: An object of type or subtype Model[Container] or Object[Container] or a prepared sample or Automatic or Null or {Index, Container} or list of one or more an object of type or subtype Model[Container] or Object[Container] or a prepared sample or Automatic or Null entries or list of one or more Automatic or Null or {Index, Container} entries or Null.
    Programmatic Pattern: (((ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null) | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null)} | {((ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null))..} | {({GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null)} | (Automatic | Null))..}) | Automatic) | Null

    AliquotPreparation

    Indicates the desired scale at which liquid handling used to generate aliquots will occur.
    Default Value: Automatic
    Default Calculation: Automatic resolution will occur based on manipulation volumes and container types.
    Pattern Description: Manual or Robotic or Null.
    Programmatic Pattern: (PreparationMethodP | Automatic) | Null

    ConsolidateAliquots

    Indicates if identical aliquots should be prepared in the same container/position.
    Default Value: Automatic
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null

Protocol Options

    Organizational Information

    Template

    A template protocol whose methodology should be reproduced in running this experiment. Option values will be inherited from the template protocol, but can be individually overridden by directly specifying values for those options to this Experiment function.
    Default Value: Null
    Pattern Description: An object of type or subtype Object[Protocol] or an object of type or subtype of Object[Protocol] with UnresolvedOptions, ResolvedOptions specified or Null.
    Programmatic Pattern: (ObjectP[Object[Protocol]] | FieldReferenceP[Object[Protocol], {UnresolvedOptions, ResolvedOptions}]) | Null

    Name

    A object name which should be used to refer to the output object in lieu of an automatically generated ID number.
    Default Value: Null
    Pattern Description: A string or Null.
    Programmatic Pattern: _String | Null

    Post Experiment

    MeasureWeight

    Indicates if any solid samples that are modified in the course of the experiment should have their weights measured and updated after running the experiment. Please note that public samples are weighed regardless of the value of this option.
    Default Value: Automatic
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null

    MeasureVolume

    Indicates if any liquid samples that are modified in the course of the experiment should have their volumes measured and updated after running the experiment. Please note that public samples are volume measured regardless of the value of this option.
    Default Value: Automatic
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null

    ImageSample

    Indicates if any samples that are modified in the course of the experiment should be freshly imaged after running the experiment. Please note that public samples are imaged regardless of the value of this option.
    Default Value: Automatic
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null

Example Calls

    Basics

    To perform a DNA sequencing experiment on samples that are already fluorescently labeled, simply run:
    Primers may be specified to indicate that they should be added to the reaction mixtures and that chain termination PCR wil be used to prepare the samples:

    PreparedPlate

    If samples will undergo chain termination PCR, add master mix containing dyes, nucleotides and buffer, as well as a diluent to prepare the samples for capillary electrophoresis

    Set PCR settings

    If a PreparedPlate is not being used, settings for the chain termination polymerase reaction can be set

    PurificationType

    Samples can be purified following chain termination PCR

    DyeSet

    The dye set used to label the DNA template samples is specified

    Capillary Electrophoresis

    Settings for the capillary gel electrophoresis run can be specified

Preferred Input Containers

    The SeqStudio accepts MicroAmp optical 96-well semi-skirted plates, which also fit in the Automated Thermocycler (ATC) for any PCR steps.

Warnings and Errors

    Messages  (36)

    DeprecatedModels  (2)

    Primer samples whose models are deprecated cannot be provided:

    Samples whose models are deprecated cannot be provided:

    DiscardedSamples  (2)

    Samples that are discarded cannot be provided:

    The primers cannot have a Status of Discarded:

    DNASequencingAnodeBufferInvalid  (1)

    An error is thrown if the AnodeBuffer specified in the SequencingCartridge Object is invalid:

    DNASequencingBufferVolumeMismatch  (2)

    The SequencingBufferVolume cannot be specified as Null when SequencingBuffer is not Null:

    The SequencingBufferVolume can only be specified when SequencingBuffer is not Null:

    DNASequencingCartridgeStorageCondition  (1)

    If the SequencingCartridgeStorageCondition is not specified as Refrigerator, a warning is thrown:

    DNASequencingCartridgeTypeInvalid  (1)

    An error is thrown if the CartridgeType specified in the SequencingCartridge Object is invalid:

    DNASequencingCathodeBufferInvalid  (1)

    An error is thrown if the CathodeSequencingBuffer specified in the BufferCartridge Object is invalid:

    DNASequencingDiluentNotSpecified  (1)

    The DiluentVolume can only be specified when Diluent is not Null:

    DNASequencingMasterMixNotSpecified  (2)

    The MasterMixVolume cannot be specified as Null when MasterMix is not Null:

    The MasterMixVolume can only be specified when MasterMix is not Null:

    DNASequencingMasterMixStorageConditionMismatch  (1)

    MasterMixStorageCondition is specified only when MasterMix is specified:

    DNASequencingPCROptionsForPreparedPlate  (1)

    A warning is thrown if PCR options are specified when PreparedPlate->True:

    DNASequencingPolymerInvalid  (1)

    An error is thrown if the Polymer specified in the SequencingCartridge Object is invalid:

    DNASequencingPreparedPlateInputInvalid  (1)

    An error is thrown if PreparedPlate->True and primer inputs are given, or PreparedPlate->False and no primer inputs are given:

    DNASequencingPreparedPlateMismatch  (2)

    If all sample preparation options are Null and there are no primer inputs, PreparedPlate should be set to True:

    If all sample preparation options are set and primers are given as inputs, PreparedPlate should be set to False:

    DNASequencingPrimerCompositionNull  (1)

    The PrimerVolume is specified or primer[Composition] is informed so PrimerVolume is automatically resolved when PreparedPlate -> False:

    DNASequencingPrimerStorageConditionMismatch  (1)

    PrimerStorageCondition is only specified when primer inputs are specified:

    DNASequencingPurificationTypeMismatch  (2)

    QuenchingReagents cannot be Null when PurificationType is BigDye XTerminator:

    SequencingBuffer cannot be Null when PurificationType is Ethanol precipitation:

    DNASequencingQuenchingReagentVolumeMismatch  (2)

    The QuenchingReagentVolumes cannot be specified as Null when QuenchingReagents is not Null:

    The QuenchingReagentVolumes can only be specified when QuenchingReagents is not Null:

    DNASequencingReadLengthNotSpecified  (1)

    If the sample composition is Null, the read length is not set based on the composition:

    DNASequencingTooManyInjectionGroups  (1)

    An error is thrown if more injection groups are formed than can fit on the plate:

    DNASequencingTooManySamples  (1)

    An error is thrown if more than 96 samples are run in a single protocol:

    DNASequencingTotalVolumeOverReactionVolume  (1)

    The total volume cannot exceed ReactionVolume:

    DuplicateName  (1)

    The Name option must not be the name of an already-existing DNASequencing protocol:

    DyeSetUnknown  (1)

    The dye sets are specified or are resolved from the name of the Master Mix:

    InvalidPreparedPlateContainer  (2)

    When using a prepared plate, all the samples must be in a 96-well Optical Semi-Skirted PCR Plate:

    When using a prepared plate, all the samples must be in a 96-well Optical Semi-Skirted PCR Plate:

    MultiplePrimerSampleOligomersSpecified  (1)

    An error is thrown if multiple oligomer molecules are specified in the primer sample composition:

    MultipleSampleOligomersSpecified  (1)

    An error is thrown if multiple oligomer molecules are specified in the sample composition:

    SolidSample  (1)

    The samples and primer samples cannot be solid:

Possible Issues

    Too much or too little DNA template

    The amount of DNA in the sample is important, and should be quantified before preparing samples for DNA sequencing. Based on the length of the DNA sample, the concentration of the DNA samples should be changed. An amount of 20 nanograms of PCR product in a 10 microliter reaction is the minimum. More DNA should be used for longer sequences.

    Dye Blobs

    Indicates poor removal of the dye terminators. If BigDye products were used, purification with BigDye XTerminator is recommended to remove unreacted dyes prior to capillary electrphoresis.

    Off scale peaks

    If the amplicon is very efficient, or if too much DNA is in the sample, off scale peaks can occur. To fix this, adjust the InjectionTime or InjectionVoltage options, or use a lower concentration of DNA in the sample, or reduce the number of cycles in the PCR experiment to prepare the samples for ExperimentDNASequencing.
Last modified on Thu 29 Dec 2022 10:45:41
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