EP-4442838-B1 - MULTIPLEXED TARGET-BINDING CANDIDATE SCREENING ANALYSIS

EP4442838B1EP 4442838 B1EP4442838 B1EP 4442838B1EP-4442838-B1

Inventors

CUNNINGHAM, Christian N.
HALLENBECK, Kenneth Karl

Dates

Publication Date: 20260513
Application Date: 20211014

Claims (15)

A computer-implemented method for normalizing a polymerase chain reaction (PCR) amplification process for a plurality of samples containing DNA, the method comprising: identifying cycle data for performing the PCR amplification process for the plurality of samples, the cycle data including a corresponding cycle count for each sample of the plurality of samples; sorting the plurality of samples into a plurality of bins based on the cycle data such that cycle count variation between bins of the plurality of bins is reduced; assigning a bin cycle count to each bin of the plurality of bins, wherein the bin cycle count is unique to each bin of the plurality of bins; generating identification information for the plurality of bins; and generating an output for performing a PCR amplification of the plurality of samples using the plurality of bins and the bin cycle count for each bin of the plurality of bins.
The method of claim 1, wherein the plurality of samples have varying amounts of DNA and wherein after the PCR amplification process is performed such that the plurality of samples become a plurality of amplified samples, the plurality of amplified samples have normalized amounts of DNA.
The method of claim 1 or claim 2, wherein generating the identification information comprises generating a bin identifier for each bin of the plurality of bins, and/or wherein generating the output comprises generating a forward worklist for a bin of the plurality of bins, the forward worklist including an order in which the set of samples in the bin is to be transferred from a source plate to a corresponding PCR plate for the bin, and/or wherein the output further comprises instructions for a thermocycler for performing the PCR amplification process using a set of PCR plates, and/or wherein generating the output comprises generating a transfer output for use in transferring the set of samples in each bin of the plurality of bins to a corresponding PCR plate of a set of PCR plates using the identification information.
The method of any preceding claim, wherein assigning the bin cycle count comprises: generating the bin cycle count for a bin of the plurality of bins using a cycle count from a group consisting of a highest cycle count of the set of samples in the bin, an average cycle count of the set of samples, a median cycle count of the set of samples, and a sum of the highest cycle count and a selected cycle count, optionally wherein the selected cycle count is changed from a first run of the PCR amplification process to a second run of the PCR amplification process.
The method of any preceding claim, wherein the sorting comprises: (a) converting any non-integer cycle count of the cycle data into an integer cycle count to form modified cycle data; assigning unique integer cycle counts of the modified cycle data that are within a selected range of cycle counts to N bins; and merging adjacent bins of the N bins using a distance equation until a target number of bins is reached, wherein the target number of bins forms at least a portion of the plurality of bins; or (b) sorting, based on the distribution for the plurality of cycle counts, the plurality of samples into the plurality of bins with a bias towards providing more bin separation between a portion of the plurality of cycle counts falling within one standard deviation of a mean of the plurality of cycle counts as compared to another portion of the plurality of cycle counts falling outside the one standard deviation of the mean.
The method of any preceding claim, wherein identifying the cycle data comprises receiving the cycle data from a quantitative PCR (qPCR) system, or wherein identifying the cycle data comprises: receiving initial cycle data from a quantitative PCR (qPCR) system, the initial cycle data including an initial cycle count for each sample of the plurality of samples; and modifying the initial cycle count for at least one of the plurality of samples to generate the cycle data.
The method of claim 6, wherein the initial cycle count for a sample of the plurality of samples is an undetermined value and wherein the modifying comprises changing the initial cycle count for the sample from the undetermined value to a preselected cycle count, and/or wherein the modifying comprises at least one of: adding two cycles to the initial cycle count for each sample of the plurality of samples; or converting each non-integer cycle count into an integer cycle count.
The method of any preceding claim, wherein the sorting comprises: (a) distributing the plurality of samples into the plurality of bins based on a selected range of cycle counts in the cycle data, optionally wherein the distributing comprises: assigning a portion of the plurality of samples having corresponding cycle counts outside of the selected range of cycle counts to a portion of the plurality of bins; and distributing, substantially evenly, a remaining portion of the plurality of samples having corresponding cycle counts within the selected range of cycle counts between a remaining portion of the plurality of bins; and/or (b) converting each non-integer cycle count in the cycle data into an integer cycle count to form modified cycle data; distributing, substantially evenly, the plurality of samples into the plurality of bins based on a selected range of cycle counts in the modified cycle data.
The method of any of claims 1-4, wherein the plurality of samples have a plurality of corresponding cycle counts and wherein the sorting comprises: assigning a first portion of the plurality of samples having a first set of cycle counts below a selected low cycle count to a first bin of the plurality of bins; assigning a second portion of the plurality of samples having a second set of cycle counts above a selected high count to a last bin of the plurality of bins; and distributing a remaining portion the plurality of samples into a set of bins between the first bin and the last bin.
The method of any of claims 1-4, wherein the plurality of samples have a plurality of corresponding cycle counts and wherein the sorting comprises: identifying a distribution for the plurality of corresponding cycle counts, optionally wherein identifying the distribution comprises: identifying a histogram distribution for the plurality of corresponding cycle counts, wherein between 60 percent and 100 percent of the plurality of corresponding cycle counts are within a selected range between and inclusive of a selected low cycle count and a selected high cycle count, optionally wherein the selected low cycle count is 4 cycle counts and wherein the selected high cycle count is 24 cycle counts.
The method of claim 10, wherein the sorting comprises: assigning any sample of the plurality of samples having the corresponding cycle count below the selected low cycle count to a first bin of the plurality of bins; and assigning any sample of the plurality of samples having the corresponding cycle count above the selected high cycle count to a last bin of the plurality of bins, optionally wherein the sorting further comprises distributing a remaining portion of the plurality of samples between a set of bins between the first bin and the last bin; or wherein 100 percent of the plurality of corresponding cycle counts are within the selected range and wherein sorting the plurality of samples into the plurality of bins comprises: distributing the plurality of samples between the plurality of bins with a bias towards providing more bin separation between lower cycle counts as compared to higher cycle counts.
The method of any of claims 1-4, wherein the sorting comprises: distributing the plurality of samples into the plurality of bins based on a selected range of cycle counts in the cycle data; and modifying the distribution of the plurality of samples in the plurality of bins such that any samples of the plurality of samples having corresponding cycle counts associated with a same integer value are grouped together in a same bin; optionally wherein the plurality of samples have a plurality of corresponding cycle counts and wherein two cycle counts of the plurality of corresponding cycle counts are associated with a same integer value if each of the two cycle counts contain a whole number that is the same integer value, or wherein the plurality of samples have a plurality of corresponding cycle counts and wherein two cycle counts of the plurality of corresponding cycle counts are associated with a same integer value if each of the two cycle counts rounds up to the same integer value.
A method comprising: performing the method of any preceding claim; transferring the set of samples in each bin of the plurality of bins to a corresponding PCR plate of a set of PCR plates based on the output; and performing the PCR amplification process using the set of PCR plates.
A system comprising: one or more data processors; and a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform the methods disclosed in claims 1-12.
A computer-program product embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform the methods disclosed in claims 1-12.

Description

FIELD Provided herein are methods and systems for improved multiplexed screening analysis. More specifically, methods and systems are provided for multiplexed screening of nucleotide-tagged peptide libraries for target-binding activity. BACKGROUND Current multiplexed target-binding candidate screening analysis systems have difficulty with the simultaneous selection of many nucleotide-containing peptide libraries for binding to a desired target due to problems such as sample-to-sample variations and data complexity. There is, therefore, a need for improved multiplexed target-binding candidate screening analysis systems and methods to help simultaneous selection of candidate binders against a desired binding target, e.g., a protein. US 2020/035329 A1 describes a computer-implemented method of generating a digital polymerase chain reaction (dPCR) result. The method comprises detecting of emission data from a plurality of samples at a first time amplification period, determining a positive or negative amplification determination for each sample of the plurality of samples based in part on the first set of emission data, and generating a dPCR result based on the positive amplification determinations. WO 98/26098 A1 relates to methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom. The methods comprise using oligonucleotides covalently linked to a solid support to isolate specific labelled nucleic acid sequences, perform quantitative comparisons, and allow for target elution and analysis. SUMMARY The embodiments described herein provide various methods, systems, and computer program products for improved multiplexed screening analysis. In some embodiments not part of the claims, a computer-implemented method is provided for normalizing a plurality of libraries of DNA-containing compositions. The method includes receiving quantification information for each of a plurality of libraries of DNA-containing compositions for a first round of selection, wherein each of the libraries comprises DNA conjugates, and wherein the first round of selection causes selection of the DNA conjugates based on binding affinity to a target protein. The method further includes determining a polymerase chain reaction ("PCR") cycle count specific for each of the libraries using the quantification information, wherein DNA-containing compositions of each of the libraries can produce or are determined to produce an associated pre-set amount of DNA after performing PCR with a corresponding PCR cycle count. The method further includes sorting the libraries of DNA-containing compositions into bins using corresponding PCR cycle counts so determined, wherein each bin comprises a different subset of the libraries of the DNA-containing compositions determined to share a common PCR cycle count for DNA amplification, and each subset of the libraries of the DNA-containing compositions in the same bin shares a common PCR cycle count different from that of other bins of libraries. The method further includes instructing a thermocycler to perform PCR, in a same run, on one of the bins with a corresponding common PCR cycle count simultaneously for a subset of the libraries of DNA-containing compositions of the same bin. The method further includes instructing the thermocycler to perform PCR on each of additional bins with a corresponding common PCR cycle count so that the plurality of libraries of DNA-containing compositions are normalized. In some embodiments not part of the claims, there is provided a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform a method for normalizing a plurality of libraries of DNA-containing compositions. The method includes receiving quantification information for each of a plurality of libraries of DNA-containing compositions for a first round of selection, wherein each of the libraries comprises DNA conjugates, and wherein the first round of selection causes selection of the DNA conjugates based on binding affinity to a target protein. The method further includes determining a polymerase chain reaction ("PCR") cycle count specific for each of the libraries using the quantification information, wherein DNA-containing compositions of each of the libraries are determined to produce an associated pre-set amount of DNA after performing PCR with a corresponding PCR cycle count. The method further includes sorting the libraries of DNA-containing compositions into bins using corresponding PCR cycle counts so determined, wherein each bin comprises a different subset of the libraries of the DNA-containing compositions determined to share a common PCR cycle count for DNA amplification, and each subset of the libraries of the DNA-containing compositions in the same bin shares a common PCR cycle count different from that of other bins of librarie