US Pat. No. 9,388,457

LOCUS SPECIFIC AMPLIFICATION USING ARRAY PROBES

Affymetrix, Inc., Santa ...

1. A method for amplifying a plurality of target nucleic acid sequences from an un-amplified nucleic acid sample and analyzing
the amplified target sequences, said method comprising:
(a) fragmenting the un-amplified nucleic acid sample to obtain un-amplified target nucleic acid fragments;
(b) mixing the un-amplified target nucleic acid fragments obtained in (a) with an array of probes arranged in features, wherein
a feature comprises multiple copies of a target specific probe, so that un-amplified target nucleic acid fragments hybridize
to complementary target specific probes to form probe:target complexes;

(c) extending the probes in the probe:target complexes using a polymerase, wherein the un-amplified target nucleic acid fragment
is used as template for extension of the probe and wherein an extended probe is generated;

(d) removing the un-amplified target nucleic acid fragment from the extended probe by denaturation;
(e) generating a plurality of complementary copies of the extended probe, wherein said copies are not covalently attached
to the surface of the solid support;

(f) allowing the copies to hybridize to the complementary target specific probes in the same feature; and
(g) analyzing the copies, wherein the plurality of target nucleic acid sequences comprises between 100 and 100,000 different
target nucleic acid sequences.

US Pat. No. 9,290,798

MANUFACTURING AND PROCESSING POLYMER ARRAYS

Affymetrix, Inc., Santa ...

1. A probe array plate comprising:
a wafer having a plurality of probe arrays bound to an active side of the wafer, each probe array surrounded by a border region
on the wafer that does not comprise any probe arrays; and

a support plate comprising structural elements;
wherein the support plate is fixedly attached to the wafer at an inactive side of the wafer opposite the active side, with
the structural elements of the support plate contacting the inactive side of the wafer at positions opposite the border regions
surrounding each one of the probe arrays, and wherein the attached wafer protrudes from the support plate.

US Pat. No. 9,085,761

METHODS AND COMPOSITIONS FOR AMPLIFICATION OF NUCLEIC ACIDS

AFFYMETRIX, INC., Santa ...

1. A composition comprising a thermostable polymerase that has been reversibly modified with a polymerase-modifying reagent
of the following structure:

wherein Y=N-hydroxysuccinimide (NHS), p-nitrophenol (pNP) or imidazole;
X=CONMe2, MeSO2, CN, NO2, CO2Me, (CH2)2SMe, N(CH3)CHO, or CH2C(O)CH3;
and a zwitterionic amine oxide.
US Pat. No. 9,074,244

ARRAY-BASED TRANSLOCATION AND REARRANGEMENT ASSAYS

Affymetrix, Inc., Santa ...

1. A method for analysis of genomic rearrangements in a sample from a genome comprising:
(a) digesting the sample with a selected restriction enzyme to obtain restriction fragments that have a first end tag from
a plurality of first end tags and a second end tag from a plurality of second end tags, with the first end tag and the second
end tag flanking a central portion, wherein the sequences of the first end tags and the sequences of the second end tags have
been determined by a computer;

(b) ligating the restriction fragments to a common backbone fragment to form first circular molecules comprising restriction
fragments and the common backbone, wherein the common backbone fragment has a first restriction enzyme recognition site for
a first type IIS restriction enzyme at its first end and a second restriction enzyme recognition site for a second type ITS
restriction enzyme at its second end;

(c) cleaving the first circular molecule using the first type IIS restriction enzyme and the second type IIS restriction enzyme
to form a plurality of first fragments, each comprising the backbone fragment flanked by a first end tag and a second end
tag and a second fragment containing the central portion of the restriction fragment;

(d) ligating the ends of the first fragments to form second circular molecules, wherein the ends of said first end tag and
said second end tag in each fragment are ligated together to form a ditag, wherein each ditag comprises a first end tag and
a second end tag that are immediately adjacent to one another in the ditag;

(e) amplifying at least a portion of the second circular molecules using a pair of primers complementary to said backbone
to obtain amplification target comprising amplified ditags;

(f) hybridizing the amplification target to an array comprising at least 100,000 different ditag probes, wherein each ditag
probe is perfectly complementary to a different first end tag from the plurality of first end tags, to obtain hybridized amplified
ditags; and,

(g) determining at least a partial sequence of the second end tag in a plurality of the hybridized amplified ditags, wherein
the presence in a hybridized amplified ditag of a first end tag from a first fragment and a second end tag from a second different
fragment indicates a genomic rearrangement.

US Pat. No. 9,388,460

COMPLEXITY MANAGEMENT OF GENOMIC DNA

Affymetrix, Inc., Santa ...

1. A method of amplifying fragments of a nucleic acid sample comprising:
fragmenting the nucleic acid sample using a first restriction enzyme and a second restriction enzyme, thereby producing a
fragmented nucleic acid sample including double stranded nucleic acid fragments having a first end cut by the first restriction
enzyme and a second end cut by the second restriction enzyme;

ligating a first double-stranded adaptor to the first end of the double stranded nucleic acid fragments and a second double-stranded
adaptor to the second end of the double stranded nucleic acid fragments, wherein one strand of the first adaptor comprises
a 5? overhang that provides a template for a primer binding site and one strand of the second adaptor comprises a 3? overhang
comprising a primer binding site, thereby producing ligated fragments comprising the 5? overhang and the 3? overhang, wherein
the 5? overhang and the 3? overhang are present on the same strand of each of the ligated fragments comprising the 5? overhang
and the 3? overhang; and

exponentially amplifying the ligated fragments comprising the 5? overhang and the 3? overhang.
US Pat. No. 9,388,459

METHODS FOR GENOTYPING

Affymetrix, Inc., Santa ...

1. A method for genotyping a plurality of polymorphisms in a nucleic acid sample, where each polymorphism has a first and
a second allele, comprising:
amplifying the nucleic acid sample using random primers and DNA polymerase in an isothermal amplification reaction to obtain
an amplified nucleic acid sample;

fragmenting the amplified nucleic acid sample to obtain a fragmented amplified nucleic acid sample;
incubating the fragmented, amplified nucleic acid sample with a plurality of beads attached to allele-specific capture probes
to allow formation of complexes between target fragments in the fragmented, amplified nucleic acid sample and allele-specific
capture probes, wherein the plurality of beads are attached to a collection of allele-specific capture probes comprising at
least 5,000different allele-specific capture probes, wherein the allele-specific capture probes comprise:

(i) a linker,
(ii) a cleavage region,
(iii) a tag region, wherein each different allele-specific capture probe has a different sequence tag region and wherein the
tag region is at least 15 bases; and,

(iv) a target-specific region that terminates at its 3? end with a base that is complementary to a polymorphic base in the
target, wherein each different allele-specific capture probe has a different target-specific region;

extending the allele-specific capture probes in the presence of labeled nucleotides using the target fragment as template
to obtain labeled allele-specific capture probes, wherein extension of the allele-specific capture probes is blocked if there
is a mismatch between the polymorphic position and the 3? end of the allele-specific capture probe;

separating the target fragment from the extended labeled allele-specific capture probes;
cleaving the extended labeled allele-specific capture probes from the beads to produce released extended labeled allele-specific
capture probes;

detecting the released extended labeled allele-specific capture probes by hybridization to an array of tag probes of known
sequence, wherein no amplification of the released extended labeled allele-specific capture probes occurs between the cleaving
and detecting steps, wherein said tag probes are present at known or determinable locations on said array and each tag probe
is complementary to a different tag region present in the allele-specific capture probes; and

determining the genotype of said plurality of polymorphisms by determining which alleles are present, wherein the presence
of hybridized labeled allele-specific capture probes is indicative of the presence of a particular allele in the nucleic acid
sample.

US Pat. No. 9,896,538

POLYFLUORENO[4,5-CDE]OXEPINE POLYMERS AND CONJUGATES THEREOF

AAT Bioquest, Inc., Sunn...

1. A polymer comprising monomers
of formula A

wherein X is the number of monomer A units in the polymer wherein the monomer units are consecutive or nonconsecutive and
wherein X is from 6 to 100; and one or more monomers selected from the group consisting of

monomer of formula B

wherein Y is the number of monomer B units in the polymer wherein the monomer units are consecutive or nonconsecutive and
wherein Y is from 0 to 99; and

monomer of formula C

wherein Z is the number of monomer C units in the polymer wherein the monomer units are consecutive or nonconsecutive and
wherein Z is from 0 to 99;

wherein R1 to R6 independently is hydrogen, an alkyl, a polyethyleneglycol (PEG), an aryl, a heteroaryl group, or a biological
substrate conjugated via a linker (L-BS);

wherein SG1 to SG6 independently is an alkyl, a water soluble group or a L-BS; and
wherein the polymer includes end groups HG1 and HG2 wherein HG1 and HG2 independently is a hydrogen, an alkyl, a halogen,
a boronyl, an aryl, a heteroaryl group or a L-BS;

wherein the ratio of X to Y+Z is >1,
wherein the sum of X+Y+Z is >10.
US Pat. No. 9,828,640

ANALYSIS OF METHYLATION USING NUCLEIC ACID ARRAYS

Affymetrix, Inc., Carlsb...

1. A method of analyzing the methylation status of at least one interrogation position in a sample of DNA, the method comprising:
treating the sample to differentiate between methylated and unmethylated sequences;
amplifying the treated sample, such that a fragment comprising the interrogation position is amplified;
contacting the amplified fragment with an array of probes, wherein at least one of the probes in the array hybridizes to the
fragment proximal to the interrogation position;

performing single base extension on the probe hybridized to the fragment to generate an extended probe; and
detecting the extended probe, wherein the nucleotide added to the extended probe indicates whether the interrogation position
was methylated in the sample of DNA,

wherein the array of probes comprises more than 250,000 different experimental probe features, wherein:
(a) each experimental probe feature comprises a plurality of copies of a nucleic acid probe that is different in sequence
from the probes of every other feature of the array;

(b) each experimental probe feature is at a known or determinable location in the array; and
(c) at least 90% of the experimental probe features comprise full length probes that are perfectly complementary to a CpG
island, wherein a CpG island is a genomic region that is at least 200 bases and has a GC dinucleotide content of at least
50% over its length.

US Pat. No. 9,273,349

DETECTION OF NUCLEIC ACIDS

Affymetrix, Inc., Santa ...

1. A method for simultaneous detection of DNA and RNA in situ, the method comprising:
providing a first set of label extenders capable of hybridizing to a DNA sequence;
providing a second set of label extenders capable of hybridizing to an RNA sequence;
wherein the first set includes at least three times as many different label extenders as the second set;
providing a sample comprising a cell, wherein the cell comprises the DNA sequence and the RNA sequence;
simultaneously hybridizing the label extenders of the first set to the DNA sequence and the label extenders of the second
set to the RNA sequence in the cell;

binding multiple copies of a first label to the first set of label extenders;
binding multiple copies of a second label to the second set of label extenders;
wherein the first label provides a first detectable signal and the second label provides a second detectable signal that is
distinguishable from the first signal; and

detecting and distinguishing the first and second signals, thereby detecting and distinguishing the DNA and RNA sequences.

US Pat. No. 9,845,494

ENZYMATIC METHODS FOR GENOTYPING ON ARRAYS

Affymetrix, Inc., Carlsb...

1. A method for genotyping a plurality of target polymorphisms in a nucleic acid sample, the method comprising:
obtaining an array comprising a plurality of array locations wherein each array location comprises a plurality of array probes
of the same sequence covalently attached to a solid support, wherein the array probes in an array location are perfectly complementary
to the sequence that is immediately 5? of a target polymorphism in a target sequence, wherein said polymorphism is a single
base change and wherein each array probe terminates at a base that is perfectly complementary to a first allele of said target
polymorphism but not to a second allele of said target polymorphism, wherein said array also comprises a second array location
comprising array probes that are perfectly complementary to the second allele;

hybridizing the nucleic acid sample to the array to allow array probes and target sequences to form complexes, wherein said
complexes comprise a double stranded duplex region comprising a target sequence hybridized to an array probe and a single
stranded region comprising target sequence, and washing to remove nucleic acid that is not hybridized specifically to an array
location of the array;

adding a collection of labeled interrogation probes to the array to allow hybridization of the labeled interrogation probes
to said single stranded region of said target sequences on the array, wherein said labeled interrogation probes are at least
6 bases in length and of random sequence;

adding a ligase to allow labeled interrogation probes to be ligated to the array probes in a template dependent manner;
washing the array to remove nucleic acids that are not covalently attached to the array;
detecting a hybridization pattern and determining from the hybridization pattern the presence or absence of labeled interrogation
probe at array locations of the array and thereby determining the genotype of a plurality of target polymorphisms.

US Pat. No. 9,359,640

METHODS FOR SYNTHESIZING POOLS OF PROBES

Affymetrix, Inc., Santa ...

1. A method for estimating the number of occurrences of a species of target molecule in a nucleic acid sample by counting
individual molecular inversion probes that are specific for that species of target molecule, the method comprising:
hybridizing molecular inversion probes (MIPs) to the sample, wherein the MIPs comprise 5? and 3? ends;
ligating the MIP ends that are hybridized to complementary targets to form circularized MIPs;
hybridizing a pool of labeled primers to one or more of the circularized MIPs, thereby providing hybridized primers; wherein
each labeled primer comprises:

(i) a primer binding site sequence,
(ii) at least one label sequence that is different between two or more members of the labeled primer pool, and
(iii) a priming sequence that is complementary to a region of at least one of the MIPs;
extending the hybridized primers using at least a portion of the MIP to which they are hybridized as a template to obtain
unique primer extension products each comprising a different label from the labeled primer and a portion that identifies the
species of the MIP to which the labeled primer was hybridized; and

determining at least a portion of the sequence of the primer extension products to determine the identity of the labels and
the identity of the species of MIPs, wherein a number of different labels identified for each species of MIP provides an estimate
of the number of the corresponding species of target in the sample.

US Pat. No. 9,816,134

MULTIPLEX AMPLIFICATION METHODS

Affymetrix, Inc., Carlsb...

1. A method for amplifying an RNA target comprising:
(a) obtaining a capture probe comprising from the 5? end a second priming sequence, a tag sequence, a first priming sequence
and a sequence complementary to the RNA target;

(b) hybridizing the RNA target to the capture probe and extending the RNA using a polymerase and using the capture probe as
template to form a duplex between the capture probe and the extended RNA target;

(c) optionally destroying or removing the capture probe; and
(d) amplifying the portion of the extended RNA comprising the first and second primer regions and the tag region.

US Pat. No. 10,108,777

METHODS FOR IDENTIFYING DNA COPY NUMBER CHANGES

Affymetrix, Inc., Carlsb...

1. A method for estimating copy numbers using a high-density nucleic acid array having a plurality of allele-specific nucleic acid probes to single nucleotide polymorphisms (SNPs), and a computer system comprising at least one processor, a memory, and an interface, the method comprising:conducting a reference assay using a first high-density microarray genotyping assay to hybridize a plurality of fragments amplified from a reference nucleic acid sample with a plurality of allele-specific perfect match nucleic acid probes in the high-density nucleic acid array, wherein each of the plurality of allele-specific perfect match probes are complementary to individual alleles of a SNP, and wherein conducting the reference assay generates reference data by:
measuring raw probe intensity data comprising observed intensity measurements for each probe;
applying a log-based transformation of the raw probe intensity data for the SNPs;
performing a standardization of the log-based transformation of the raw probe intensity data based on background intensity signals measured from a plurality of background probes to establish comparability across samples;
identifying, using a target set of allelic copy numbers, a plurality of sets of nucleic acid probes by calculating a linear correlation between the standardized log-based transformation of the raw probe intensity data, and the copy numbers from the target set of allelic copy numbers,
and selecting for the plurality of sets of nucleic acid probes, those sets of probes that have a linear correlation that meets or exceeds a threshold amount;
determining and storing in the computer system a plurality of allele-specific reference models of allele-specific relationships between copy number and standarized log-based transformation of the raw probe intensity data of the selected plurality of sets of nucleic acid probes for the reference assay;
conducting an experimental assay using the first high-density microarray genotyping assay to hybridize a plurality of fragments amplified from an experimental nucleic acid sample with the selected plurality of sets of nucleic acid probes to generate experimental data including raw experimental probe intensity data comprising observed intensity measurements for the selected plurality of sets of nucleic acid probes; and
using the computer system to process the experimental data to genotype and determine estimated copy numbers for a plurality of alleles by applying respective allele-specific reference models corresponding to the selected plurality of sets of nucleic acid probes to respective portions of the raw experimental probe intensity data that correspond to the selected plurality of sets of nucleic acid probes.

US Pat. No. 9,933,365

METHODS, SYSTEMS AND APPARATUSES FOR TESTING AND CALIBRATING FLUORESCENT SCANNERS

Affymetrix, Inc., Carlsb...

1. A system for evaluating optical performance by a fluorescent microscopy instrument, the system comprising:a calibration target, wherein the calibration target comprises a substrate, a reflective layer on the substrate, an outer fluorescent layer, and an optically transparent spacing layer between the reflective layer and the outer fluorescent layer, wherein the outer fluorescent layer is configured to produce fluorescent emissions upon illumination with a first set of one or more excitation wavelengths, and wherein the reflective layer is configured to produce a reflection upon illumination with a second set of one or more wavelengths; and
a fluorescent microscopy instrument, wherein the fluorescent microscopy instrument is configured to illuminate the calibration target with the first and second sets of one or more excitation wavelengths and to detect the fluorescent emissions and the reflection from the calibration target.
US Pat. No. 9,523,122

MULTIPLEX TARGETED AMPLIFICATION USING FLAP NUCLEASE

Affymetrix, Inc., Santa ...

1. A method for amplifying a plurality of target sequences from a complex mixture of nucleic acid comprising:
a. fragmenting the nucleic acid to obtain a fragmented nucleic acid sample;
b. mixing the fragmented nucleic acid sample with a plurality of dU probes to the fragmented nucleic acid sample, wherein
there is a dU probe for each target sequence in the plurality and wherein each dU probe comprises:

i. a central target region that is perfectly complementary to a target region in a target fragment in the nucleic acid sample,
wherein the target fragment comprises a target sequence and a 5? non-target sequence and wherein the target fragment hybridizes
to the dU probe so that the target sequence is hybridized to the dU probe and the 5? non-target sequences forms a single stranded
5? flap structure;

ii. a 5? first common sequence; and
iii. a 3? second common sequence;
c. mixing the fragmented nucleic acid sample with a first oligonucleotide that is complementary to said first common sequence
and a second oligonucleotide that is complementary to said second common sequence;

d. adding a 5? flap nuclease to the mixture from (c) to cleave the 5? flap from the target fragment to generate a new 5? end
in the target;

e. adding a ligase to the reaction to ligate the first oligonucleotide to the new 5? end in the target and the second oligonucleotide
to the 3? end of the target;

f. cleaving the dU probes; and
g. amplifying the target sequences using primers for said first and second common sequences.
US Pat. No. 9,388,461

METHODS FOR GENOTYPING SELECTED POLYMORPHISM

Affymetrix, Inc., Santa ...

1. A method for amplifying a collection of target sequences from a nucleic acid sample wherein each target sequence comprises
a common sequence and a consensus sequence immediately adjacent to the common sequence, the method comprising:
fragmenting the nucleic acid sample into fragments with a selected restriction enzyme;
ligating an adaptor to the fragments to obtain adaptor-ligated fragments, wherein said adaptor comprises a first priming sequence;
contacting the adaptor-ligated fragments with probes, wherein each probe comprises a second priming sequence and a target
specific sequence that comprises a region that is complementary to the common sequence and a region that is complementary
to the consensus sequence;

extending at least some of the probes; and
amplifying the extended probes with primers to said first and second priming sequences.

US Pat. No. 10,019,620

METHODS AND DEVICES FOR READING MICROARRAYS

Affymetrix, Inc., Santa ...

1. A method for improving image flatness of a surface image of a probe array having an array surface roughness, the method comprising:imaging one or more fiducials of the probe array at a plurality of positions along an axis of translation and determining a best position measurement for each of the fiducials at which the imaging is sharpest;
generating a surface fit profile based on a plurality of the best position measurements; and
imaging the probe array and adjusting one or more surface non-flatness parameters based on the surface fit profile to improve the image flatness of the surface image of the probe array.

US Pat. No. 9,334,537

METHOD FOR DETECTING TRANSCRIPTION FACTOR-PROTEIN INTERACTIONS

Affymetrix, Inc., Santa ...

1. A method of identifying one or more an active transcription factors, the method comprising:
providing a first library of two different transcription factor probes each comprising a double stranded nucleic acid and
a first detectable marker;

contacting the probes with a first biological sample comprising at least one putative active transcription factor protein,
thus forming at least one transcription factor complex;

separating the at least one transcription factor complex from the sample;
separating the transcription factor probe in the complex from the transcription factor;
identifying at least one transcription factor probe from the at least one complex; and
determining which at least one active transcription factor was in the sample based on the at least one transcription factor
probe identified from the at least one complex;

wherein transcription factor probes in the library of probes are adapted to bind to at least two active transcription factors
selected from the group consisting of: AP1, AP-2, ARE, Brn-3, C/EBP, CBF, CDP, c-Myb, CREB, E2F-1, EFR, ERE, Ets, Ets-1/PEA3,
FAST-1, GAS/ISRE, GATA, GRE, HNF-4, IRF-1, MEF-1, MEF-2, Myc-Max, NF-1, NFATc, NF-E1, NF-E2, NF?B, Oct-1, p53, Pax-5, Pbx1,
Pit 1, PPAR, PRE, RAR (DR-5), SIE, Smad SBE, Smad3/4, SP1, SRE, Stat1, Stat3, Stat4, Stat5, Stat6, TFIID, TR, TR (DR-4), USF-1,
VDR (DR-3), HSE, and MRE.

US Pat. No. 9,147,103

FEATURE INTENSITY RECONSTRUCTION OF BIOLOGICAL PROBE ARRAY

Affymetrix, Inc., Santa ...

1. A computer-implemented method of modifying image data comprising:
accessing image data of a feature of a biological probe array hybridized with nucleic acids, wherein said image data of the
feature comprises a set of observed pixel values;

determining a theoretical pixel intensity with a transfer function representing the proportion of the intensity in a set of
pixels due to a feature, a feature intensity of said feature, and a background value of said feature;

determining an optimized multiplicative error function using a multiplicative error function and a standard deviation function,
wherein said multiplicative error function comprises said theoretical pixel intensity and said set of observed pixel values;
and

determining said feature intensity with an update rule for said feature intensity using a weight function representing the
weight of said pixel and the optimized multiplicative error function where said update rule for said feature intensity iteratively
generates a unique value to which said feature intensity converges

obtaining an observed pixel intensity chart comprised of cells including a plurality a pixels for each cell wherein each pixel
has an observed pixel intensity and wherein each cell has a center pixel,

producing a feature intensity chart by averaging the pixel intensities of the plurality of pixels within each cell,
producing a transfer function intensity chart having pixel intensities calculated using a transfer function that relates the
distance of each pixel from the center pixel with an intensity value that is proportional to the observed feature intensity,

multiplying the transfer function intensity chart by the feature intensity chart to form product image files, and
summing the product image files together to produce a summed image file.

US Pat. No. 9,556,360

SILANE MIXTURES

Affymetrix, Inc., Santa ...

1. An array of polymers on a silanated glass or polymer substrate including a silica surface thereon, wherein the silica surface
has covalently attached thereto a silanation coating comprising a mixture of a first compound and a second compound, and wherein
the first and second compounds are each covalently attached to the silica surface;
the first compound comprising a trialkoxy silyl group and wherein the first compound further includes a reactive functional
group capable of supporting polymer synthesis selected from the group consisting of hydroxyl, amino and carboxyl, and wherein
the polymers of the array are attached to the silanation coating via the active functional group of the first compound;

the first compound being selected from the group consisting of N-(3-(triethoxysilyl)-propyl)-4-hydroxybutyramide; N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane;
3-((2-hydroxyethyl)methylamino)propyl-trimethoxysilane, N-(2-hydroxy-ethyl)-N,N-bis(trimethoxy-silylpropyl)-amine; 1,2-bis((2-hydroxyethyl)-(3-trimethoxysilylpropyl)aminoethane;
3-acetoxy-propyltrimethoxysilane; (trimethoxysilyloxy)-(C3-C12-alkyl)-(triethoxysilane); and 3-glycidoxypropyltriethoxysilane;
and

the second compound comprising a trialkoxy silyl group and wherein the second compound does not include a reactive functional
group of the first compound;

the second compound being selected from the group consisting of 1,2-bis(trimethoxysilyl)-ethane; 1,3-bis(trimethoxysilyl)propane;
1,6-bis(trimethoxysilyl)hexane; 3-(N,N-dimethyl-amino)-propyl-trimethoxysilane; and N,N-bis(3-trimethoxysilylpropyl)-methylamine;

wherein the molar ratio of the first compound to the second compound ranges from about 1 to 50 to 1 to 500;
and wherein the mixture of the first and second compounds in the silanation coating imparts increased hydrolytic stability
to the coating.

US Pat. No. 9,445,025

SYSTEM, METHOD, AND PRODUCT FOR IMAGING PROBE ARRAYS WITH SMALL FEATURE SIZES

Affymetrix, Inc., Santa ...

1. A method for resolving features on a probe array, comprising:
acquiring a set of micro-shifted images of a region of a probe array comprising features, wherein the images are acquired
using an image acquisition device comprising an objective lens and a sensing element having pixels, and wherein each image
in the set is shifted from other images in the set by less than one half a size of a pixel of the sensing element;

reconstructing an image of the probe array using the set of micro-shifted images, wherein reconstructing the image comprises:
creating a single image from the set of micro-shifted images by taking a pixel from a common location within each of the micro-shifted
images and tiling the pixels in the single image according to the spatial orientation corresponding to the shift direction
and magnitude used during image acquisition, and repeating for each pixel associated with the set of micro-shifted images;
and

creating an unboxed image from the single image, wherein the unboxed image is made up of subpixels defined by the shifts used
to produce the micro-shifted images, and wherein creating the unboxed image comprises solving a system of linear algebraic
equations to determine the value of each subpixel from the values of the pixels in the single image; and

deriving intensity values for one or more probe features on the region of the probe array from a reconstructed image, wherein
the reconstructed image is created from at least one of the unboxed image or a deconvolved image.

US Pat. No. 9,708,643

CIRCULATING MIRNA BIOMAKER SIGNATURES

AFFYMETRIX, INC., Santa ...

1. A method for analyzing circulating miRNAs within a sample of blood comprising segregating the circulating miRNAs from cell-associated
miRNAs by: (a) spinning the sample of blood to separate it into a cloudy supernatant fraction of plasma, a white blood cell
fraction and a red blood cell fraction; (b) removing cell-associated RNAs from the cloudy supernatant fraction by spinning
the cloudy supernatant fraction to separate it into a first supernatant primarily including cell-free circulating miRNAs and
a first pellet enriched in cell-associated miRNAs; (c) optionally spinning the first supernatant to separate it into a second
supernatant primarily including cell-free circulating miRNAs and a second pellet enriched in cell-associated miRNAs; (d) extracting
total RNA from either the first supernatant, if step (c) has not been performed, or the second supernatant if step (c) has
been performed, wherein the resulting total RNA is enriched in circulating miRNAs as compared to
the starting blood sample, wherein the spin of step (a) is at about 1700 g for 10 minutes and the spin of step (b) is at about
2000 g for 10 minutes and the spin of step (c) is at about 12000 g for 10 minutes;

analyzing the total RNA for circulating miRNA content; and
extracting out interfering signals from contaminant cells in the total RNA, wherein the extracting is performed by a computer
upon data comprising the interfering signals from the contaminant cells and signals from the circulating miRNA content, wherein
the extracting comprises background subtraction based on GC content of anti-genomic probes.

US Pat. No. 9,599,561

METHODS, SYSTEMS AND APPARATUSES FOR TESTING AND CALIBRATING FLUORESCENT SCANNERS

Affymetrix, Inc., Santa ...

1. A method of evaluating fluorescence detection performance by a fluorescent microscopy instrument, the method comprising:
illuminating a calibration target, wherein the calibration target comprises a substrate, a reflective layer on the substrate,
an outer fluorescent layer, and an optically transparent spacing layer between the reflective layer and the outer fluorescent
layer, and wherein the outer fluorescent layer is illuminated with light configured to produce fluorescent emissions;

detecting the fluorescent emissions with a fluorescent microscopy instrument; and
analyzing the fluorescent emissions with a computer, wherein the computer comprises a computer software program, and wherein
analyzing comprises comparing the fluorescent emissions to one or more pre-established criteria.

US Pat. No. 9,932,636

ARRAY-BASED TRANSLOCATION AND REARRANGEMENT ASSAYS

AFFYMETRIX, INC., Carlsb...

1. A method for analysis of genomic rearrangements in a sample from a genome, the method comprising:digesting the sample with a selected restriction enzyme to obtain restriction fragments, with each restriction fragment having a first end tag including a predicted sequence of the genome and a second end tag including a predicted sequence of the genome, with the first end tag and the second end tag flanking a central portion;
generating a population of paired-end tags wherein the first end tag and the second end tag of each restriction fragment are directly coupled;
hybridizing the first end tags of the population of paired-end tags to an array having at least 100,000 different probes attached to a substrate; and,
determining at least a partial sequence of the second end tags in the population of paired-end tags, wherein the presence of a first end tag from a first fragment and a second end tag from a second different fragment indicates a genomic rearrangement.
US Pat. No. 9,528,148

MULTIPLEX TARGETED AMPLIFICATION USING FLAP NUCLEASE

Affymetrix, Inc., Santa ...

1. A method for amplifying a plurality of target sequences comprising:
fragmenting a nucleic acid to obtain a plurality of target fragments including the plurality of target sequences;
mixing the plurality of target fragments with (1) a plurality of probes, wherein each probe comprises a central target region,
a 5? first common sequence and a 3? second common sequence, wherein the central target region is complementary to a target
region in a target fragment, wherein the target fragment hybridizes to the central target region of the probe and one or more
of a single stranded 5? flap structure or a single stranded 3? flap structure is formed; and (2) a first oligonucleotide that
is complementary to the first common sequence and hybridizes thereto and a second oligonucleotide that is complementary to
the second common sequence and that hybridizes thereto, to form a mixture;

adding one or more of a 5? flap nuclease or a 3? flap nuclease to the mixture, wherein one or more of the 5? flap structure
or the 3? flap structure is cleaved;

adding a ligase to the mixture to ligate the first oligonucleotide to a 5? end of the target fragment and the second oligonucleotide
to a 3? end of the target fragment;

removing the probes; and
amplifying the target sequences using primers for the first and second common sequences.
US Pat. No. 9,798,855

DIFFERENTIAL FILTERING OF GENETIC DATA

Affymetrix, Inc., Santa ...

1. A computer implemented method of differentially filtering genetic data, which comprises:
accessing, by a computer comprising a processor and a memory, data of intensity measurements corresponding to hybridization
of target nucleic acids to an array of single nucleotide polymorphism nucleic acid probes and copy number nucleic acid probes,
wherein the single nucleotide polymorphism nucleic acid probes are designed to identify one or more single nucleotide polymorphisms
in the target nucleic acids and the copy number nucleic acid probes are designed to identify one or more copy number variations
in the target nucleic acids;

applying, by the processor, one or more algorithms stored in memory to the data of the intensity measurements to analyze and
convert the data of the intensity measurements to genetic data selectable by a user on at least one input interface window;

displaying the genetic data on a visual display device connected to the computer, wherein the computer comprises a computer
program configured to visually display the genetic data to a user on the visual display device in a display area comprising
a plurality of user configurable windows displayed on the visual display device, wherein the plurality of user configurable
windows comprises a multiple chromosomes view, a selected chromosome view, and a segment filters view, wherein each view comprises
at least one genomic map obtained from the genetic data;

presenting the user with an input interface via the computer, wherein the input interface is presented on the visual display
device and receives input from the user via an interface including the at least one genomic map to modify the views of the
user configurable windows;

receiving, by the computer and from the input interface, a selection of a subset of genetic data designated as one or more
regions of genetic data characterized by loss of heterozygosity, long continuous stretches of homozygosity, copy number mosaicism,
or copy number variation;

receiving, by the computer and from the user through the at least one input interface window, a first set of parameters selected
by the user for filtering the one or more regions of genetic data;

differentially filtering, by a processor of the computer, the subset of genetic data in response to receiving the first set
of parameters by determining a first filtered subset of the genetic data that corresponds to the first set of parameters;
and

displaying on the visual display device, the first filtered subset of the genetic data corresponding to the first set of parameters
nearly simultaneously as the first set of parameters is received by the computer, wherein the first filtered subset of genetic
data is visualized using various colors, icons or other visual markers in the user configurable windows to distinguish from
the genetic data that does not correspond to the first set of parameters.

US Pat. No. 9,982,293

AMPLIFICATION AND ANALYSIS OF SELECTED TARGETS ON SOLID SUPPORTS

Affymetrix, Inc., Carlsb...

1. A method for amplifying a plurality of target sequences from a nucleic acid sample and analyzing the amplified target sequences, the method comprising:(a) fragmenting the nucleic acid sample to obtain target fragments comprising target sequences;
(b) mixing the target fragments obtained in (a) with an array of probes arranged in features on a solid support, wherein each feature comprises multiple copies of a target specific array probe having a first probe region that is perfectly complementary to a first target region and a second probe region that is perfectly complementary to a second target region, wherein the first probe region is 5? of the second probe region and wherein the first target region is 5? of the second target region, and wherein the probes are attached to the solid support at the 5? end of the probe, wherein target fragments hybridize to complementary target specific array probes to form probe:target fragment complexes wherein the target fragment hybridizes to the target specific array probe so that the first target region is hybridized to the first probe region and the second target region is hybridized to the second probe region and so that a single stranded overhang of the target fragment is generated and wherein a nuclease that recognizes the overhang is used to remove the overhang;
(c) joining a 5? end of each target fragment to the 3? end of the same target fragment to form target circles;
(d) extending the array probes using the target circles as a template in a rolling circle amplification reaction, thereby obtaining extended array probes comprising a plurality of copies of the complement of a plurality of the target fragment; and
(e) analyzing the copies.
US Pat. No. 9,845,490

COMPLEXITY MANAGEMENT OF GENOMIC DNA

Affymetrix, Inc., Carlsb...

1. A method of producing a second nucleic acid sample from a first nucleic acid sample, wherein said second nucleic acid sample
is enriched with a collection of adaptor ligated target sequences, the method comprising:
fragmenting the first nucleic acid sample to produce sample fragments in a selected size range that comprises a collection
of target sequences, wherein the fragmenting includes digestion with an enzyme or enzyme combination, wherein the collection
of target sequences comprises between 10,000 and 3,000,000 single nucleotide polymorphisms, wherein greater than 80% of the
single nucleotide polymorphisms in the collection of target sequences are at least 10 kilo bases and less than 500 kilo bases
from another single nucleotide polymorphism, and wherein the average minimum distance between single nucleotide polymorphisms
is between 10 kilo bases and 800 kilo bases;

ligating at least one synthetic adaptor to the sample fragments to produce the collection of adaptor ligated target sequences;
and

generating the second nucleic acid sample by amplifying the sample fragments, wherein the collection of adaptor ligated target
sequences is enriched in the second nucleic acid sample.

US Pat. No. 9,767,342

METHODS AND DEVICES FOR READING MICROARRAYS

Affymetrix, Inc., Santa ...

1. A method for improving image flatness of a surface image of a probe array having an array surface roughness, the method
comprising:
imaging a plurality of fiducials at a plurality of z positions and determining a best z position measurement for each of the
plurality of fiducials at which the imaging is sharpest;

generating a surface fit profile based on the best z position measurement for each of the plurality of fiducials; and
imaging the probe array and adjusting one or more surface non-flatness parameters based on the surface fit profile to improve
the image flatness of the surface image of the probe array.

US Pat. No. 9,760,675

SYSTEM, METHOD, AND COMPUTER SOFTWARE PRODUCT FOR GENOTYPE DETERMINATION USING PROBE ARRAY DATA

Affymetrix, Inc., Santa ...

1. A method for genotyping a plurality of single nucleotide polymorphisms (SNPs) in a nucleic acid sample using seed genotype
cluster estimates derived without requiring mismatch probe data, the method comprising:
hybridizing a nucleic acid sample with a plurality of allele-specific perfect-match probes provided in an array of perfect-match
probes for a plurality of target sequences which the array is designed to genotype, wherein, for substantially all of the
plurality of target sequences, the array is without corresponding mismatch probes;

acquiring intensity data associated with the hybridizing, wherein the intensity data comprises intensity values;
summarizing the intensity values to obtain a signal value for each allele for each of the plurality of SNPs;
transforming the signal values by discarding size information from the signal values, thereby generating transformed signal
values represented in one-dimensional contrast space;

evaluating all plausible divisions of the transformed signal values into seed genotypes by applying a Gaussian likelihood
model;

averaging the plausible divisions over most likely plausible divisions to derive a plurality of seed genotype clusters; and
genotyping the plurality of SNPs, wherein genotyping comprises a comparison of the transformed signal values with a set of
typical values for each genotype, wherein the set of typical values comprises prior values, wherein the prior values further
comprise estimates of genotype cluster center locations and genotype cluster center variances of the plurality of seed genotype
clusters determined from the clustering properties of the transformed signal values;

wherein the steps of summarizing, transforming, evaluating, averaging, and genotyping are performed on a computer, and wherein
the computer comprises a computer processor.

US Pat. No. 9,689,867

ASSAYS FOR AFFINITY PROFILING OF NUCLEIC ACID BINDING PROTEINS

Affymetrix, Inc., Santa ...

1. A method for measuring an affinity level of one or more binding proteins for one or more recognition sites, the method
comprising:
hybridizing one or more target nucleic acids to one or more probes, which probes each comprise a first homology region and
a second homology region, wherein the first homology region hybridizes to a first region of a target nucleic acid, wherein
the second homology region hybridizes to a second region of the same target nucleic acid, and wherein the first and second
homology regions hybridize to the same target nucleic acid so that a gap of one or more bases separates the probe first and
second homology regions;

enzymatically extending one or more hybridized probes from the first or second homology region across the gap to synthesize
a complementary nucleic acid, thereby creating a recognition site for a binding protein;

introducing one or more putative binding proteins to the recognition site; and,
measuring an affinity level of the one or more putative binding proteins for the recognition site.
US Pat. No. 9,663,822

MULTIPLEX CAPTURE OF NUCLEIC ACIDS

Affymetrix, Inc., Santa ...

1. A method of capturing ten or more different target nucleic acids, the method comprising:
providing a sample comprising or suspected of comprising the ten or more different target nucleic acids (TNA);
providing a solid support having associated therewith a support capture probe (SCP);
providing ten or more subsets of n different target capture probes (TCP), wherein n is at least two, wherein a different subset
of target capture probes is provided for each different target nucleic acid, wherein the target capture probes in each subset
are capable of hybridizing to nonoverlapping polynucleotide sequences in the corresponding target nucleic acid, and wherein
the target capture probes in each subset are capable of hybridizing to the support capture probe;

hybridizing any target nucleic acid present in the sample to its corresponding subset of n target capture probes and hybridizing
the subset of n target capture probes to the support capture probe, thereby capturing the target nucleic acid on the solid
support in a TNA/TCP/SCP complex,

wherein the hybridizing the subset of n target capture probes to the support capture probe is performed at a hybridization
temperature which is greater than a melting temperature Tm of a TCP/SCP complex between each individual target capture probe and the support capture probe;

after hybridizing any target nucleic acid present in the sample to its corresponding subset of n target capture probes and
hybridizing the subset of n target capture probes to the support capture probe, removing the captured target nucleic acids
from the TNA/TCP/SCP complex on the solid support; and

after removing the captured target nucleic acids from the solid support, manipulating the captured target nucleic acids;
wherein said manipulating at least comprises sequencing.
US Pat. No. 10,119,162

MULTIPLEX TARGETED AMPLIFICATION USING FLAP NUCLEASE

AFFYMETRIX, INC., Carlsb...

1. A method for amplifying a plurality of target sequences from a complex mixture of nucleic acid comprising:(a) fragmenting the nucleic acid to obtain a fragmented nucleic acid sample;
(b) adding a plurality of circularization probes to the fragmented nucleic acid sample to form a mixture, wherein there is a circularization probe for each target sequence and wherein each circularization probe comprises:
(i) a 5? target region that is perfectly complementary to a first region in a complementary target sequence,
(ii) a 3? target region that is perfectly complementary to a second region in the complementary target sequence and
(iii) a central region immediately 3? of the 5? target region and immediately 5? of the 3? target region that is perfectly complementary to a third region in the complementary target sequence and also perfectly complementary to a fourth region in the complementary target sequence wherein said third region is immediately 3? of the second region and said fourth region is immediately 5? of said first region and wherein said third and fourth regions are distinct regions but share a common sequence of at least 2 contiguous bases;
(c) generating circularized target sequences by contacting the circularization probes with an exonuclease, a flap endonuclease and a ligase;
(g) optionally amplifying at least some of the circularized target sequences; and
(f) sequencing at least some of the circularized target sequences or their amplification products.

US Pat. No. 10,422,750

METHODS, SYSTEMS AND APPARATUSES FOR TESTING AND CALIBRATING FLUORESCENT SCANNERS

Affymetrix, Inc., Carlsb...

1. A method of evaluating fluorescence detection performance by a fluorescent microscopy instrument comprising a fluorescent channel and a reflective channel, the method comprising:illuminating a calibration target, wherein the calibration target comprises a substrate, a reflective layer, and a fluorescent layer, and wherein the fluorescent layer is illuminated with light configured to produce fluorescent emissions, and the reflective layer is illuminated with light configured to produce reflective illuminations;
detecting the fluorescent emissions with the fluorescent channel;
detecting the reflective illuminations with the reflective channel; and
analyzing the fluorescent emissions and the reflective illuminations with a computer, wherein the computer comprises a computer software program, and wherein analyzing comprises comparing the fluorescent emissions and the reflective illuminations to one or more pre-established criteria.

US Pat. No. 10,329,600

LOCUS SPECIFIC AMPLIFICATION USING ARRAY PROBES

AFFYMETRIX, INC., Carlsb...

1. A method for amplifying and analyzing a plurality of target sequences from a nucleic acid sample, said method comprising:(a) fragmenting the nucleic acid sample with a restriction enzyme to obtain fragments with known sequences at the 5? and 3? ends of the fragments, wherein said fragments comprise a plurality of target fragments comprising target sequences;
(b) mixing the fragments obtained in (a) with a plurality of target specific splint oligonucleotides, wherein each splint oligonucleotide comprises a first target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at the 3? end of a corresponding target fragment, and a second target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at and including the 5? end of said corresponding target fragment, and wherein said first sequence is 5? of said second sequence in said splint oligonucleotide, wherein target specific splint oligonucleotides further comprises a first common priming sequence at the 5? end and a second common priming sequence at the 3? end;
(c) adding first and second primers to the mixture wherein said first primer is complementary to said first common priming sequence and said second primer is complementary to said second common priming sequence and wherein said first and second primers hybridize to said splint oligonucleotides so the first primer is adjacent to the 3? end of the target fragment and the second primer is adjacent to the 5? end of the target fragment;
(d) ligating the first primer to the 3? end of the target fragment and the second primer to the 5? end of the target fragment to obtain ligated target fragments comprising a first common priming site at the 3? end and a second common priming sites at the 5? end;
(e) after said ligating step (d) fragmenting said splint oligonucleotides;
(f) amplifying the ligated target fragments from (d) to obtain amplified target fragments; and
(g) analyzing the amplified target fragments by a method comprising hybridization to an array comprising a plurality of oligonucleotide probes present at known or determinable locations in the array.

US Pat. No. 10,316,136

POLYFLUORENO[4,5-CDE]OXEPINE CONJUGATES AND THEIR USE IN METHODS OF ANALYTE DETECTION

AAT Bioquest, Inc., Sunn...

1. A method of detecting an analyte in a sample, comprisinga) combining said sample with a detection reagent comprising a polymer under conditions under which said detection reagent will bind said analyte; and
b) detecting the detection reagent bound analyte by fluorescence
wherein the polymer comprises
monomer of formula A

wherein X is the number of monomer A units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein X is from 6 to 100; and one or more monomers selected from the group consisting of
monomer of formula B

wherein Y is the number of monomer B units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein Y is from 0 to 99, and
monomer of formula C

wherein Z is the number of monomer C units in the polymer wherein the monomer units are consecutive or nonconsecutive and wherein Z is from 0 to 99;
wherein R1 to R6 independently is hydrogen, an alkyl, a polyethyleneglycol (PEG), an aryl, a heteroaryl group, or a biological substrate conjugated via a linker (L-BS);
wherein SG1 to SG6 independently is an alkyl, a water soluble group or a L-BS; and
wherein the polymer includes end groups HG1 and HG2 wherein HG1 and HG2 independently is a hydrogen, an alkyl, a halogen, a boronyl, an aryl, a heteroaryl group or a L-BS;
wherein the ratio of X to Y+Z is >1,
wherein the sum of X+Y+Z is >10; wherein the polymer comprises at least one L-BS wherein L is a linker and BS is a biological substrate wherein the biological substrate is a complementary binding member capable of specifically binding to the analyte to be detected.

US Pat. No. 10,407,717

METHODS OF ANALYSIS OF METHYLATION

Affymetrix, Inc., Carlsb...

1. A method for determining the methylation status of a plurality of cytosines in a plurality of restriction sites for a methylation-sensitive restriction enzyme (MSRE) in a genomic DNA sample comprising:(a) fragmenting the genomic DNA sample with the MSRE;
(b) filling in the ends generated by cleavage with the MSRE using a DNA polymerase;
(c) fragmenting the product of (b) with a methylation-insensitive restriction enzyme (MIRE) that recognizes the same restriction site as the MSRE;
(d) hybridizing the products of (c) with methylation-specific dU probes, wherein each dU probe comprises a barcode tag sequence, a 5? first common priming sequence and a 3? second common priming sequence;
(e) adding a ligase and oligonucleotides that are complementary to the barcode tag sequences, the first common priming sequence and the second common priming sequence under conditions to allow ligation of the oligonucleotides to a product of (c) hybridized to a dU probe;
(f) digesting the products of (e) with uracil DNA glycosylase;
(g) amplifying the product of (f) by PCR using primers to the first and second common priming sequences; and,
(h) detecting the presence or absence of amplified barcode tag sequences from (g) by hybridization to determine the methylation status of said plurality of cytosines.

US Pat. No. 10,213,762

SILANE MIXTURES

AFFYMETRIX, INC., Carlsb...

1. A method of preparing an array of monomers or polymers comprising:providing a substrate including a silica surface or other silicon oxide-containing surface thereon;
contacting the silica or the other silicon oxide-containing surface of the substrate with a silanation composition comprising a mixture of a first compound and a second compound such that the first and the second compounds are covalently attached to the surface to form a silanation coating;
the first compound comprising a trialkoxy silyl group and a functional group, wherein said functional group is capable of supporting polymer synthesis and is selected from the group consisting of hydroxyl, amino and carboxyl; the first compound being selected from the group consisting of N-(3-(triethoxysilyl)-propyl)-4-hydroxybutyrarnide; N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane; 3-((2 hydroxyethyl)methylamino)propyl-trimethoxysilane; N-(2-hydroxy-ethyl)-N,N-bis(trimethoxy-silylpropyl)-amine; 1,2-bis((2-hydroxyethyl)-(3 trimethoxysilylpropyl)aminoethane; 3-acetoxy-propyltrimethoxysilane; (trimethoxysilyloxy)-(C3-C12-alkyl)-(triethoxysilane); and 3-glycidoxypropyltriethoxysilane; and
the second compound comprising a trialkoxy silyl group and wherein the second compound in the mixture does not include the functional group of the first compound; the second compound being selected from the group consisting of 1,2-bis(trimethoxysilyl)-ethane; 1,3-bis(trimethoxysilyl)propane; 1,6-bis(trimethoxysilyl)hexane; 3-(N,N-dimethyl-amino)-propyl-trimethoxysilane; and N,N-bis(3-trimethoxysilylpropyl)-methylamine;
wherein the molar ratio of the first compound to the second compound in the mixture ranges from about 1:50 to 1:500;
and wherein the mixture of the first and second compounds in the silanation coating imparts increased hydrolytic stability to the coating;
protecting the functional group capable of supporting polymer synthesis of the first compound with a protecting group or reacting the functional group capable of supporting polymer synthesis of the first compound with a compound having a reactive group protected by a protecting group to provide the first compound of the silanation coating protected with the protecting group;
removing the protecting group of the first compound in selected regions of the silanation coating to provide an exposed functional or reactive group;
reacting said exposed functional or reactive group with the monomer or the polymer whereby the monomer or the polymer is coupled to the exposed group; and
repeating the steps of removing and reacting to produce the array of monomers or polymers.

US Pat. No. 10,586,095

METHODS AND DEVICES FOR READING MICROARRAYS

Affymetrix, Inc., Carlsb...

1. A method for improving image flatness of a surface image of a probe array, the method comprising:providing a probe array and one or more fiducials, said probe array comprising a plurality of sub-arrays;
imaging the one or more fiducials at one or more of positions;
determining a position measurement for each of the one or more fiducials;
generating a surface fit profile based on a plurality of the position measurements; and
imaging a sub-array and adjusting one or more surface non-flatness parameters based on the surface fit profile to improve the image flatness of the surface image of the sub-array.
US Pat. No. 10,275,568

METHODS AND COMPUTER SOFTWARE FOR DETECTING SPLICE VARIANTS

Affymetrix, Inc., Carlsb...

1. A method of identifying one or more alternative splicing events for one or more genes of a target genome using a plurality of exon array, each array comprising a plurality of nucleic acid probe sets having one or more nucleic acid probes that are complementary to at least a portion of an exon and do not comprise junction probes, and a computer system comprising at least one processor, a memory, and an interface, the method comprising:providing the plurality of exon arrays, wherein a nucleic acid probe set in each exon array is designed to probe for at least a portion of a first probe selection region (PSR) corresponding to an exon derived from one or more input annotations for the target genome, wherein a nucleic acid probe set associated with the first PSR is included in a first gene expression level grouping of a first gene on the target genome and the exon corresponding to the PSR maps to the first gene;
hybridizing the plurality of nucleic acid probes of a first exon array to nucleic acids derived from one or more nucleic acid samples from a first tissue sample to generate first probe selection region (PSR) intensity data corresponding to the nucleic acid probe set associated with the first;
calculating a first normalized exon intensity comprising a ratio of first exon intensity data to a first gene expression level, wherein the first exon intensity data comprises data from the first PSR intensity data corresponding to the exon, and the first gene expression level comprises a first gene intensity value obtained from PSR intensity data assembled from nucleic acid probes associated with the first gene expression level grouping;
hybridizing the plurality of nucleic acid probes of a second exon array to nucleic acids derived from one or more nucleic acid samples from a second tissue sample to generate second probe selection region (PSR) intensity data corresponding to the nucleic acid probe set associated with the first PSR;
calculating a second normalized exon intensity comprising a ratio of second exon intensity data to a second gene expression level, wherein the second exon intensity data comprises data from the second PSR intensity data, and the second gene expression level comprises a second gene intensity value obtained from PSR intensity data assembled from nucleic acid probes associated with the first gene expression level grouping; and
detecting differential exon expression of the gene using the first and second normalized exon intensities.
US Pat. No. 10,229,244

METHODS FOR IDENTIFYING DNA COPY NUMBER CHANGES USING HIDDEN MARKOV MODEL BASED ESTIMATIONS

Affymetrix, Inc., Carlsb...

1. A computer-implemented method for estimating a copy number of each of a plurality of genomic regions in a nucleic acid sample comprising a plurality of nucleic acid molecules, each genomic region containing at least one single nucleotide polymorphism (SNP), the method comprising:hybridizing the plurality of nucleic acid molecules with a plurality of allele-specific perfect match probes for at least one SNP on a nucleic acid array comprising a plurality of perfect match probes wherein each of the plurality of perfect match probes does not have a corresponding mismatch probe;
obtaining, by a computer comprising a processor and a memory, an initial intensity measurement for each of the plurality of allele-specific perfect match probes wherein the initial intensity measurements obtained for the plurality of allele-specific perfect match probes for the SNP do not include intensities for any mismatch probes;
normalizing, by the processor, the initial intensity measurement for each of the plurality of allele-specific perfect match probes, resulting in normalized intensity measurements, wherein the normalized intensity measurements are determined for the plurality of allele-specific perfect match probes without utilizing data derived from any mismatch probes;
determining and applying a cross-talk correction without use of mismatch probe data, by performing a rotation transformation to obtain cross-talk corrected normalized intensity measurements of the plurality of allele-specific perfect match probes, wherein the cross-talk corrected normalized intensity measurements include a cross-talk component from another allele that is determined to correct for probes for one allele of an SNP hybridizing with another allele of the SNP;
calculating, by the processor, an initial copy number estimate for each of the plurality of genomic regions, wherein the initial copy number estimate is based upon the cross-talk corrected normalized intensity measurements and the cross-talk corrected normalized intensity measurements of a reference sample;
performing, by the processor, data smoothing on the initial copy number estimates, wherein the data smoothing reduces noise within the initial copy number estimates to generate smoothed copy number estimates; and
estimating, by the processor, the copy number of each of the plurality of genomic regions using a Hidden Markov Model to assign the smoothed copy number estimates to different copy number states.

US Pat. No. 10,648,021

DNA LIGATION ON RNA TEMPLATE

AFFYMETRIX, INC., Carlsb...

1. A method for detecting a plurality of target nucleic acids from a nucleic acid sample, comprising:a) contacting a DNA array-bound probe with:
(i) a DNA interrogation probe, and
(ii) a nucleic acid sample comprising at least one target nucleic acid of RNA,
wherein the array-bound probe and the interrogation probe hybridize to the target nucleic acid such that the array-bound probe and the interrogation probe are directly adjacent to one another, and wherein the interrogation probe contains a detectable label;
b) adding an effective amount of ATP to a solution of at least two different DNA ligase enzymes,
wherein the at least two different DNA ligase enzymes comprise a first ligase, which can form a DNA ligase-AMP intermediate complex, and a second ligase, which is a mutant ligase unable to form a DNA ligase-AMP intermediate complex;
c) depleting ATP from the solution of at least two different DNA ligase enzymes;
d) adding the solution of at least two different DNA ligase enzymes to step a), thereby ligating the array-bound probe and the interrogation probe; and
e) detecting the ligated product.
US Pat. No. 10,535,420

SYSTEMS AND METHODS FOR PROBE DESIGN TO DETECT THE PRESENCE OF SIMPLE AND COMPLEX INDELS

Affymetrix, Inc., Carlsb...

1. A method of providing a genotyping array for genotyping a plurality of complex or multi-base indels within one or more sample genomes, the method comprising:accessing a data set of one or more genome databases, said data set comprising information regarding genomic locations of the plurality of complex or multi-base indels (insertion/deletion) and sequences flanking at least one direction of the indels;
designing a plurality of indel probes to generate a plurality of designed indel probes for inclusion on a genotyping array by using one or more computer processors coupled to one or more memories configured to provide the one or more computer processors with instructions to, for each complex or multi-base indel of the plurality of complex or multi-base indels:
(a) access or generate a probe sequence that is complementary to a target sequence flanking the complex or multi-base indel;
(b) compare a deletion allele interrogation base to an insertion allele interrogation base in an indel identification sequence, the deletion allele interrogation base and the insertion allele interrogation base being referred to collectively as the interrogation bases, and,
(c) in response to the interrogation bases being different and being in different detection channels, create and output a single indel probe adapted for using the interrogation bases to differentiate the deletion allele and the insertion allele; and
(d) in response to the compared interrogation bases being a same interrogation base, incorporate the same interrogation base into the probe sequence and set new interrogation bases to be compared based on a next downstream base in a deletion allele sequence and a next downstream base in an insertion allele sequence and repeat (b)-(c) until either an interrogation distance threshold would be exceeded or the interrogation bases are different and are in different detection channels, and, when the interrogation bases are different and are in different detection channels, create and output a single indel probe adapted for using the interrogation bases to differentiate the deletion allele and the insertion allele;
synthesizing a plurality of indel probes of the genotyping array according to the plurality of designed indel probes; and
providing, with the genotyping array, labelled nucleotide bases corresponding to the interrogation bases for adding to a corresponding probe of the plurality of synthesized probes after the corresponding probe is hybridized with a sample during a genotyping assay, interrogation bases corresponding to some of the plurality of synthesized indel probes having different interrogation distances than do interrogation bases corresponding to others of the plurality of synthesized indel probes, a complex or multi-base indel of the plurality of complex or multi-base indels being genotyped by detecting labels corresponding to the labelled nucleotide bases.

US Pat. No. 10,920,269

AMPLIFICATION AND ANALYSIS OF SELECTED TARGETS ON SOLID SUPPORTS

AFFYMETRIX, INC., Carlsb...

1. A method for amplifying a plurality of target sequences from a nucleic acid sample and analyzing the amplified target sequences, the method comprising:(a) mixing the nucleic acid sample with (a1) an array of probes arranged in features and (a2) a plurality of oligonucleotides,
wherein the nucleic acid sample comprises target fragments comprising target sequences,
each feature comprises multiple copies of a target specific array probe having (i) a first probe region that is complementary to a first target region, (ii) a second probe region that is complementary to a second target region, and (iii) a central probe region that is complementary to one of the oligonucleotides, said central probe region being between the first and second probe regions,
the target fragments hybridize to complementary target specific array probes to form probe:target fragment complexes, and
in the probe:target fragment complexes, the first target region hybridizes to the first probe region, the second target region hybridizes to the second probe region, and the oligonucleotide hybridizes to the central probe region;
(b) joining the first target region, the oligonucleotide, and the second target region that hybridize to the same array probe to form a target circle;
(c) extending the array probe using the target circle as a template, thereby obtaining an extended array probe comprising a plurality of copies of the complement of the target sequences; and
(d) analyzing the copies.

US Pat. No. 10,515,447

ANALYSIS OF DATA OBTAINED FROM MICROARRAYS

Affymetrix, Inc., Santa ...

1. A computer-implemented method of correcting a data set comprising one or more intensity values from a microarray experiment to account for one or more independent variables, the method comprising:scanning, by a scanning device, a nucleic acid sample hybridized to a nucleic acid probe mounted on a microarray used for the microarray experiment such that a data set comprising one or more intensity values from a microarray experiment is obtained;
defining, by a processor, a plurality of discrete bins for intensity values of the data set, wherein each discrete bin comprises a different range of intensity values with respect to the other discrete bins, and wherein defining the plurality of discrete bins includes selecting a quantity of two or more discrete bins based on one or more independent variables associated with the data set and partitioning the two or more discrete bins according to one or more spatial dimensions defined by the one or more independent variables;
dividing, by the processor, the one or more intensity values from the data set into the plurality of discrete bins;
determining, by the processor, a median value of the one or more intensity values in each of the plurality of discrete bins;
determining, by the processor, an overall median value of the one or more intensity values of the data set;
determining covariate adjustor code for each of the plurality of discrete bins using a processor configured to track a ratio of the overall median value to the median value for a respective bin of the plurality of discrete bins, wherein the covariate adjustor code comprises code for creating or not creating at least one of a fragment GC, local GC, probe GC, and super GC covariate adjuster adjustor for one or more intensity values determined to need correction in the data set obtained, and code for determining whether to tag intensity values to undergo no correction for a given covariate;
adjusting, by the processor, the one or more intensity values in each of the one or more discrete bins by applying a corresponding one of the covariate adjustors such that the median value of the one or more intensity values determined to need correction in each of the plurality of discrete bins is adjusted to a same target median value, thereby generating a corrected data set; and
displaying at least a portion of the corrected data set as a graphical presentation within an electronic user interface.

US Pat. No. 10,303,922

METHODS AND DEVICES FOR READING MICROARRAYS

Affymetrix, Inc., Carlsb...

1. A method for improving image flatness of a surface image of a probe array having an array surface roughness, the method comprising:imaging one or more fiducials of the probe array and determining a position measurement for each of the one or more fiducials based on at least image sharpness;
generating a surface fit profile based on one or more of the position measurements; and
imaging the probe array and adjusting one or more surface non-flatness parameters based on the surface fit profile to improve the image flatness of the surface image of the probe array.
US Pat. No. 10,648,030

METHODS OF DETERMINING THE PRESENCE OR ABSENCE OF A PLURALITY OF TARGET POLYNUCLEOTIDES IN A SAMPLE

Affymetrix, Inc., Carlsb...

1. A method of determining the allele frequency of one or more target polynucleotides of a plurality of target polynucleotides in a sample, the method comprising:a) combining a sample comprising one or more of the plurality of target polynucleotides with a plurality of sets of complementary polynucleotides, said one or more of the plurality of target polynucleotides suspected to have a site of a single nucleotide polymorphism (SNP) containing a polymorphic nucleotide;
wherein each of the plurality of sets of complementary polynucleotides comprises:
(i) a first complementary polynucleotide comprising a complementary sequence to a first target sequence of a target polynucleotide, a first allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a first polymorphic nucleotide,
(ii) a second complementary polynucleotide comprising a complementary sequence to the first target sequence, a second allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a second polymorphic nucleotide; and
(iii) a third complementary polynucleotide comprising a complementary sequence to a second target sequence of the target polynucleotide,
wherein the allele-specific barcodes are not complementary to the first target sequence, and
wherein the allele-specific barcodes allow up to 4 sequencing errors while still allowing identification of the specific allele ;
b) incubating the plurality of sets of complementary polynucleotides with the plurality of target polynucleotides under conditions that allow hybridization of complementary sequences;
c) joining a pair of the first and third complementary polynucleotides and/or joining a pair of the second and third complementary polynucleotides by a ligation reaction when both complementary polynucleotides of each pair are hybridized to the target polynucleotide to form one or more product polynucleotides; and
d) detecting the presence of one or more product polynucleotides to determine the allele frequency of the target polynucleotide, wherein the detecting step is accomplished by sequencing all or part of one or more of the product polynucleotides or a complement thereof;
e) dividing the number of sequence reads of one polymorphism by the total number of sequence reads for the target polynucleotide; and
f) determining the target polynucleotide is homozygous if the frequency of one allele is 0.7 or greater, or 0.3 or lower, and determining the target polynucleotide is heterozygous if the frequency of one allele is between 0.3 and 0.7.

US Pat. No. 10,872,681

DIFFERENTIAL FILTERING OF GENETIC DATA

Affymetrix, Inc., Carlsb...

1. A computer-implemented method of differentially filtering genetic data, comprising:accessing, by a computer comprising a processor and a memory, intensity measurement data obtained from hybridization of one or more target nucleic acid molecules to at least one of a plurality of single nucleotide polymorphism probes and a plurality of copy number variation nucleic acid probes of an array, wherein the single nucleotide polymorphism nucleic acid probes are designed to identify one or more single nucleotide polymorphisms in the target nucleic acids and the copy number variation nucleic acid probes are designed to identify one or more copy number variations in the target nucleic acids;
converting the intensity measurement data at the processor to genetic data selectable by a user on at least one input interface window of a visual display device connected to the computer;
displaying the genetic data on the visual display device to the user in a display area of the visual display device, wherein the display area comprises one or more user configurable windows comprising at least one of a multiple chromosomes view, a selected chromosome view, and a segment filters view;
receiving from the user at the computer through the at least one input interface window a first set of parameters selected by the user for filtering the genetic data;
differentially filtering at the processor, a first subset of genetic data using the first set of parameters to form a first filtered subset of the genetic data; and
displaying on the visual display device the first filtered subset of the genetic data nearly simultaneously as the first set of parameters is received by the computer, wherein the first filtered subset of the genetic data is visualized using various colors, icons or other visual markers in the user configurable windows to distinguish from the genetic data that does not correspond to the first set of parameters.
US Pat. No. 10,832,796

SYSTEM, METHOD, AND COMPUTER SOFTWARE PRODUCT FOR GENOTYPE DETERMINATION USING PROBE ARRAY DATA

Affymetrix, Inc., Carlsb...

1. A method for genotyping a plurality of polymorphisms in a nucleic acid sample, the method comprising:hybridizing a nucleic acid sample with a plurality of allele-specific perfect-match probes provided in an array of perfect-match probes for a plurality of target sequences which the array is designed to genotype, wherein, for substantially all of the plurality of target sequences, the array is without corresponding mismatch probes;
summarizing intensity values associated with the hybridizing to obtain a plurality of signal values, the plurality of signal values comprising a respective signal value for each respective allele of the plurality of polymorphisms;
transforming the respective signal value for each allele into a space that includes a contrast dimension to obtain a plurality of transformed signal values;
evaluating likelihood of all plausible divisions of the transformed signal values into seed genotypes to identify most likely plausible divisions;
averaging over the most likely plausible divisions to derive a plurality of seed genotype clusters; and
genotyping the plurality of polymorphisms, wherein genotyping comprises comparing the transformed signal values with a set of typical values for each genotype, wherein the set of typical values comprises prior values, wherein the prior values comprise estimates of genotype cluster center locations and genotype cluster center variances of the plurality of seed genotype clusters determined from clustering properties of the transformed signal values;
wherein the steps of summarizing, transforming, evaluating, averaging, and genotyping are performed on a computer, and wherein the computer comprises a computer processor.
US Pat. No. 10,822,642

METHODS OF ANALYSIS OF METHYLATION

Affymetrix, Inc., Carlsb...

1. A method for analyzing the methylation of a plurality of cytosines in a plurality of target sequences, said method comprising:(a) obtaining a genomic DNA sample;
(b) fragmenting the genomic DNA sample with a methylation-sensitive restriction enzyme (MSRE) to obtain fragments, wherein said fragments comprise a mixture of target fragments and non-target fragments;
(c) filling in the ends generated by cleavage with the MSRE using a DNA polymerase;
(d) fragmenting the product of (c) with a methylation-insensitive restriction enzyme (MIRE) that recognizes the same restriction site as the MSRE;
(e) mixing the fragments of (d) with (i) a plurality of template probes, each template probe comprising a target complementarity region, a first common priming sequence and a second common priming sequence, wherein said first common priming sequence is 3? of the target complementarity region and said second common priming sequences is 5? of the target complementarity region, (ii) an oligonucleotide complementary to said first common priming sequence and an oligonucleotide that is complementary to said second common priming sequence, wherein at least one of the oligonucleotides is exonuclease-resistant, and (iii) a ligase, to obtain ligation products;
(f) treating the products of (e) with an exonuclease to digest the template probes;
(g) treating the products of (f) with bisulfite;
(h) amplifying the products of (g) by PCR using primers to the common priming sequences to obtain an amplification product;
(i) hybridizing the amplification product from step (h) to an array of probes to obtain a hybridization pattern; and
(j) analyzing the hybridization pattern to detect the presence or absence of methylation at a plurality of cytosines in the target sequences.
US Pat. No. 10,822,649

MULTIPLEX TARGETED AMPLIFICATION USING FLAP NUCLEASE

AFFYMETRIX, INC., Carlsb...

1. A method for amplifying a plurality of target sequences from a complex mixture of nucleic acid comprising:(a) fragmenting the nucleic acid by a method that generates fragments of known sequences at the 5? and 3? ends to obtain a fragmented nucleic acid sample including the plurality of target sequences;
(b) adding a plurality of circularization probes to the fragmented nucleic acid sample to form a mixture, wherein there is a circularization probe for each target sequence and wherein each circularization probe comprises:
(i) a first 5? region that is complementary to a first sequence in the target wherein said first sequence comprises the known sequence at the 5? end of the target and includes the 5? end of the target,
(ii) a second 3? region that is complementary to a second sequence in the target wherein said second sequence comprises the known sequence at the 3? end of the target and includes the 3? end of the target, and wherein the first sequence is separated from the second sequence by a third sequence,
wherein said circularization probe hybridizes to said target to form a structure wherein the first sequence and the second sequence are brought into juxtaposition by hybridization of the circularization probe so that the 5? end of the first sequence and the 3? end of the second sequence are separated by a nick or a gap,
(c) adding a DNA polymerase and at least one species of dNTP to fill the gap if the gap is present,
(d) adding a ligase to form ligated targets; and
(e) amplifying the ligated targets.
US Pat. No. 10,822,659

ANALYSIS OF METHYLATION USING NUCLEIC ACID ARRAYS

AFFYMETRIX, INC., Carlsb...

1. An array of probes comprising: more than 250,000 different experimental probe features, wherein(a) each experimental probe feature comprises a plurality of copies of a nucleic acid probe that is different in sequence from the probes of every other feature of the array;
(b) each probe feature is at a known or determinable location in the array; and
(c) at least 90% of the probe features contain full length probes that are perfectly complementary to a CpG island, wherein a CpG island is a genomic region that is at least 200 bases and has a GC content of at least 50% over its length.