0001
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`GUARDANT - EXHIBIT 2030
`Foundation Medicine, Inc. v. Guardant Health, Inc.
`IPR2019-00634
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`Atty Dockct No. 42534-704601
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`sequence; d) quantifying/counting mappedreads in two or more predefined regions of the
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`reference sequence; e) determining a copy numbervariation in one or moreof the predefined
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`regions by (i) normalizing the numberof reads in the predefined regions to each other and/or the
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`number of unique barcodesin the predefined regions to each other; and (ii) comparing the
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`normalized numbersobtainedin step (i) to normalized numbers obtained from a control sample.
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`[0005] The disclosure also provides for a method for detecting a rare mutation in a cell-free or
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`substantially ccll free sample obtained from a subject comprising: a) sequencing extraccllular
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`polynucleotides from a bodily sample from a subject, wherein each of the extracellular
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`polynucleotide generate a plurality of sequencing reads; b) sequencing extracellular
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`polynucleotides from a bodily sample from a subject, wherein each of the extracellular
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`polynucleotide generate a plurality of sequencing reads; sequencing extracellular polynucleotides
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`from a bodily sample from a subject, wherein each of the extracellular polynucleotide generate a
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`plurality of sequencing reads; c) filtering out reads that fail to meet a set threshold; d) mapping
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`sequence reads derived from the sequencing onto a reference sequence; e) identifying a subset of
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`mapped sequencereads that align with a variant of the reference sequence at each mappable base
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`position; f) for cach mappable basc position, calculating a ratio of (a) a number of mapped
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`sequencereads that include a variant as comparedto the reference sequence, to (b) a number of
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`total sequence reads for each mappable base position; g) normalizing the ratios or frequency of
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`variance for each mappable base position and determining potential rare variant(s) or mutation(s);
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`h) and comparing the resulting number for each of the regions with potential rare variant(s) or
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`mutation(s) to similarly derived numbers from a reference sample.
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`[0006] Additionally, the disclosure also provides for a method of characterizing the heterogeneity
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`of an abnormal condition in a subject, the method comprising generating a genetic profile of
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`extracellular polynucleotides in the subject, wherein the genetic profile comprises a plurality of
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`data resulting from copy numbervariation and/or other rare mutation (c.g., genctic altcration)
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`analyses.
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`[0007] In some embodiments, the prevalence/concentration of each rare variant identified in the
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`subject is reported and quantified simultaneously. In other embodiments, a confidence score,
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`regarding the prevalence/concentrations of rare variants in the subject, is reported.
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`[0008] In some embodiments, extracellular polynucleotides comprise DNA. In other
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`embodiments, extracellular polynucleotides comprise RNA. Polynucleotides may be fragments or
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`fragmented after isolation. Additionally, the disclosure provides for a methodfor circulating
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`nucleic acid isolation and extraction.
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`[0009] In some embodiments, extracellular polynucleotides are isolated from a bodily sample that
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`may be selected from a group consisting of blood, plasma, serum, urine, saliva, mucosal
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`excretions, sputum, stool andtears.
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`[0010] In some embodiments, the methods of the disclosure also comprise a step of determining
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`the percent of sequences having copy numbervariation or other rare genctic altcration (c.g.,
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`sequencevariants) in said bodily sample.
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`[0011] In some embodiments, the percent of sequences having copy numbervariation in said
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`bodily sample is determined by calculating the percentage of predefined regions with an amount
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`of polynucleotides above or below a predetermined threshold.
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`[0012] In some embodiments, bodily fluids are drawn from a subject suspected of having an
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`abnormal condition which may beselected from the group consisting of, mutations, rare
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`mutations, single nucleotide variants, indels, copy numbervariations, transversions,
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`translocations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal
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`instability, chromosomalstructure altcrations, gene fusions, chromosomefusions, gence
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`truncations, gene amplification, gene duplications, chromosomal lesions, DNAlesions, abnormal
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`changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns,
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`abnormal changes in nucleic acid methylation infection and cancer.
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`[0013] In some embodiments, the subject may be a pregnant female in which the abnormal
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`condition maybe a fetal abnormality selected from the group consisting of, single nucleotide
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`variants, indels, copy numbervariations, transversions, translocations, inversion, deletions,
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`aneuploidy, partial aneuploidy, polyploidy, chromosomalinstability, chromosomal structure
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`alterations, gene fusions, chromosomefusions, genetruncations, gene amplification, gene
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`duplications, chromosomal Icsions, DNA Icsions, abnormal changesin nucleic acid chemical
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`modifications, abnormal changes in epigenetic patterns, abnormal changes in nucleic acid
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`methylation infection and cancer
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`[0014] In some embodiments, the method may comprise comprising attaching one or more
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`barcodesto the extracellular polynucleotides or fragments thereof prior to sequencing, in which
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`the barcodes comprise are unique. In other embodiments barcodes attached to extracellular
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`polynucleotides or fragments thereof prior to sequencingare not unique.
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`[0015]
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`In some embodiments, the methods of the disclosure may comprise selectively enriching
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`regions from the subject’s genomeor transcriptomeprior to sequencing. In other embodiments
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`the methods of the disclosure comprise selectively enriching regions from the subject’s genome or
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`transcriptome prior to sequencing. In other embodiments the methodsofthe disclosure comprise
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`non-selectively enriching regions from the subject’s genomeortranscriptome prior to sequencing.
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`[0016] Further, the methods of the disclosure comprise attaching one or more barcodesto the
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`extraccllular polynucleotides or fragments thercof prior to any amplification or cnrichmentstcp.
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`[0017] In some embodiments, the barcode is a polynucleotide, which may further comprise
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`random sequenceor a fixed or semi-randomset of oligonucleotides that in combination with the
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`diversity of molecules sequenced from a select region enables identification of unique molecules
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`andbeat least a 3, 5, 10, 15, 20 25, 30, 35, 40, 45, or 5Omerbase pairs in length.
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`[0018] In some embodiments, extracellular polynucleotides or fragments thereof may be
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`amplified. In some embodiments amplification comprises global amplification or whole genome
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`amplification.
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`[0019] In some embodiments, sequence reads of unique identity may be detected based on
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`sequence information at the beginning(start) and cnd (stop) regions of the sequenceread and the
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`length of the sequence read. In other embodiments sequence molecules of unique identity are
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`detected based on sequence information at the beginning (start) and end (stop) regions of the
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`sequenceread, the length of the sequence read and attachmentof a barcode.
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`[0020] In some embodiments, amplification comprises selective amplification, non-selective
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`amplification, suppression amplification or subtractive enrichment.
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`[0021] In some embodiments, the methods of the disclosure comprise removing a subset of the
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`reads from further analysis prior to quantifying or enumerating reads.
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`[0022] In some embodiments, the method may comprise filtering out reads with an accuracy or
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`quality score of less than a threshold, c.g., 90%, 99%, 99.9%, or 99.99% and/or mapping score
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`less than a threshold, e.g., 90%, 99%, 99.9% or 99.99%. In other embodiments, methods of the
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`disclosure comprise filtering reads with a quality score lower than a set threshold.
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`[0023] In some embodiments, predefined regions are uniform or substantially uniform in size,
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`about 10kb, 20kb, 30kb 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, or 100kb in size. In some
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`embodiments, at least 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, or 50,000 regionsare
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`analyzed.
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`[0024] In some embodiments, a genetic variant, rare mutation or copy numbervariation occurs in
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`a region of the genomeselected from the group consisting of gene fusions, gene duplications,
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`gene deletions, gene translocations, microsatellite regions, gene fragments or combination thereof.
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`In other embodimentsa genetic variant, rare mutation, or copy numbervariation occurs in a
`
`region of the genomeselected from the group consisting of genes, oncogenes, tumor suppressor
`
`genes, promoters, regulatory sequence elements, or combination thereof. In some embodiments
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`the variant is a nucleotide variant, single basc substitution, or small indcl, transversion,
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`translocation, inversion, deletion, truncation or genetruncation about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15
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`or 20 nucleotides in length.
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`[0025] In some embodiments, the method comprises correcting/normalizing/adjusting the
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`quantity of mapped reads using the barcodes or unique properties of individualreads.
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`[0026] In some embodiments, enumerating the reads is performed through enumeration of unique
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`barcodesin each of the predefined regions and normalizing those numbersacross at least a subset
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`of predefined regions that were sequenced. In some embodiments, samples at succeeding time
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`intervals from the same subject are analyzed and compared to previous sample results. The
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`method ofthe disclosure may furthcr comprise determining partial copy numbervariation
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`frequency, loss of heterozygosity, gene expression analysis, epigenetic analysis and
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`hypermethylation analysis after amplifying the barcode-attached extracellular polynucleotides.
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`[0027] In some embodiments, copy numbervariation and rare mutation analysis is determined in
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`a cell-free or substantially cell free sample obtained from a subject using multiplex sequencing,
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`comprising performing over 10,000 sequencing reactions; simultaneously sequencingat least
`
`10,000 different reads; or performing data analysis on at least 10,000 different reads across the
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`genome. The method may comprise multiplex sequencing comprising performing data analysis
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`on at least 10,000 different reads across the genome. The method may further comprise
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`cnumcrating sequencedreads that are uniqucly identifiable.
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`[0028] In some embodiments, the methods of the disclosure comprise normalizing and detection
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`is performed using one or more of hidden markov, dynamic programming, support vector
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`machine, Bayesian network,trellis decoding, Viterbi decoding, expectation maximization,
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`Kalmanfiltering, or neural network methodologies.
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`[0029] In some embodiments the methods of the disclosure comprise monitoring disease
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`progression, monitoring residual disease, monitoring therapy, diagnosing a condition, prognosing
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`a condition, or selecting a therapy based on discovered variants.
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`[0030] In some embodiments, a therapy is modified based on the most recent sample analysis.
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`Further, the methodsof the disclosure comprise inferring the genetic profile of a tumor, infection
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`or other tissue abnormality. In some embodiments growth, remission or evolution of a tumor,
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`infection or other tissue abnormality is monitored. In some embodiments the subject’s immune
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`system are analyzed and monitoredat single instancesor over time.
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`[0031] In some embodiments, the methods of the disclosure comprise identification of a variant
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`that is followed up through an imagingtest (c.g., CT, PET-CT, MRI, X-ray, ultrasound) for
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`localization of the tissue abnormality suspected of causing the identified variant.
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`[0032] In some embodiments, the methods of the disclosure comprise use of genetic data obtained
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`from a tissue or tumor biopsy from the samepatient. In some embodiments, whereby the
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`phylogenetics of a tumor, infection or other tissue abnormality is inferred.
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`[0033] In some embodiments, the methodsof the disclosure comprise performing population-
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`based no-calling and identification of low-confidence regions. In some embodiments, obtaining
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`the measurementdata for the sequence coverage comprises measuring sequence coverage depth at
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`every position of the genome. In some embodiments correcting the measurementdata for the
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`sequence coverage bias comprises calculating window-avecraged coverage. In some embodiments
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`correcting the measurementdata for the sequence coverage bias comprises performing
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`adjustments to account for GC bias in the library construction and sequencing process. In some
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`embodiments correcting the measurement data for the sequence coverage bias comprises
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`performing adjustments based on additional weighting factor associated with individual mappings
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`to compensatefor bias.
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`[0034] In some embodiments, the methods of the disclosure comprise extracellular polynucleotide
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`derived from a diseased cell origin. In some embodiments, the extracellular polynucleotide is
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`derived from a healthy cell origin.
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`[0035] The disclosure also provides for a system comprising a computer readable medium for
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`performing the following steps: selecting predefined regions in a genome; enumerating number of
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`sequencereads in the predefined regions; normalizing the number of sequence reads acrossthe
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`predefined regions; and determining percent of copy numbervariation in the predefined regions.
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`In some embodiments,the entirety of the genomeorat least 10%, 20%, 30%, 40%, 50%, 60%,
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`70%, 80%, or 90% of the genomeis analyzed. In some embodiments, computer readable medium
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`provides data on percent cancer DNA or RNAin plasma or serum to the end user.
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`[0036] In some embodiments, the amount of genetic variation, such as polymorphismsor causal
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`variants is analyzed. In some embodiments, the presence or absenceof genetic alterations is
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`detected.
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`[0037] The disclosure also provides for a method for detecting a rare mutation in a cell-free or a
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`substantially cell free sample obtained from a subject comprising: a) sequencing extracellular
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`polynucleotides from a bodily sample from a subject, wherein each of the extracellular
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`polynuclcotides gencrate a plurality of sequencing reads; b) filtcring out reads that fail to mect a
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`set threshold; c) mapping sequence reads derived from the sequencing onto a reference sequence;
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`d) identifying a subset of mapped sequencereads that align with a variant of the reference
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`sequence at each mappable base position; e) for each mappable baseposition, calculating a ratio
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`of (a) a number of mapped sequence readsthat include a variant as comparedto the reference
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`sequence, to (b) a numberof total sequence reads for each mappable baseposition; f) normalizing
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`the ratios or frequency of variance for each mappable base position and determining potential rare
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`variant(s) or other genetic alteration(s); and g) comparing the resulting number for each of the
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`regions
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`[0038] This disclosure also provides for a method comprising: a. providing at lcast one sct of
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`tagged parent polynucleotides, and for each set of tagged parent polynucleotides; b. amplifying
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`the tagged parent polynucleotides in the set to produce a correspondingset of amplified progeny
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`polynucleotides; c. sequencing a subset (including a proper subset) of the set of amplified progeny
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`polynucleotides, to produce a set of sequencing reads; and d. collapsing the set of sequencing
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`reads to generate a set of consensus sequences, each consensus sequence corresponding to a
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`unique polynucleotide amongtheset of tagged parent polynucleotides. In certain embodiments
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`the method further comprises: e. analyzing the set of consensus sequences for each set of tagged
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`parent molecules.
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`[0039] In some cmbodiments cach polynucleotide in a sct is mappable to a reference sequence.
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`[0040] In some embodiments the method comprises providinga plurality of sets of tagged parent
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`polynucleotides, wherein each set is mappable to a different reference sequence.
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`[0041] In some embodiments the method further comprises converting initial starting genetic
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`material into the tagged parent polynucleotides.
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`[0042] In some embodiments the initial starting genetic material comprises no more than 100 ng
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`of polynucleotides.
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`[0043] In some embodiments the method comprises bottlenecking the initial starting genetic
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`material prior to converting.
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`[0044] In some embodiments the method comprises converting the initial starting genetic material
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`into tagged parent polynucleotides with a conversion efficiency of at least 10%, at least 20%, at
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`least 30%, at least 40%, at least 50%, at least 60%, at least 80% or at least 90%.
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`[0045] In some embodiments converting comprises any of blunt-endligation, sticky end ligation,
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`molecular inversion probes, PCR, ligation-bascd PCR,single strand ligation and single strand
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`circularization.
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`[0046] In some embodiments the initial starting genetic material is cell-free nucleic acid.
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`[0047] In some embodiments a plurality of the reference sequences are from the same genome.
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`[0048] In some embodiments each tagged parent polynucleotide in the set is uniquely tagged.
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`[0049] In some embodiments the tags are non-unique.
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`[0050] In some embodiments the generation of consensus sequencesis based on information from
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`the tag and/orat least one of sequence information at the beginning(start) region of the sequence
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`read, the end (stop) regions of the sequence read and the length of the sequenceread.
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`[0051] In some cmbodiments the method comprises sequencing a subsct of the sct of amplificd
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`progeny polynucleotides sufficient to produce sequencereads for at least one progeny from of
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`each ofat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least
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`80%, at least 90% at least 95%, at least 98%, at least 99%, at least 99.9% or at least 99.99% of
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`unique polynucleotides in the set of tagged parent polynucleotides.
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`[0052] In some embodiments the at least one progenyis a plurality of progeny,e.g., at least 2, at
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`least 5 or at least 10 progeny.
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`[0053] In some embodiments the number of sequence reads in the set of sequence reads is greater
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`than the numberof unique tagged parent polynucleotides in the set of tagged parent
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`polynucleotides.
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`[0054] In some embodiments the subset of the set of amplified progeny polynucleotides
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`sequencedis of sufficient size so that any nucleotide sequence represented in the set of tagged
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`parent polynucleotides at a percentage that is the same as the percentage per-base sequencing
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`error rate of the sequencing platform used, has at least a 50%, at least a 60%, at least a 70%, at
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`least a 80%, at least a 90% at least a 95%, at least a 98%, at least a 99%, at least a 99.9% or at
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`least a 99.99% chance of being represented amongthe set of consensus sequences.
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`[0055] In some embodiments the method comprises enriching the set of amplified progeny
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`polynucleotides for polynucleotides mapping to one or more selected reference sequencesby:(i)
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`selective amplification of sequences from initial starting genetic material converted to tagged
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`parent polynucleotides; (11) selective amplification of tagged parent polynucleotides; (iii) selective
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`sequence capture of amplified progeny polynucleotides; or (iv) selective sequence capture of
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`initial starting genetic material.
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`[0056] In some cmbodiments analyzing compriscs normalizing a measure (c.g., number) taken
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`from a set of consensus sequences against a measure taken from a set of consensus sequences
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`from a control sample.
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`[0057] In some embodiments analyzing comprises detecting mutations, rare mutations, single
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`nucleotide variants, indels, copy numbervariations, transversions, translocations, inversion,
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`deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomalinstability, chromosomal
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`structure alterations, gene fusions, chromosomefusions, gene truncations, gene amplification,
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`gene duplications, chromosomal lesions, DNA lesions, abnormal changesin nucleic acid chemical
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`modifications, abnormal changes in epigenetic patterns, abnormal changesin nucleic acid
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`methylation infection or canccr.
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`[0058] In some embodiments the polynucleotides comprise DNA, RNA, a combination of the two
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`or DNA plus RNA-derived cDNA.
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`[0059] In some embodiments a certain subset of polynucleotides is selected for or is enriched
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`based on polynucleotide length in base-pairs from the initial set of polynucleotides or from the
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`amplified polynucleotides.
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`[0060] In some embodiments analysis further comprises detection and monitoring of an
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`abnormality or disease within an individual, such as, infection and/or cancer.
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`[0061] In some embodiments the method is performed in combination with immunerepertoire
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`profiling.
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`[0062] In some embodiments the polynucleotides are extract from the group consisting of blood,
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`plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears.
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`[0063] In some embodiments collapsing comprising detecting and/or correcting errors, nicks or
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`lesions present in the sense or anti-sense strand of the tagged parent polynucleotides or amplified
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`progeny polynucleotides.
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`[0064] This disclosure also provides for a method comprising detecting genetic variation in initial
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`starting genetic material with a sensitivity of at least 5%, at least 1%, at least 0.5%, at least 0.1%
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`or at least 0.05%. In some embodiments the initial starting genetic material is provided in an
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`amount less than 100 ng of nucleic acid, the genetic variation is copy number/heterozygosity
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`variation and detecting is performed with sub-chromosomalresolution; e.g., at least 100 megabase
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`resolution, at least 10 megabase resolution, at least 1 megabase resolution, at least 100 kilobase
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`resolution, at least 10 kilobase resolution or at least 1 kilobase resolution. In another embodiment
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`the method comprises providing a plurality of sets of tagged parent polynucleotides, wherein each
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`sect is mappable to a different reference sequence. In another embodiment the reference sequence
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`is the locus of a tumor marker, and analyzing comprises detecting the tumor markerin the set of
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`consensus sequences. In another embodiment the tumor marker is present in the set of consensus
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`sequencesat a frequency less than the error rate introduced at the amplifying step. In another
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`embodimentthe at least oneset is a plurality of sets, and the reference sequences comprise a
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`plurality of reference sequences, each of which is the locus of a tumor marker. In another
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`embodiment analyzing comprises detecting copy numbervariation of consensus sequences
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`betweenat least two sets of parent polynucleotides. In another embodiment analyzing comprises
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`detecting the presence of sequence variations compared with the reference sequences. In another
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`embodiment analyzing comprises detecting the presence of sequence variations compared with
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`the reference sequences and detecting copy numbervariation of consensus sequences between at
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`least two sets of parent polynucleotides. In another embodimentcollapsing comprises: 1. grouping
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`sequences reads sequenced from amplified progeny polynucleotides into families, each family
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`amplified from the same tagged parent polynucleotide; and ii. determining a consensus sequence
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`based on sequencereads in a family.
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`[0065] This disclosure also provides for a system comprising a computer readable medium for
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`performing the following steps: a. providing at least one set of tagged parent polynucleotides, and
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`for each set of tagged parent polynucleotides; b. amplifying the tagged parent polynucleotides in
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`the set to produce a corresponding sct of amplificd progeny polynucleotides; c. sequencing a
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`subset (including a proper subset) of the set of amplified progeny polynucleotides, to produce a
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`set of sequencing reads; and d. collapsing the set of sequencing reads to generate a set of
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`consensus sequences, each consensus sequence corresponding to a unique polynucleotide among
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`the set of tagged parent polynucleotides and, optionally, ce. analyzing the set of consensus
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`sequencesfor each set of tagged parent molecules.
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`[0066] This disclosure also provides a method comprising: a. providing at least one set of tagged
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`parent polynucleotides, and for each set of tagged parent polynucleotides; b. amplifying the
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`tagged parent polynucleotides in the set to produce a corresponding set of amplified progeny
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`polynucleotides; c. sequencing a subset (including a propersubset) of the set of amplified progeny
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`polynucleotides, to produce a set of sequencing reads; d. collapsing the set of sequencing reads to
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`generate a set of consensus sequences, each consensus sequence corresponding to a unique
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`polynucleotide amongthe set of tagged parent polynucleotides; and e. filtering out from among
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`the consensus sequencesthose that fail to meet a quality threshold. In one embodimentthe
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`quality threshold considers a number of sequence reads from amplified progeny polynucleotides
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`collapsed into a consensus sequence. In another embodiment the quality threshold considers a
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`number of sequence reads from amplified progeny polynucleotides collapsed into a consensus
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`sequence. This disclosure also provides a system comprising a computer readable medium for
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`performing the aforesaid method.
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`[0067] This disclosure also provides a method comprising: a. providing at least one set of tagged
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`parent polynucleotides, wherein each set maps to a different reference sequence in one or more
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`genomes,and, for each set of tagged parent polynucleotides; i. amplifying the first
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`polynucleotides to produce a set of amplified polynucleotides; ii. sequencing a subset ofthe set of
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`amplificd polynucleotides, to produce a sct of sequencing reads; and iii. collapsing the sequence
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`reads by: 1. grouping sequences reads sequenced from amplified progeny polynucleotides into
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`families, each family amplified from the same tagged parent polynucleotide. In one embodiment
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`collapsing further comprises: 2. determining a quantitative measure of sequence reads in each
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`family. In another embodiment the method further comprises (including a) includinga): b.
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`determining a quantitative measure of unique families; and c. based on (1) the quantitative
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`measure of unique families and (2) the quantitative measure of sequence reads in each group,
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`inferring a measure of unique tagged parent polynucleotides in the set. In another embodiment
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`inferring is performed usingstatistical or probabilistic models. In another embodiment wherein
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`the at lcast onc sct is a plurality of scts. In another cmbodiment the method further comprises
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`correcting for amplification or representational bias between the two sets. In another embodiment
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`the method further comprises using a control or set of control samples to correct for amplification
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`or representational biases between the two sets. In another embodiment the method further
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`comprises determining copy numbervariation betweenthe sets. In another embodiment the
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`method further comprises (includinga, b, c): d. determining a quantitative measure of
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`polymorphic forms amongthe families; and e. based on the determined quantitative measure of
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`polymorphic forms, inferring a quantitative measure of polymorphic forms in the number of
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`inferred unique tagged parent polynucleotides. In another embodiment wherein polymorphic
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`formsinclude but are not limited to: substitutions, insertions, deletions, inversions, microsatellite
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`changes, transversions, translocations, fusions, methylation, hypermethylation,
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`hyrdroxymethylation, acetylation, epigenetic variants, regulatory-associated variants or protein
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`binding sites. In another embodiment wherein the sets derive from a common sample, the method
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`further comprising: a. inferring copy numbervariation for the plurality of sets based on a
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`comparison of the inferred numberof tagged parent polynucleotides in cach sct mapping to cach
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`of a plurality of reference sequences. In another embodimentthe original number of
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`polynucleotides in each set is further inferred. This disclosure also provides a system comprising
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`a computer readable medium for performing the aforesaid methods.
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`[0068] This disclosure also provides a method of determining copy numbervariation in a sample
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`that includes polynucleotides, the method comprising: a. providing at least two sets of first
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`polynucleotides, wherein each set maps to a different reference sequence in a genome, and, for
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`eachset of first polynucleotides; i. amplifying the polynucleotides to produce a set of amplified
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`polynucleotides; ii. sequencing a subset of the set of amplified polynucleotides, to producea set of
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`sequencing reads; ili. grouping scquences reads sequenced from amplificd polynucleotides into
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`families, each family amplified from the samefirst polynucleotide in the set; iv. inferring a
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`quantitative measure of families in the set; b. determining copy numbervariation by comparing
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`the quantitative measure of families in each set. This disclosure also provides a system
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`comprising a computer readable medium for performing the aforesaid methods.
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`[0069] This disclosure also provides a methodof inferring frequency of sequencecalls in a
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`sample of polynucleotides comprising: a. providing at least oneset of first polynucleotides,
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`wherein each set maps to a different reference sequence in one or more genomes, and, for each set
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`of first polynucleotides; 1. amplifying the first polynucleotides to producea set of amplified
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`polynucleotides; 11. sequencing a subsct of the sct of amplificd polynucleotides, to producea sct of
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`sequencingreads; iii. grouping the sequencereadsinto families, cach family comprising sequence
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`reads of amplified polynucleotides amplified from the samefirst polynucleotide; b. inferring, for
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`each set of first polynucleotides, a call frequency for one or more bases in thesetoffirst
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`polynucleotides, wherein inferring comprises: 1. assigning, for each family, confidence score for
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`each of a plurality of calls, the confidence score taking into consideration a frequencyofthe call
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`among members of the family; and ii. estimating a frequency of the one or morecalls taking into
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`consideration the confidence scores of the one or more calls assigned to each family. This
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`Atty Dockct No. 42534-704601
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`disclosure also provides a system comprising a computer readable medium for performing the
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`aforesaid methods.
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`[0070] This disclosure also provides a method of communicating sequence information about at
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`least one individual polynucleotide molecule comprising: a. providing at least one individual
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`polynucleotide molecule; b. encoding sequence information in the at least one individual
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`polynucleotide molecule to produce a signal; c. passing at least part of the signal through a
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`channel to produce a received signal comprising nucleotide sequence information about the at
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`least one individual polynucleotide molecule, wherein the received signal comprises noise and/or
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`distortion; d. decoding the received signal to produce a message comprising sequence information
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`about the at least one individual polynucleotide molecule, wherein decoding reduces noise and/or
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`distortion in the message; and e. providing the messageto a recipient. In one embodiment the
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`noise comprises incorrect nucleotide calls. In another embodimentdistortion comprises uneven
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`amplification of the individual polynucleotide molecule compared with other individual
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`polynucleotide molecules. In another embodimentdistortion results from amplification or
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`sequencing bias. In another embodimentthe at least one individual polynucleotide moleculeis a
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`plurality of individual polynuclcotide molecules, and decoding produccs a message about cach
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`molecule in the plurality. In another embodiment encoding comprises amplifying the at least
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`individual polynucleotide molecule which has optionally been tagged, wherein the signal
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`comprises a collection of amplified molecules. In another embodiment the channel comprises a
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`polynucleotide sequencer andthe received signal comprises sequence reads ofa plurality of
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`polynucleotides amplified from the at least one individual polynucleotide molecule. In another
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`embodiment decoding comprises grouping sequence reads of amplified