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`AMENDMENTS TO THE CLAIMS
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`This listing of claims will replace all prior versions andlistings in the above-referenced
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`patent application.
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`The foregoing amendmentsare without prejudice and do not constitute an
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`admission regarding the patentability of the amended subject matter and should not so be
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`construed. Applicant reserves the right to pursue the subject matter of the canceled claims in
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`this or any other appropriate patent application.
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`Listing of Claims:
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`1. — 60. (Cancelled).
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`61.
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`(a)
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`(New): A method, comprising:
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`providing a population of cell-free deoxyribonucleic acid (cfDNA) molecules
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`having first and second complementary strands;
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`(b)
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`tagging a plurality of the cfDNA molecules in the population with a set of duplex
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`tags comprising molecular barcodes from a set of molecular barcodes to produce tagged parent
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`polynucleotides, wherein the duplex tags are attached at both ends of a molecule of the cfDNA
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`molecules;
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`(c)
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`amplifying a plurality of the tagged parent polynucleotides to produce amplified
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`progeny polynucleotides;
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`(d)
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`sequencing at least a subset of the amplified progeny polynucleotides to produce a
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`set of sequence reads; and
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`(e)
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`reducing and/or tracking redundancy in the set of sequence reads to generate a
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`plurality of consensus sequences representative of original cfDNA molecules from among the
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`tagged parent polynucleotides, wherein the plurality of consensus sequences are generated from
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`(i) paired reads corresponding to sequence reads generated fromafirst tagged strand and a
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`second tagged complementary strand derived from a cfDNA molecule from amongthe tagged
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`parent polynucleotidesor(11) unpaired reads corresponding to sequence reads generated from a
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`first tagged strand having no second tagged complementary strand derived from a cfDNA
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`molecule from amongthe tagged parent polynucleotides.
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`62.
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`(New): The method of claim 61, wherein the sample is obtained from a subject
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`having cancer.
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`63.
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`(New): The method of claim 61, wherein the plurality of cfDNA molecules
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`comprises | nanogram (ng) to 100 ng of cfDNA molecules.
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`64.
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`(New): The method of claim 61, wherein the molecular barcodesare ligated to the
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`cfDNA molecules using more than a 10X molar excess of duplex tags as compared to the cfDNA
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`molecules.
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`65.
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`(New): The method of claim 64, wherein at least 20% of the cfDNA molecules
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`from the sample are tagged with the duplex tags.
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`66.
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`(New): The method of claim 61, wherein tagging comprises non-uniquely tagging
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`the plurality of the cfDNA molecules with the set of duplex tags comprising molecular barcodes
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`from the set of molecular barcodes, wherein the cfDNA molecules that map to a mappable base
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`position of a reference sequence are tagged with a numberofdifferent molecular barcodes
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`ranging from at least 2 and fewer than a number of cfDNA molecules that map to the mappable
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`base position
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`67.
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`(New): The method of claim 61, wherein the molecular barcodesin the set of
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`molecular barcodes have predetermined sequences.
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`68.
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`(New): The method of claim 61, wherein the molecular barcodesin the set of
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`molecular barcodes have 5 to 10,000 different molecular barcode sequences and are 5 to 20 base
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`pairs in length.
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`69.
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`(New): The method of claim 61, further comprising enriching the amplified
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`progeny polynucleotides for target regions of interest prior to sequencing.
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`70.
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`(New): The method of claim 69, wherein the target regions of interest comprise
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`genetic sequencesofa plurality of genes selected from the group consisting of ALK, APC,
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`BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN,
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`RB1, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSF1IR, CTNNB1, ERBB4, EZH2,
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`FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2,
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`JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET, SMAD4,
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`SMARCBI, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1,
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`CCND2, CDK6, NF1, TP53, ARIDIA, BRCA2, CCNEI, ESR1, RIT1, GATA3, MAP2K1,
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`RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA, and NTRK1.
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`71.
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`(New): The method of claim 61, further comprising amplifying a plurality of the
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`enriched progeny polynucleotides prior to sequencing.
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`72.
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`(New): The method of claim 61, wherein the molecular barcodesare part of
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`sequencing adapters.
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`73.
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`(New): The method of claim 72, wherein the adapter is a Y-shaped adapter.
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`74.—(New): The method of claim 61, wherein reducing and/or tracking redundancy in
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`the set of sequence reads comprises mapping a plurality of the sequence readsto a reference
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`sequence.
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`75.
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`(f)
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`(New): The method of claim 61, further comprising:
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`determining quantitative measures of at least two of (i) paired reads,(ii) unpaired
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`reads, (111) read depth of the paired reads and (iv) read depth of the unpaired reads.
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`76.
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`(g)
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`(New): The method of claim 75, further comprising:
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`estimating with a programmed computer processor a quantitative measure oftotal
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`cfDNA molecules based on said quantitative measuresof at least two of(1) paired reads, (11)
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`unpaired reads,(111) read depth of the paired reads and(iv) read depth of the unpaired reads.
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`77.
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`(New): The method of claim 76, wherein (f) comprises determining quantitative
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`measures of paired reads and unpaired reads, and wherein in (g), the quantitative measureoftotal
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`cfDNA molecules is determined based on the quantitative measures of paired reads and unpaired
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`reads.
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`78.
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`(a)
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`(New): A method, comprising:
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`providing a population of double-stranded cell-free deoxyribonucleic acid
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`(cfDNA) molecules having first and second complementary strands;
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`(b)—non-uniquely tagging a plurality of the double-stranded cfDNA molecules in the
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`population with a set of duplex tags comprising molecular barcodes from a set of molecular
`
`barcodes to produce non-uniquely tagged parent polynucleotides,
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`wherein the double-stranded cfDNA molecules that map to a mappable base position of a
`
`reference sequence are tagged with a numberofdifferent molecular barcodes ranging from at
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`least 2 and fewer than a numberof double-stranded cfDNA molecules that map to the mappable
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`base position;
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`(c)
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`amplifying a plurality of the non-uniquely tagged parent polynucleotides to
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`produce amplified progeny polynucleotides;
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`(d)
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`sequencing at least a subset of the amplified progeny polynucleotides to produce a
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`set of sequencereads;
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`(e)
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`(f)
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`reducing and/or tracking redundancy in the set of sequencereads;
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`sorting sequence reads into paired reads and unpaired reads, wherein (1) a paired
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`read corresponds to sequence reads generated fromafirst tagged strand and a second tagged
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`complementary strand derived from a double-stranded cfDNA molecule from among the non-
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`uniquely tagged parent polynucleotides, and (11) an unpaired read corresponds to sequence reads
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`generated fromafirst tagged strand having no second tagged complementary strand derived
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`from a double-stranded cfDNA molecule from among the non-uniquely tagged parent
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`polynucleotides; and
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`(g)
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`determining quantitative measures of at least two of (i) paired reads,(ii) unpaired
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`reads, (111) read depth of the paired reads and (iv) read depth of the unpaired reads; and
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`79.
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`(New): The method of claim 78, wherein the sample is blood, plasma, or serum.
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`80.|(New): The method of claim 78, wherein the plurality of double-stranded cfDNA
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`molecules comprises 1 nanogram (ng) to 100 ng of double-stranded cfDNA molecules.
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`81.
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`(New): The method of claim 78, wherein the tagging comprisesligating the
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`molecular barcodes to double-stranded cfDNA molecules.
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`82.
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`(New): The method of claim 78, wherein the molecular barcodesin the set have 2
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`to 10,000 different molecular barcode sequences.
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`83.|(New): The method of claim 78, wherein the molecular barcodesin the set have 5
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`to 10,000 different molecular barcode sequencesand are 5 to 20 basepairs in length.
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`84.
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`(New): The method of claim 78, further comprising enriching the amplified
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`progeny polynucleotides for target regions of interest prior to sequencing.
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`85,
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`(New): The method of claim 84, wherein the target regions of interest comprise
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`genetic sequencesofa plurality of genes selected from the group consisting of ALK, APC,
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`BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN,
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`RB1, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSF1IR, CTNNB1, ERBB4, EZH2,
`
`FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2,
`
`JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET, SMAD4,
`
`SMARCBI, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1,
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`CCND2, CDK6, NF1, TP53, ARIDIA, BRCA2, CCNEI, ESR1, RIT1, GATA3, MAP2K1,
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`RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA, and NTRK1.
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`86.|(New): The method of claim 78, further comprising amplifying a plurality of the
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`enriched progeny polynucleotides prior to sequencing.
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`87.
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`(New): The method of claim 78, wherein reducing and/or tracking redundancy in
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`the set of sequence reads comprises collapsing a plurality of the sequence reads to generate
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`consensus sequencesrepresentative of original double-stranded cfDNA molecules from among
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`the non-uniquely tagged parent polynucleotides.
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`88.
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`(New): The method of claim 87, further comprising mapping a plurality of the
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`sequence reads and/or consensus sequencesto a reference sequence.
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`89.
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`(h)
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`(New): The methodof claim 78, further comprising:
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`estimating with a programmed computer processor a quantitative measureoftotal
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`double-stranded polynucleotide molecules based on said quantitative measuresof at least two of
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`(i) paired reads,(11) unpaired reads, (1i1) read depth of the paired reads and(iv) read depth of the
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`unpaired reads.
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`90.
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`(New): The method of claim 89, wherein (g) comprises determining quantitative
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`measures of paired reads and unpaired reads, and wherein in (h), the quantitative measureoftotal
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`double-stranded cfDNA molecules is determined based on the quantitative measures of paired
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`reads and unpaired reads.
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`