Application No. 15/952,203
`Amendment dated: August 10, 2018
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`AMENDMENTS TO THE CLAIMS
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`This listing of claims will replace all prior versions, and listings of claims in this application.
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`Applicant reserves the right to pursue any subject matter of any canceled claims in this or any other
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`appropriate patent application. Support for these claims is provided in the remarks following the listing of
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`claims.
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`Listing of the Claims
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`1-114.
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`(Cancelled)
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`115.
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`(New) A method for performing a primer extension reaction on RNA and DNA, comprising:
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`a) providing a sample comprising a mixture of single-stranded DNA and single-stranded
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`RNA,
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`adapter,
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`DNA, and
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`b) attaching a first adapter to said single-stranded DNA,
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`c) attaching a second adapter to said single-stranded RNA,
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`d) annealing a first primer to said first adapter and annealing a second primer to said second
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`e) extending said annealed f1rst primer on said single-stranded DNA to form double-stranded
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`f) extending said annealed second primer on said single-stranded RNA to form a double-
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`stranded DNA-RNA hybrid.
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`116.
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`(New) The method of claim 115, wherein said attaching said first adapter comprises ligating
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`said first adapter to a 3’ end of said single-stranded DNA.
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`117.
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`(New) The method of claim 116, wherein said ligating said first adapter is performed by a
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`ligase selected from CircLigase ll, Thermostable App-DNA/RNA ligase, T4 RNA ligase 1, T4 RNA Ligase 2
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`truncated, and any combination thereof.
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`118.
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`(New) The method of claim 115, wherein said attaching said second adapter comprises
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`ligating said second adapter to a 3’ end of said single-stranded RNA.
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`119.
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`(New) The method of claim 118, wherein said ligating said second adapter is performed
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`using an RNA ligase.
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`120.
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`(New) The method of claim 118, wherein said ligating said second adapter is performed
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`using T4 RNA ligase 2 or T4 DNA ligase.
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`121.
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`122.
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`(New) The method of claim 115, wherein said single-stranded DNA is cell-free DNA.
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`(New) The method of claim 115, wherein said sample is selected from the group consisting
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`of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-alveolar lavage, urine, stool, saliva,
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`nasal swab, and any combination thereof.
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`Application No. 15/952,203
`Amendment dated: August 10, 2018
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`123.
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`(New) The method of claim 115, wherein said extending said annealed first primer on said
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`single-stranded DNA is performed by a DNA polymerase.
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`124.
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`(New) The method of claim 115, wherein said extending said annealed first primer on said
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`single-stranded DNA is performed by Bst 2.0 DNA polymerase.
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`125.
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`(New) The method of claim 115, wherein said extending said annealed second primer on
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`said single-stranded RNA is performed by a polymerase selected from Moloney Murine Leukemia Virus (M-
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`MLV) reverse transcriptase, and a SMARTer reverse transcriptase.
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`126.
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`(New) The method of claim 115, further comprising adding at least one non-templated
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`nucleotide to a first primer extension strand.
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`127.
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`(New) The method of claim 126, wherein said at least one non-templated nucleotide is a
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`deoxycytidine.
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`128.
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`to a 3’ end.
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`129.
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`eight nucleotides.
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`(New) The method of claim 126, wherein said at least one non-templated nucleotide is added
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`(New) The method of claim 126, wherein said at least one non-templated nucleotide is up to
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`(New) The method of claim 126, wherein said at least one non-templated nucleotide is three,
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`four, or five non-templated nucleotides.
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`(New) The method of claim 126, wherein said at least one non-templated nucleotide is one
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`130.
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`131.
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`132.
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`first overhang.
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`133.
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`non-templated nucleotide.
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`(New) The method of claim 126, wherein said at least one non-templated nucleotide forms a
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`(New) The method of claim 132, further comprising adding at least one second non-
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`templated nucleotide to a second primer extension strand forming a second overhang.
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`134.
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`(New) The method of claim 133, further comprising hybridizing a third adapter to said
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`first overhang and a fourth adapter to said second overhang.
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`135.
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`(New) The method of claim 134, further comprising sequencing said third adapter and
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`said fourth adapter and sequences attached to said third adapter and said fourth adapter.
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`136.
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`(New) The method of claim 134, further comprising (1) identifying sequences associated
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`with said third adapter as originating from said DNA in said mixture of single-stranded DNA and single-
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`stranded RNA and (ii) identifying sequences associated with said fourth adapter as originating from said
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`RNA in said mixture of single-stranded DNA and single-stranded RNA.
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`Application No. 15/952,203
`Amendment dated: August 10, 2018
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`137.
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`(New) A method of performing an amplification reaction on a first RNA and a first DNA,
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`comprising:
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`a) providing a sample comprising a mixture of said first DNA and said first RNA, wherein
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`said first DNA does not comprise a sequence complementary to said first RNA,
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`b) tagging said first DNA with a first tag without using a transposase,
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`c) tagging said first RNA with a second tag,
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`d) performing an amplification or primer extension reaction on said first DNA with a
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`polymerase that is selective for DNA templates; and
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`e) synthesizing a complementary cDNA strand from said first RNA with a reverse
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`transcriptase.
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`138.
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`(New) The method of claim 137, wherein said first DNA is single-stranded DNA, double-
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`stranded DNA, triple-stranded DNA, or a Holliday junction.
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`139.
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`(New) The method of claim 137, wherein said first RNA is single-stranded RNA, double-
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`stranded RNA, or a ribozyme.
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`140.
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`(New) The method of claim 137, wherein said first DNA is cell-free DNA.
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`141.
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`(New) The method of claim 137, wherein said sample is selected from the group consisting
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`of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-alveolar lavage, urine, stool, saliva,
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`nasal swab, and any combination thereof.
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`142.
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`(New) The method of claim 137, comprising performing said amplification to generate
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`amplified products.
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`143.
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`144.
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`acids,
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`(New) The method of claim 142, further comprising sequencing said amplified products.
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`(New) A method of sequencing nucleic acids comprising:
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`a) providing a sample comprising double-stranded nucleic acids and single-stranded nucleic
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`b) ligating a first adapter to an end of said double-stranded nucleic acids,
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`c) denaturing said double-stranded nucleic acids into single-stranded nucleic acids, and
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`d) sequencing nucleic acids ligated to said first adapter and identifying sequences associated
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`with said first adapter as being double-stranded.
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`145.
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`(New) A method for concurrent processing of different nucleic acid forms in a sample
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`comprising:
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`a) denaturing said nucleic acid forms in a sample,
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`Application No. 15/952,203
`Amendment dated: August 10, 2018
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`b) ligating a first adapter to one end of a first nucleic acid form using a ligase that has a
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`preference for said first nucleic acid form and ligating a second adapter to one end of a second nucleic acid
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`form using a ligase that has preference for said second nucleic acid form,
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`c) primer extending said first and said second ligated nucleic acid forms,
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`d) ligating a third adapter comprising a priming element, and
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`e) amplifying said first and second nucleic forms.
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`146.
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`(New) A reaction mixture composition comprising:
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`a)
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`b)
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`c)
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`d)
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`an adapter,
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`a first ligase that has a preference for a first nucleic acid form,
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`a second ligase that has a preference for a second nucleic acid form, and
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`a buffer.
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`147.
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`(New) A reaction mixture comprising:
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`a) a ligase,
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`b) a DNA-dependent polymerase that has non-templated activity, wherein said non-
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`templated base is N1, and
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`c) a RT polymerase that has non-templated activity, wherein said non-templated base is N2,
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`wherein N1 and N2 are different nucleic acid bases.
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`148.
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`(New) A kit comprising:
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`a)
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`an adapter,
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`b)
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`a first ligase that has a preference for a first nucleic acid form,
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`c)
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`d)
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`a second ligase that has a preference for a second nucleic acid form, and
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`a buffer.
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`149.
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`(New) A kit comprising:
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`a) a ligase,
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`b) a DNA-dependent polymerase that has non-templated activity, wherein the non-templated
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`base is N1, and
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`c) a RT polymerase that has non-templated activity, wherein the non-templated base is N2,
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`wherein N1 and N2 are different nucleic acid bases.
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`150.
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`(New) A method of sequencing different nucleic acids forms comprising:
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`a) providing a sample comprising different nucleic acid forms,
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`b) denaturing said nucleic acid forms in a sample,
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`c) ligating a first adapter to one end of a first nucleic acid form using a ligase that has a
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`preference for said first nucleic acid form, and ligating a second adapter to one end of a second nucleic acid
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`Application No. 15/952,203
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`form using a ligase that has preference for said second nucleic acid form, wherein said first and said second
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`adapter comprise different identifying sequences; and
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`d) sequencing said ligated nucleic acids, thereby identifying said different nucleic acid forms
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`in said sample.
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`151.
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`(New) A method for processing different nucleic acid forms in a sample comprising:
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`a) denaturing said different nucleic acid forms in a sample, wherein said different nucleic
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`acid forms comprise a first nucleic acid form and a second nucleic acid form,
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`b) attaching a first adapter to said first nucleic acid form and a second adapter to said second
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`nucleic acid form,
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`c) amplifying said first nucleic acid form using a DNA-dependent polymerase that has non-
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`templated activity, wherein said non-templated activity comprises adding at least one Nl nucleotide or a first
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`sequence to amplified products of said amplification of said first nucleic acid form, and
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`d) amplifying said second nucleic acid form using a reverse transciptase polymerase that has
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`non-templated activity, wherein said non-templated activity comprises adding at least one N2 nucloetide or a
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`second sequence to amplified products of said amplification of said second nucleic acid form, wherein said
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`Nl nucleotide and said N2 nucleotide are different nucleotides or said first sequence is different from said
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`second sequence.
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