WHAT IS CLAIMED IS:
`
`CLAIMS
`
`1.
`
`A method for performing a primer extension reaction on RNA and DNA;
`
`comprising:
`
`stranded RNA;
`
`a) providing a sample comprising a mixture of single-stranded DNA and single-
`
`b) attaching a first adapter to said single-stranded DNA;
`
`c) attaching a second adapter to said single-stranded RNA;
`
`d) annealing a first primer to said first adapter and annealing a second primer to said
`
`second adapter;
`
`e) extending said annealed first primer on said single-stranded DNA to form double-
`
`stranded DNA; and
`
`f) extending said annealed second primer on said single-stranded RNA to form a
`
`double-stranded DNA-RNA hybrid.
`
`2.
`
`The method of claim 1; wherein said attaching of said first adapter and said attaching
`
`of said second adapter occurs concurrently or within a single reaction mixture.
`
`3.
`
`The method of claim 1 or 2; wherein said attaching of said first adapter to said
`
`single-stranded DNA comprises performing a ligation reaction or a primer extension reaction.
`
`4.
`
`The method of any one of the preceding claims; wherein said attaching of said
`
`second adapter to said single-stranded RNA comprises performing a ligation reaction or a primer
`
`extension reaction.
`
`5.
`
`The method of claim 1; wherein said first adapter is ligated to a 3’ end of said single-
`
`stranded DNA or said second adapter is ligated to a 3’end of said single-stranded RNA.
`
`6.
`
`The method of any one of the preceding claims; wherein said extending said first
`
`primer on said single-stranded DNA to form double-stranded DNA occurs prior to said annealing
`
`of said second primer to said second adapter ligated to said 3’ end of said single-stranded RNA.
`
`7.
`
`The method of any one of the preceding claims ; wherein said extending said first
`
`primer on said single-stranded DNA to form double-stranded DNA occurs at a same time as said
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`extending said second primer on said single-stranded RNA to form a double-stranded DNA-RNA
`
`hybrid.
`
`8.
`
`The method of any one of the preceding claims, wherein said extending of said first
`
`primer is performed using a first polymerase that adds at least one first non-templated nucleotide to
`
`a 3’ end of a first primer extension strand, thereby generating a first overhang.
`
`9.
`
`The method of any one of the preceding claims, wherein said extending of said
`
`second primer is performed using a second polymerase that adds at least one second non-templated
`
`nucleotide to a 3’ end of a second primer extension strand, wherein said at least one second non-
`
`templated nucleotide is different from said at least one first non-templated nucleotide, thereby
`
`generating a second overhang.
`
`10.
`
`The method of any one of the preceding claims, further comprising hybridizing a
`
`third adapter to said first overhang and a fourth adapter to said second overhang.
`
`11.
`
`The method of any one of the preceding claims, further comprising sequencing said
`
`third and fourth adapters and sequences attached to said third and fourth adapters.
`
`12.
`
`The method of any one of the preceding claims, further comprising (i) identifying
`
`sequences associated with said third adapter as originating from said DNA in said initial mixture of
`
`single-stranded DNA and single-stranded RNA and (ii) identifying sequences associated with said
`
`fourth adapter as originating from said RNA in said initial mixture of single-stranded DNA and
`
`single-stranded RNA.
`
`13.
`
`A method of performing an amplification reaction on a first RNA and a first DNA,
`
`comprising:
`
`a) providing a sample comprising a mixture of a first DNA and a first RNA, wherein said
`
`first DNA does not comprise a sequence complementary to said first RNA,
`
`b) tagging said first DNA with a first tag without using a transposase,
`
`c) tagging said first RNA with a second tag,
`
`d) performing an amplification or primer extension reaction on said first DNA with a
`
`polymerase that is selective for DNA templates, and
`
`e) synthesizing a complementary cDNA strand from said first RNA with a reverse
`
`transcriptase.
`
`14.
`
`The method of claim 13, wherein said first and second tags are identical.
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`15.
`
`16.
`
`The method of claim 23, wherein said first and second tags are not identical.
`
`The method of claim 13, wherein a 3’ end of said first DNA is tagged with said first
`
`tag or wherein a 3’ end of said first RNA is tagged with said second tag.
`
`17.
`
`The method of any one of the preceding claims, wherein said first DNA is derived
`
`from a bacterium and said first RNA is derived from a virus.
`
`18.
`
`The method of any one of the preceding claims, further comprising sequencing said
`
`first DNA and said first RNA.
`
`19.
`
`A method of sequencing nucleic acids comprising:
`
`a) providing a sample comprising double-stranded nucleic acids and single-stranded
`
`nucleic acids,
`
`and
`
`b) attaching a first adapter to an end of said double-stranded nucleic acids,
`
`c) denaturing said double-stranded nucleic acids into single-stranded nucleic acids,
`
`d) sequencing said nucleic acids ligated to said first and second adapters and
`
`identifying sequences associated with said first adapter as being double-stranded.
`
`20.
`
`The method of any one of the preceding claims wherein said attaching of said first
`
`adapter to said double-stranded nucleic acids comprises performing a ligation reaction or a primer
`
`extension reaction.
`
`21.
`
`The method of any one of the preceding claims, wherein said attaching said first
`
`adapter comprises ligating said first adapter to a 3’ end of said double-stranded nucleic acids.
`
`22.
`
`The method of any one of the preceding claims, wherein the method further
`
`comprises ligating a second adapter to said denatured nucleic aicds, wherein said second adapter has
`
`a different sequence than said first adapter.
`
`23.
`
`The method of any one of the preceding claims, wherein the method further
`
`comprises identifying sequences associated with said second adapter as being single-stranded
`
`24.
`
`The method of any one of the preceding claims, wherein said double-stranded
`
`nucleic acids are DNA or RNA.
`
`25.
`
`The method of any one of the preceding claims, wherein said single-stranded nucleic
`
`acids are DNA or RNA.
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`26.
`
`The method of any one of the preceding claims, further comprising reducing
`
`concatemerization of short sequences.
`
`27.
`
`The method of any one of the preceding claims, wherein said DNA is single-stranded
`
`DNA, double-stranded DNA, triple-stranded DNA, or a Holliday junction.
`
`28.
`
`The method of any one of the preceding claims, wherein said RNA is single-stranded
`
`RNA, double-stranded RNA, or a ribozyme.
`
`29.
`
`30.
`
`The method of any one of the preceding claims, wherein said DNA is cell-free DNA.
`
`The method of any one of the preceding claims, wherein said sample is selected from
`
`the group consisting of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-alveolar
`
`lavage, urine, stool, saliva, nasal swab, and any combination thereof.
`
`3 l.
`
`The method of any one of the preceding claims, wherein said extending said primer
`
`on said single-stranded DNA is performed by a DNA polymerase.
`
`32.
`
`The method of any one of the preceding claims, wherein said extending said primer
`
`on said single-stranded DNA is performed by Bst 2.0 DNA polymerase.
`
`33.
`
`The method of any one of the preceding claims, wherein said extending said primer
`
`on said single-stranded RNA is performed by a polymerase selected from Moloney Murine
`
`Leukemia Virus (M-MLV) reverse transcriptase, and a SMARTer reverse transcriptase.
`
`34.
`
`The method of any one of the preceding claims, further comprising sequencing said
`
`amplified products.
`
`35.
`
`The method of any one of the preceding claims, wherein said ligating said first
`
`adapter is performed by a ligase selected from CircLigase II, Thermostable App-DNA/RNA ligase,
`
`T4 RNA ligase 1, T4 RNA Ligase 2 truncated, and any combination thereof.
`
`36.
`
`The method of any one of the preceding claims, wherein said ligating said second
`
`adapter is performed using a double-stranded RNA ligase.
`
`37.
`
`The method of any one of the preceding claims, wherein said ligating said second
`
`adapter is performed using T4 RNA ligase 2 or T4 DNA ligase.
`
`38.
`
`The method of any one of the preceding claims, further comprising adding at least
`
`one non-templated nucleotide to a primer extension strand.
`
`39.
`
`The method of any one of the preceding claims, wherein said at least one non-
`
`templated nucleotide is a deoxyadenosine.
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`40.
`
`The method of any one of the preceding claims, wherein said at least one non-
`
`templated nucleotide is one non-templated nucleotide.
`
`41.
`
`The method of any one of the preceding claims, wherein said third adapter ligated
`
`comprises an overhang containing at least one deoxythymidine.
`
`42.
`
`The method of any one of the preceding claims, further comprising adding at least
`
`one non-templated nucleotide to a primer extension strand of said double-stranded DNA-RNA
`
`hybrid.
`
`43.
`
`The method of any one of the preceding claims, wherein said at least one non-
`
`templated nucleotide is a deoxycytidine.
`
`44.
`
`The method of any one of the preceding claims wherein said at least one non-
`
`templated nucleotide is added to a 3’ end.
`
`45.
`
`The method of any one of the preceding claims, wherein said at least one non-
`
`templated nucleotide is up to eight nucleotides.
`
`46.
`
`The method of any one of the preceding claims wherein said at least one non-
`
`templated nucleotide is three, four, or five non-templated nucleotides.
`
`47.
`
`The method of any one of the preceding claims, wherein said fourth adapter contains
`
`an overhang comprising at least one deoxyguanosine residue.
`
`48.
`
`The method of any one of the preceding claims, wherein said overhang comprises at
`
`least three deoxyguanosine residues.
`
`49.
`
`The method of any one of the preceding claims 2, wherein said sequencing is
`
`performed by a next generation sequencing, a massively-parallel sequencing, a pyrosequencing, a
`
`sequencing-by-synthesis, a single molecule real-time sequencing, a polony sequencing, a DNA
`
`nanoball sequencing, a heliscope single molecule sequencing, a nanopore sequencing, a Sanger
`
`sequencing, a shotgun sequencing, or a Gilbert's sequencing assay.
`
`50.
`
`A method for concurrent processing of different nucleic acid forms in a sample
`
`comprising:
`
`(a) denaturing said nucleic acid forms in a sample,
`
`(b) ligating a first adapter to one end of a first nucleic acid form using a ligase that
`
`has a preference for said first nucleic acid form and ligating a second adapter to one end of a
`
`second nucleic acid form using a ligase that has preference for said second nucleic acid form,
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`(c) primer extending said first and second ligated nucleic acid forms,
`
`(d) ligating a third adapter comprising a priming element, and
`
`(e) amplifying said first and second nucleic forms.
`
`51.
`
`The method of any one of the preceding claims, wherein said ligating of said first
`
`adapter to one end of a first nucleic acids form occurs concurrently with said ligating of said
`
`second adapter to said second nucleic acid form.
`
`52.
`
`The method of any one of the preceding claims, wherein said first nucleic acid form
`
`is a DNA molecule and said second nucleic acid form is RNA a molecule.
`
`53.
`
`The method of any one of the preceding claims, wherein said first nucleic acid form
`
`is ssDNA and said second nucleic acid form is ssRNA.
`
`54.
`
`The method of any one of the preceding claims, wherein said polymerase comprises
`
`a DNA-dependent polymerase and a RT polymerase.
`
`55.
`
`The method of any one of the preceding claims, wherein said ligase is selected from
`
`a T4 DNA Ligase, a T3 DNA Ligase, a T7 DNA Li gase, a E. coli DNA Ligase, a HiFi Taq DNA
`
`Ligase, a 9°NTM DNA Ligase, a Taq DNA Ligase, a SplintR® Ligase, a Thermostable 5'
`
`AppDNA/RNA Ligase, a T4 RNA Ligase, a T4 RNA Ligase 2, a T4 RNA Ligase 2 Truncated, a T4
`
`RNA Ligase 2 Truncated K227Q, a T4 RNA Ligase 2, a Truncated KQ, a thB Ligase, a
`
`CircLigase II, a CircLigase ssDNA Ligase, a CircLigase RNA Ligase, a Ampligase® Thermostable
`
`DNA Ligase, and any combination thereof.
`
`56.
`
`The method of any one of the preceding claims, wherein said polymerase is selected
`
`from a Bst DNA Polymerase, a Full Length, a Bst DNA Polymerase, a Large Fragment, a Bsu DNA
`
`Polymerase, a Crimson Taq DNA Polymerase, a Large Fragment, Deep VentRTM, a DNA
`
`Polymerase, a Deep VentRTM (exo—), a DNA Polymerase, a E. coli DNA Polymerase I, a Klenow
`
`Fragment (3'—>5' exo-), a DNA Polymerase I, a Large (Klenow) Fragment, a LongAmp® Taq DNA
`
`Polymerase or Hot Start, a M-MuLV Reverse Transcriptase, a OneTaq® DNA Polymerase or Hot
`
`Start, a phi29 DNA Polymerase, a Phusion® Hot Start FleX DNA Polymerase, a Phusion® High-
`
`Fidelity DNA Polymerase, a Q5® + Q5® Hot Start DNA Polymerase, a Sulfolobus DNA
`
`Polymerase IV, a T4 DNA Polymerase, a T7 DNA Polymerase, a Taq DNA Polymerase, a
`
`TherrninatorTM DNA Polymerase, a VentR® DNA Polymerase, a VentR® (exo—) DNA
`
`Polymerase, and any combination thereof.
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`57.
`
`The method of any one of the preceding claims, wherein said RT polymerase
`
`selected from a WarmStart RTX Reverse Transcriptase, a AMV Reverse Transcriptase, a
`
`Superscript IV RT, a M-MLV Rnase H(—), a SMARTer reverse transcriptase, a RevertAid RnaseH(-
`
`) RT, a ProtoScript® II Reverse Transcriptase, and any combination thereof.
`
`58.
`
`59.
`
`The method of any one of the preceding claims, further comprising a detecting step.
`
`The method of any one of the preceding claims, wherein said detecting is performed
`
`by a real-time PCR, sequencing, a digital droplet PCR, or a microarray detection assay.
`
`60.
`
`The method of any one of the preceding claims, wherein said sequencing is a next
`
`generation sequencing, a massively-parallel sequencing, a pyrosequencing, a sequencing-by-
`
`synthesis, a single molecule real-time sequencing, a polony sequencing, a DNA nanoball
`
`sequencing, a heliscope single molecule sequencing, a nanopore sequencing, a Sanger sequencing, a
`
`shotgun sequencing, or a Gilbert's sequencing assay.
`
`61.
`
`A method for concurrent processing of different nucleic acid forms in a sample
`
`comprising:
`
`(a) denaturing said nucleic acid forms in a sample,
`
`(b) ligating a first adaptor to one end of a first nucleic acid form using a f1rstligase
`
`that has a preference for said first nucleic acid form and ligating a second adapter to one end of a
`
`second nucleic acid form using a second ligase that has a preference for said second nucleic acid
`
`form, wherein said first adapter and said second adapter comprises an identifying sequence that is
`
`different from each other, and
`
`(c) detecting said ligated nucleic acid forms.
`
`62.
`
`The method of claim 61, wherein said first nucleic acid form is a DNA molecule and
`
`said second nucleic acid form is RNA a molecule.
`
`63.
`
`The method of claim 61, wherein said first nucleic acid form is ssDNA and said
`
`second nucleic acid form is ssRNA.
`
`64.
`
`The method of claim 61, wherein said ligase is selected from T4 DNA Ligase, T3
`
`DNA Ligase, T7 DNA Li gase, E. coli DNA Ligase, HiFi Taq DNA Ligase, 9°NTM DNA Ligase, Taq
`
`DNA Ligase, SplintR® Ligase, Thermostable 5' AppDNA/RNA Ligase, T4 RNA Ligase, T4 RNA
`
`Ligase 2, T4 RNA Ligase 2 Truncated, T4 RNA Ligase 2 Truncated K227Q, T4 RNA Ligase 2,
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`Truncated KQ, thB Ligase, CircLigase II, CircLigase ssDNA Ligase, CircLigase RNA Ligase,
`
`Ampligase® Thermostable DNA Ligase, and any combination thereof.
`
`65.
`
`The method of claim 61, wherein said polymerase is selected from Bst DNA
`
`Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bsu DNA Polymerase, Crimson
`
`Taq DNA Polymerase, Large Fragment, Deep VentRTM, DNA Polymerase, Deep VentRTM (exo—),
`
`DNA Polymerase, E. coli DNA Polymerase I, Klenow Fragment (3'—>5' exo-), DNA Polymerase 1,
`
`Large (Klenow) Fragment, LongAmp® Taq DNA Polymerase or Hot Start, M-MuLV Reverse
`
`Transcriptase, OneTaq® DNA Polymerase or Hot Start, phi29 DNA Polymerase, Phusion® Hot
`
`Start Flex DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, Q5® + Q5® Hot Start
`
`DNA Polymerase, Sulfolobus DNA Polymerase IV, T4 DNA Polymerase, T7 DNA Polymerase,
`
`Taq DNA Polymerase, or TherminatorTM DNA Polymerase, VentR® DNA Polymerase, VentR®
`
`(exo—) DNA Polymerase, and any combination thereof.
`
`66.
`
`The method of claim 61, said RT polymerase is selected from WarmStart RTx
`
`Reverse Transcriptase, AMV Reverse Transcriptase, Superscript IV RT, M-MLV Rnase H(-),
`
`SMARTer reverse transcriptase, and RevertAid RnaseH(-) RT, and ProtoScript® II Reverse
`
`Transcriptase, and any combination thereof.
`
`67.
`
`The method of claim 61, wherein said detecting is by a real-time PCR, a sequencing,
`
`a digital droplet PCR, or a microarray assay.
`
`68.
`
`The method of claim 67, wherein said sequencing is a next generation sequencing, a
`
`massively-parallel sequencing, a pyrosequencing, a sequencing-by-synthesis, a single molecule
`
`real-time sequencing, a polony sequencing, a DNA nanoball sequencing, a heliscope single
`
`molecule sequencing, a nanopore sequencing, a Sanger sequencing, a shotgun sequencing, or a
`
`Gilbert's sequencing assay.
`
`69.
`
`A reaction mixture composition comprising:
`
`(a)
`
`(b)
`
`(c)
`
`(d)
`
`an adapter,
`
`a first ligase that has a preference for a first nucleic acid form,
`
`a second ligase that has a preference for a second nucleic acid form, and
`
`a buffer.
`
`70.
`
`The reaction mixture of any one of the preceding claims, further comprising a
`
`polymerase and/or a RT polymerase.
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`71.
`
`The reaction mixture of any one of the preceding claims, wherein the components
`
`are liquid, dry, or a combination thereof.
`
`72.
`
`The reaction mixture of any one of the preceding claims, wherein said ligase is
`
`selected from T4 DNA Ligase, T3 DNA Ligase, T7 DNA Ligase, E. coli DNA Ligase, HiFi Taq
`
`DNA Ligase, 9°NTM DNA Ligase, Taq DNA Ligase, SplintR® Ligase, Thermostable 5'
`
`AppDNA/RNA Ligase, T4 RNA Ligase, T4 RNA Ligase 2, T4 RNA Ligase 2 Truncated, T4 RNA
`
`Ligase 2 Truncated K227Q, T4 RNA Ligase 2, Truncated KQ, thB Ligase, CircLigase II,
`
`CircLigase ssDNA Ligase, CircLigase RNA Ligase, Ampligase® Thermostable DNA Ligase, and
`
`any combination thereof.
`
`73.
`
`The reaction mixture of any one of the preceding claims, wherein said polymerase is
`
`selected from Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bsu DNA
`
`Polymerase, Crimson Taq DNA Polymerase, Large Fragment, Deep VentRTM, DNA Polymerase,
`
`Deep VentRTM (exo—), DNA Polymerase, E. coli DNA Polymerase I, Klenow Fragment (3'—>5'
`
`exo-), DNA Polymerase 1, Large (Klenow) Fragment, LongAmp® Taq DNA Polymerase or Hot
`
`Start, M-MuLV Reverse Transcriptase, OneTaq® DNA Polymerase or Hot Start, phi29 DNA
`
`Polymerase, Phusion® Hot Start Flex DNA Polymerase, Phusion® High-Fidelity DNA Polymerase,
`
`Q5® + Q5® Hot Start DNA Polymerase, Sulfolobus DNA Polymerase IV, T4 DNA Polymerase,
`
`T7 DNA Polymerase, Taq DNA Polymerase, or TherminatorTM DNA Polymerase, VentR® DNA
`
`Polymerase, VentR® (exo—) DNA Polymerase, and any combination thereof.
`
`74.
`
`The reaction mixture of any one of the preceding claims, wherein said RT
`
`polymerase is selected from WarmStart RTx Reverse Transcriptase, AMV Reverse Transcriptase,
`
`Superscript IV RT, M-MLV Rnase H(-), SMARTer reverse transcriptase, and RevertAid RnaseH(-)
`
`RT, and ProtoScript® II Reverse Transcriptase, and any combination thereof.
`
`75.
`
`A reaction mixture comprising:
`
`(a) a ligase,
`
`(b) a DNA-dependent polymerase that has non-templated activity, wherein said non-
`
`templated base is N1, and
`
`(c) a RT polymerase that has non-templated activity, wherein said non-templated base
`
`is N2, wherein N1 and N2 are different nucleic acid bases.
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`76.
`
`The reaction mixture of any one of the preceding claims, wherein said DNA-
`
`dependent polymerase is selected from a A- and B-family DNA polymerases, a KOD XL, KOD
`
`(exo-), a Bst 2.0, a Therminator, a Deep Vent (exo-), a Pfu DNA polymerase, and aTaq.
`
`77.
`
`The reaction mixture of any one of the preceding claims, wherein said reverse
`
`transcriptase, is selected from HIV reverse transcriptase, Moloney murine leukemia Virus,
`
`SuperScript IITM (ThermoFisher), and SuperScript IIITM.
`
`78.
`
`A kit comprising:
`
`(a)
`
`(b)
`
`(c)
`
`(d)
`
`an adapter,
`
`a first ligase that has a preference for a first nucleic acid form,
`
`a second ligase that has a preference for a second nucleic acid form, and
`
`a buffer.
`
`79.
`
`80.
`
`The kit of any one of the preceding claims, further comprising instructions.
`
`The kit of any one of the preceding claims, further comprising components for
`
`detection, wherein said detecting is by a real-time PCR, a sequencing, a digital droplet PCR, or a
`
`microarray assay.
`
`81.
`
`The kit of any one of the preceding claims, wherein said sequencing is a next
`
`generation sequencing, a massively-parallel sequencing, a pyrosequencing, a sequencing-by-
`
`synthesis, a single molecule real-time sequencing, a polony sequencing, a DNA nanoball
`
`sequencing, a heliscope single molecule sequencing, a nanopore sequencing, a Sanger sequencing, a
`
`shotgun sequencing, or a Gilbert's sequencing assay.
`
`82.
`
`The kit of any one of the preceding claims, wherein said ligase is selected from T4
`
`DNA Ligase, T3 DNA Ligase, T7 DNA Ligase, E. coli DNA Ligase, HiFi Taq DNA Ligase, 9°NTM
`
`DNA Ligase, Taq DNA Ligase, SplintR® Ligase, Thermostable 5' AppDNA/RNA Ligase, T4 RNA
`
`Ligase, T4 RNA Ligase 2, T4 RNA Ligase 2 Truncated, T4 RNA Ligase 2 Truncated K227Q, T4
`
`RNA Ligase 2, Truncated KQ, thB Ligase, CircLigase II, CircLigase ssDNA Ligase, CircLigase
`
`RNA Ligase, Ampligase® Thermostable DNA Ligase, and any combination thereof.
`
`83.
`
`The kit of any one of the preceding claims, further comprising a said polymerase,
`
`wherein said polymerase is a DNA-dependent polymerase and/or an RT polymerase.
`
`84.
`
`The kit of any one of the preceding claims, wherein said polymerase is selected from
`
`Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bsu DNA Polymerase,
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`Crimson Taq DNA Polymerase, Large Fragment, Deep VentRTM, DNA Polymerase, Deep VentRTM
`
`(exo—), DNA Polymerase, E. coli DNA Polymerase I, Klenow Fragment (3'—>5' exo-), DNA
`
`Polymerase 1, Large (Klenow) Fragment, LongAmp® Taq DNA Polymerase or Hot Start, M-MuLV
`
`Reverse Transcriptase, OneTaq® DNA Polymerase or Hot Start, phi29 DNA Polymerase,
`
`Phusion® Hot Start FleX DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, Q5® +
`
`Q5® Hot Start DNA Polymerase, Sulfolobus DNA Polymerase IV, T4 DNA Polymerase, T7 DNA
`
`Polymerase, Taq DNA Polymerase, or TherminatorTM DNA Polymerase, VentR® DNA
`
`Polymerase, VentR® (exo—) DNA Polymerase, and any combination thereof.
`
`85.
`
`The kit of any one of the preceding claims, wherein said RT polymerase is selected
`
`from WarmStart RTX Reverse Transcriptase, AMV Reverse Transcriptase, Superscript IV RT, M-
`
`MLV Rnase H(—), SMARTer reverse transcriptase, and RevertAid RnaseH(-) RT, and ProtoScript®
`
`II Reverse Transcriptase, and any combination thereof.
`
`86.
`
`87.
`
`The kit of any one of the preceding claims, further comprising a control.
`
`The kit of any one of the preceding claims, wherein said instructions are directed to
`
`the concurrent detection of different nucleic acid forms in a sample.
`
`88.
`
`The kit of any one of the preceding claims, wherein said instructions are directed to
`
`the detecting a DNA and/or a RNA forms in a sample.
`
`89.
`
`The kit of any one of the preceding claims, wherein said instructions are directed to
`
`detecting different nucleic acid forms in a low-quality sample.
`
`90.
`
`The kit of any one of the preceding claims wherein said instructions are directed to
`
`detecting a disease or a condition in a subject.
`
`91.
`
`The kit of any one of the preceding claims, wherein said instructions are directed to
`
`detecting an infection in a subject.
`
`92.
`
`A kit comprising:
`
`(a) a ligase,
`
`(b) a DNA-dependent polymerase that has non-templated activity, wherein the non-
`
`templated base is N1, and
`
`(c) a RT polymerase that has non-templated activity, wherein the non-templated base
`
`is N2, wherein N1 and N2 are different nucleic acid bases.
`
`93.
`
`The kit of claim 92, further comprising instructions.
`
`- l 07-
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`WSGR Docket No. 47697-709201
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`94.
`
`The kit of any one of the preceding claims, wherein said DNA-dependent
`
`polymerase is selected from a A- and B-family DNA polymerases, a KOD XL, KOD (exo-), a Bst
`
`2.0, a Therminator, a Deep Vent (exo-), a Pfu DNA polymerase, and aTaq.
`
`95.
`
`The kit of any one of the preceding claims, said reverse transcriptase, is selected
`
`from HIV reverse transcriptase, Moloney murine leukemia virus, SuperScript 11““, and SuperScript
`
`IIITM.
`
`96.
`
`97.
`
`The kit of any one of the preceding claims, further comprising a control.
`
`The kit of any one of the preceding claims, wherein said instructions are directed to
`
`the concurrent detection of different nucleic acid forms in a sample.
`
`98.
`
`The kit of c any one of the preceding claims, wherein said instructions are directed to
`
`detecting a DNA and/or a RNA form in a sample.
`
`99.
`
`The kit of any one of the preceding claims, wherein said instructions are directed to
`
`detecting different nucleic acid forms in a low-quality sample.
`
`100.
`
`The kit of any one of the preceding claims, wherein said instructions are directed to
`
`detecting a disease or a condition in a subject.
`
`101.
`
`The kit of any one of the preceding claims, wherein said instructions are directed to
`
`detecting an infection in a subject.
`
`102.
`
`A method of sequencing different nucleic acids forms comprising:
`
`(a) providing a sample comprising different nucleic acid forms,
`
`(b) denaturing said nucleic acid forms in a sample,
`
`(c) ligating a first adapter to one end a first nucleic acid form using a ligase that has
`
`a preference of said first nucleic acid form, and ligating a second adapter to one end of a second
`
`nucleic acid form using a ligase that has preference of said second nucleic acid form, wherein said
`
`first and said second adapter comprise different identifying sequences, and
`
`(d) sequencing said ligated nucleic acids, thereby identifying said different nucleic
`
`acid forms in said sample.
`
`103.
`
`The sequencing method of any one of the preceding claims, further comprising
`
`amplification by a polymerase, wherein said polymerase is a DNA-dependent polymerase and/or an
`
`RT polymerase.
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`WSGR Docket No. 47697-709201
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`

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`104.
`
`The sequencing method of claim 102, wherein said ligase is selected from T4 DNA
`
`Ligase, T3 DNA Ligase, T7 DNA Ligase, E. coli DNA Ligase, HiFi Taq DNA Ligase, 9°NTM DNA
`
`Ligase, Taq DNA Ligase, SplintR® Ligase, Thermostable 5' AppDNA/RNA Ligase, T4 RNA
`
`Ligase, T4 RNA Ligase 2, T4 RNA Ligase 2 Truncated, T4 RNA Ligase 2 Truncated K227Q, T4
`
`RNA Ligase 2, Truncated KQ, thB Ligase, CircLigase II, CircLigase ssDNA Ligase, CircLigase
`
`RNA Ligase, Ampligase® Therrnostable DNA Ligase, and any combination thereof.
`
`105.
`
`The sequencing method of claim 102, wherein said polymerase is selected from
`
`Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bsu DNA Polymerase,
`
`Crimson Taq DNA Polymerase, Large Fragment, Deep VentRTM, DNA Polymerase, Deep VentRTM
`
`(exo—), DNA Polymerase, E. coli DNA Polymerase I, Klenow Fragment (3'—>5' exo-), DNA
`
`Polymerase 1, Large (Klenow) Fragment, LongAmp® Taq DNA Polymerase or Hot Start, M-MuLV
`
`Reverse Transcriptase, OneTaq® DNA Polymerase or Hot Start, phi29 DNA Polymerase,
`
`Phusion® Hot Start FleX DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, Q5® +
`
`Q5® Hot Start DNA Polymerase, Sulfolobus DNA Polymerase IV, T4 DNA Polymerase, T7 DNA
`
`Polymerase, Taq DNA Polymerase, or TherminatorTM DNA Polymerase, VentR® DNA
`
`Polymerase, VentR® (exo—) DNA Polymerase, and any combination thereof.
`
`106.
`
`The sequencing method of claim 102, wherein said RT polymerase is selected from
`
`WarmStart RTX Reverse Transcriptase, AMV Reverse Transcriptase, Superscript IV RT, M-MLV
`
`Rnase H(-), SMARTer reverse transcriptase, and RevertAid RnaseH(-) RT, and ProtoScript® II
`
`Reverse Transcriptase, and any combination thereof.
`
`107.
`
`The sequencing method of claim 102, wherein said sequencing is performed by a
`
`next generation sequencing, a massively-parallel sequencing, a pyrosequencing, a sequencing-by-
`
`synthesis, a single molecule real-time sequencing, a polony sequencing, a DNA nanoball
`
`sequencing, a heliscope single molecule sequencing, an nanopore sequencing, a Sanger sequencing,
`
`a shotgun sequencing, or a Gilbert's sequencing assay.
`
`108. A method for processing different nucleic acid forms in a sample comprising:
`
`(a) denaturing said different nucleic acid forms in a sample, wherein said different
`
`nucleic acid forms comprise a first nucleic acid form and a second nucleic acid form,
`
`(b) attaching a first adapter to said first nucleic acid form and a second adapter to said
`
`second nucleic acid form,
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`- l 09-
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`WSGR Docket No. 47697-709201
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`(c) amplifying said first nucleic acid form using a DNA-dependent polymerase that
`
`has non-templated activity, wherein said non-templated activity comprises adding at least
`
`one Nl nucleotide or a first sequence to amplified products of said amplification of said first
`
`nucleic acid form, and
`
`(d) amplifying said second nucleic acid form using a reverse transciptase polymerase
`
`that has non-templated activity, wherein said non-templated activity comprises adding at
`
`least one N2 nucloetide or a second sequence to amplified products of said amplification of
`
`said second nucleic acid form, wherein said Nl nucleotide and said N2 nucleotide are
`
`different nucleotides or said first sequence is different from said second sequence.
`
`109.
`
`The method of any one of the preceding claims, wherein said first nucleic acid form
`
`is a DNA molecule and said second nucleic acid form is RNA a molecule.
`
`110.
`
`The method of any one of the preceding claims, wherein said first nucleic acid form
`
`is ssDNA and said second nucleic acid form is ssRNA.
`
`111.
`
`The method of any one of the preceding claims, wherein said DNA-dependent
`
`polymerase is selected from A- and B-family DNA polymerases, KOD XL, KOD (exo-), Bst 2.0,
`
`Therminator, Deep Vent (exo-), Pfu DNA polymerase, and Taq.
`
`112.
`
`The method of any one of the preceding claims,wherein said reverse transcriptase, is
`
`selected from HIV reverse transcriptase, Moloney murine leukemia virus, SuperScript 11““, and
`
`SuperScript IIITM.
`
`113.
`
`The method of any one of the preceding claims, further comprising distinguishing
`
`said first nucleic acid form from said second nucleic acid form based on said non-templated activity
`
`of said reverse transcriptase or based on said non-templated activity of said DNA-dependent
`
`polymerase.
`
`114.
`
`The method of any one of the preceding claims, further comprising distinguishing
`
`said first nucleic acid form from said second nucleic acid form based on said Nl or N2 nucleotides
`
`or said first or second sequences.
`
`-1 lO-
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`WSGR Docket No. 47697-709201
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`