WSGR Docket No. 47697-709201
`
`PATENT APPLICATION
`
`SAMPLE PREPARATION METHODS, SYSTEMS AND COMPOSITIONS
`
`Inventor(s):
`
`Timothy A. BLAUWKAMP,
`Citizen of United States, residing at
`4020 Sutherland Dr
`
`Palo Alto, CA 94303
`
`Rene SIT,
`Citizen of United States, Residing at
`1085 Reed Avenue, Unit C
`Sunnyvale, CA 94086
`
`Igor D. VILFAN,
`Citizen of Slovenia, Residing at
`1677 Woodland Ave
`
`East Palo Alto, CA 943 03
`
`Assignee:
`
`Karius, Inc.
`975 Island Drive, Ste. 101
`Redwood City, CA 94065
`
`a Delaware Corporation
`
`Entity:
`
`Large business concern
`
`xii/12R
`
`Wilson Sunsini Goodrich ES? Rosai‘i
`PR C- ,t‘ I.- \- \.
`\7 If :3 i.
`i} C) R. 1’ if} R. .KIE O N
`
`650 Page Mill Road
`Palo Alto, CA 94304
`(650) 493—9300 (Main)
`(650) 493-6811 (Facsimile)
`
`Filed Electronically on: April 12, 2018
`
`

`

`SAMPLE PREPARATION METHODS, SYSTEMS AND COMPOSITIONS
`
`CROSS-REFERENCE
`
`[0001] This application claims the benefit of US. Provisional Application No. 62/484,856, filed
`
`April 12, 2017 which is incorporated herein by reference in its entirety.
`
`BACKGROUND
`
`[0002] The analysis of genetic material in a sample has numerous potential uses and applications,
`
`including the identification of genetic indicators of disease (e.g., cancer), infection, disease
`
`progression, and fetal health. Advances in high-throughput sequencing technologies and PCR—
`
`based approaches have permitted more accurate identification of such genetic material. Before
`
`these approaches can be used, usually a starting sample is processed in some manner. For example,
`
`nucleic acids may be extracted or purified from the sample. The nucleic acids may then be tagged in
`
`some manner. Tagging may aid the detection of the sequence of the nucleic acids in a downstream
`
`application, such as by making the nucleic acid compatible for use in a particular type of sequencer.
`
`SUMMARY
`
`[0003] The application of current technologiesfor genetic analysis is often impeded by inefficient
`
`sample processing techniques. Also, most nucleic acid sample preparation methods have limited
`
`uses in that they only can detect one nucleic acid form at a time. For example, most sample
`
`preparation methods require that a sample be divided so that RNA and DNA can be processed in
`
`parallel. Samples containing low quantities of nucleic acids, or low quality nucleic acids, may thus
`
`not have sufficient material to permit detection of both RNA and DNA, resulting in the possible loss
`
`of valuable information about the sample.
`
`[0004] The present disclosure overcomes these challenges and others. Many of the methods,
`
`compositions, systems, and kits provided herein enables the concurrent processing and detection of
`
`multiple different types of nucleic acids, generally without the need of physically separating or
`
`dividing a sample. Such concurrent analysis of different nucleic acid forms in a sample permits
`
`more efficient detection of genetic material, and for more accurate and useful genetic analyses.
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`[0005] Provided herein are methods, systems, processes, kits, and reagent compositions useful for
`
`carrying out sample preparation processes for the analysis of different forms of nucleic acids in a
`
`sample. The methods include methods of processing nucleic acids of multiple forms (e.g., single-
`
`stranded DNA, double-stranded DNA, single-stranded RNA, and/or double-stranded RNA) within
`
`samples to identify the nucleic acids present within the sample. The methods, systems, processes,
`
`kits, and reagent compositions provided herein can often be practiced or used in a single reaction
`
`mixture, without the need to separate or divide a sample into different portions.
`
`[0006] In some embodiments, the methods can be applied to samples that comprise both DNA and
`
`RNA fragments of interest, and result in the analysis of both of those nucleic acid forms from a
`
`single reaction mixture. Further, these methods may identify fragments in accordance with their
`
`originating form in the sample, e.g., as DNA or RNA and/or as single-stranded or double-stranded,
`
`such that downstream analysis may yield both sequence identification and identification of the
`
`chemical and/or structural form of the original nucleic acid in the sample.
`
`[0007] In one aspect, provided herein is a method of performing a primer extension reaction on
`
`RNA and DNA, comprising: (a) providing a sample comprising a mixture of single-stranded DNA
`
`and single-stranded RNA, (b) attaching a first adapter to the single-stranded DNA, (c) attaching a
`
`second adapter to the single-stranded RNA, (d) annealing a first primer to the first adapter and
`
`annealing a second primer to the second adapter, (e) extending the annealed first primer on the
`
`single-stranded DNA to form double-stranded DNA, and/or (f) extending the annealed second
`
`primer on the single-stranded RNA to form a double-stranded DNA-RNA hybrid. In some cases,
`
`the attaching the first adapter to the single-stranded DNA comprises ligating the first adapter to the
`
`single-stranded DNA. In some cases, the attaching of the first adapter to the single-stranded DNA
`
`comprises performing a primer extension reaction. In some cases, the attaching the first adapter to
`
`the single-stranded rNA comprises ligating the first adapter to the single-stranded rNA. In some
`
`cases, the attaching of the first adapter to the single-stranded RNA comprises performing a primer
`
`extension reaction.
`
`[0008] In some cases, the first adapter is ligated or attached to the 3’ end of the single-stranded
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`DNA. In some cases, the second adapter is ligated or attached to the 3’ end of the single-stranded
`
`RNA. In some cases, the ligating or attaching of said first adapter and said second adapter occurs
`
`concurrently or within a single reaction mixture. In some cases, extending the first primer on the
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`single-stranded DNA to form double-stranded DNA occurs prior to the annealing of the second
`
`primer to the second adapter ligated to the end (e.g., 3’ end) of the single-stranded RNA. In some
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`cases, the extending the first primer on the single-stranded DNA to form double-stranded DNA
`
`occurs at the same time as the extending the second primer on the single-stranded RNA to form a
`
`double-stranded DNA-RNA hybrid. In some cases, the first adapter and the second adapter have
`
`different sequences. In some cases, the first adapter and the second adapter have the same sequence.
`
`In some cases, the first primer and the second primer have different sequences. In some cases, the
`
`first primer and the second primer have the same sequence. In some cases, the extending of the first
`
`primer can be performed using a first polymerase that adds at least one first non-templated
`
`nucleotide to an end (e.g,. 3’ end) of a first primer extension strand, thereby generating a first
`
`overhang. In some cases, the extending of the second primer is performed using a second
`
`polymerase that adds at least one second non-templated nucleotide to an end (e. g, 3’ end) of a
`
`second primer extension strand, wherein the at least one second non-templated nucleotide is
`
`different from the at least one first non-templated nucleotide, thereby generating a second overhang.
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`[0009] In some cases, the methods further comprise hybridizing a third adapter to the first overhang
`
`and a fourth adapter to the second overhang. In some cases, the method further comprises
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`sequencing the third and fourth adapters and sequences attached to the third and fourth adapters. In
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`some cases, the method further comprises (i) identifying sequences associated with the third adapter
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`as originating from the DNA in the initial mixture of single-stranded DNA and single-stranded
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`RNA and (ii) identifying sequences associated with the fourth adapter as originating from the RNA
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`in the initial mixture of single-stranded DNA and single-stranded RNA.
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`[0010] In one aspect, provide herein is a method of performing an amplification reaction on a first
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`RNA and a first DNA, comprising: (a) providing a sample comprising a mixture of a first DNA and
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`a first RNA, wherein the first DNA does not comprise a sequence complementary to the first RNA,
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`(b) tagging an end (e. g, 3’ end) of the first DNA with a first tag without using a transposase, (c)
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`tagging a an end (e.g.,. a 3’ end) of the first RNA such that the first RNA comprises a tag that is
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`identical to the first tag or is not identical to the first tag, (d) performing an amplification or primer
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`extension reaction on the first DNA with a polymerase that is selective for DNA templates, and (e)
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`synthesizing a complementary cDNA strand from the first RNA with a reverse transcriptase. In
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`some cases, the first DNA is derived from a bacterium and the first RNA is derived from a virus. In
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`some cases, the method further comprises sequencing the first DNA and the first RNA.
`
`[0011] In one aspect, provide herein is a method of sequencing nucleic acids, comprising: (a)
`
`providing a sample comprising a mixture of double-stranded nucleic acids and single-stranded
`
`nucleic acids, (b) attaching (e.g., by ligation or primer extension reaction) the first adapter to the
`
`double-stranded nucleic acids (e. g, at the 3’ end of the double-stranded nucleic acids), (c)
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`denaturing the double-stranded nucleic acids into single-stranded nucleic acids, (d) ligating a second
`
`adapter to the denatured nucleic acids of step c, wherein the second adapter has a different sequence
`
`than the first adapter or has a sequence that is identical to that of the first adapter, and/or (e)
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`sequencing the nucleic acids ligated to the first and second adapters and/or identifying sequences
`
`associated with the first adapter as being double-stranded and/or sequences associated with the
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`second adapter as being single-stranded.
`
`[0012] In some cases, the double-stranded nucleic acids are DNA. In some cases, the double-
`
`stranded nucleic acids are RNA. In some cases, the single-stranded nucleic acids are RNA. In some
`
`cases, the single-stranded nucleic acids are DNA. In some cases, the method further comprises
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`reducing concatemerization of short sequences. In some cases, the DNA is single-stranded DNA,
`
`double-stranded DNA, triple-stranded DNA, or a Holliday junction. In some cases, the RNA is
`
`single-stranded RNA, double-stranded RNA, or a ribozyme. In some cases, the DNA is cell-free
`
`DNA. In some cases, the RNA is cell-free RNA. In some cases, the sample is selected from the
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`group consisting of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-alveolar
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`lavage, urine, stool, saliva, nasal swab, and any combination thereof.
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`[0013] In some cases, extending the primer on the single-stranded DNA can be performed by a
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`DNA polymerase. In some cases, the extending the primer on the single-stranded DNA is
`
`performed by Bst 2.0 DNA polymerase. In some cases, the extending the primer on the single-
`
`stranded RNA can be performed by a polymerase selected from Moloney Murine Leukemia Virus
`
`(M-MLV) reverse transcriptase, and a SMARTer reverse transcriptase.
`
`[0014] In some cases, a method described herein further comprises sequencing the amplified
`
`products.
`
`[0015] In some cases, the ligating the first adapter is performed by a ligase selected from
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`CircLigase II, Thermostable App-DNA/RNA ligase, T4 RNA ligase 1, T4 RNA Ligase 2 truncated,
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`and any combination thereof. In some cases, the ligating the second adapter is performed using a
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`double-stranded RNA ligase. In some cases, the ligating the second adapter is performed using T4
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`RNA ligase 2 or T4 DNA ligase.
`
`[0016] In some cases, a method described herein further comprises adding at least one non-
`
`templated nucleotide to a primer extension strand. In some cases, the at least one non-templated
`
`nucleotide is a deoxyadenosine. In some cases, the at least one non-templated nucleotide is one non-
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`templated nucleotide. In some cases, the third adapter ligated comprises an overhang containing at
`
`least one deoxythymidine. In some cases, the method further comprises adding at least one non-
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`templated nucleotide to a primer extension strand of the double-stranded DNA-RNA hybrid. In
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`some cases, the at least one non-templated nucleotide is a deoxycytidine. In some cases, the at least
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`one non-templated nucleotide is added to a 3’ end. In some cases, the at least one non-templated
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`nucleotide is up to eight nucleotides. In some cases, the at least one non-templated nucleotide is
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`three, four, or five non-templated nucleotides. In some cases, the fourth adapter contains an
`
`overhang comprising at least one deoxyguanosine residue. In some cases, the overhang comprises at
`
`least three deoxyguanosine residues.
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`[0017] In one aspect, provide herein is 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 the first DNA is derived from a bacterium and the first RNA is derived from a
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`virus, (b) amplifying the first RNA with a reverse transcriptase that selectively amplifies RNA, and
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`(c) amplifying the first DNA with a polymerase that selectively amplifies DNA.
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`[0018] In one aspect, provided herein is a method of performing an amplification reaction on a first
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`RNA and a first DNA, comprising: (a) providing a sample comprising a mixture of a first DNA and
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`a first RNA, wherein the first DNA is genomic DNA derived from a first organism and the first
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`RNA is genomic RNA derived from a second organism, (b) amplifying the first RNA with a reverse
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`transcriptase that selectively amplifies RNA, and (c) amplifying the first DNA with a polymerase
`
`that selectively amplifies DNA. In some cases, the first organism can be a bacterium and the second
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`organism can be a virus.
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`[0019] Provided herein are methods for concurrent processing of different nucleic acid forms in a
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`sample. The method can comprise (a) denaturing the nucleic acid forms in a sample, (b) ligating a
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`first adapter to one end a first nucleic acid form using a ligase that has a preference for a first
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`nucleic acid form and ligating a second adapter to one end of a second nucleic acid form using a
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`ligase that has preference for a second nucleic acid form,
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`(c) primer extending a first and second
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`ligated nucleic acid forms,
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`(d) ligating a third adapter comprising a priming element, and (e)
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`amplifying. In some cases, the ligating of the first adapter to the first nucleic acidf form occurs
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`concurrently with the ligating of the second adapter to the second nucleic acid form, or in the same
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`reaction mixture. In a method disclosed herein, a first nucleic acid form can be a DNA molecule and
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`a second nucleic acid form can be RNA a molecule. In other cases, a first nucleic acid form can be
`
`ssDNA and a second nucleic acid form can be ssRNA. A polymerase can comprise a DNA-
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`dependent polymerase and a RT polymerase. The polymerase can be selected from a Bst DNA
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`Polymerase, a Full Length, a Bst DNA Polymerase, a Large Fragment, a Bsu DNA Polymerase, a
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`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
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`Start, a M-MuLV Reverse Transcriptase, a OneTaq® DNA Polymerase or Hot Start, a phi29 DNA
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`Polymerase, a Phusion® Hot Start FleX DNA Polymerase, a Phusion® High-Fidelity DNA
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`Polymerase, a Q5® + Q5® Hot Start DNA Polymerase, a Sulfolobus DNA Polymerase IV, a T4
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`DNA Polymerase, a T7 DNA Polymerase, a Taq DNA Polymerase, a TherrninatorTM DNA
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`Polymerase, a VentR® DNA Polymerase, a VentR® (exo—) DNA Polymerase, and any
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`combination thereof. In some cases, a RT polymerase can be selected from a WarmStart RTX
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`Reverse Transcriptase, a AMV Reverse Transcriptase, a Superscript IV RT, a M-MLV Rnase H(-),
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`a SMARTer reverse transcriptase, a RevertAid RnaseH(-) RT, a ProtoScript® II Reverse
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`Transcriptase, and any combination thereof. Whereas a ligase can be selected from a T4 DNA
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`Ligase, a T3 DNA Ligase, a T7 DNA Ligase, a E. coli DNA Ligase, a HiFi Taq DNA Ligase, a
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`9°NTM DNA Ligase, a Taq DNA Ligase, a SplintR® Ligase, a Thermostable 5' AppDNA/RNA
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`Ligase, a T4 RNA Ligase, a T4 RNA Ligase 2, a T4 RNA Ligase 2 Truncated, a T4 RNA Ligase 2
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`Truncated K227Q, a T4 RNA Ligase 2, a Truncated KQ, a thB Ligase, a CircLigase II, a
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`CircLigase ssDNA Ligase, a CircLigase RNA Ligase, a Ampligase® Thermostable DNA Ligase,
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`and any combination thereof. The method described herein can further comprise a detecting step,
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`wherein the detecting can be performed by a real-time PCR, sequencing, a digital droplet PCR, or a
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`microarray detection assay. Sequencing can comprise a next generation sequencing, a massively-
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`parallel sequencing, a pyrosequencing, a sequencing-by-synthesis, a single molecule real-time
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`sequencing, a polony sequencing, a DNA nanoball sequencing, a heliscope single molecule
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`sequencing, a nanopore sequencing, a Sanger sequencing, a shotgun sequencing, or a Gilbert's
`
`sequencing assay.
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`[0020] Provided herein are methods for concurrent processing of different nucleic acid forms in a
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`sample. In some cases, the method can comprise: (a) denaturing a nucleic acid forms in a sample;
`
`(b) ligating a first adaptor to one end of a first nucleic acid form using a first ligase that has a
`
`preference for the first nucleic acid form and ligating a second adapter to one end of a second
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`nucleic acid form using a second ligase that has a preference for the second nucleic acid form,
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`wherein the first adapter and the second adapter comprise an identifying sequence that is different
`
`from each other; and (c) detecting the ligated nucleic acid forms.
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`[0021] Further provided are reaction mixtures comprising: 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
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`acid form, and a buffer. The reaction mixture can further comprise a polymerase and/or a RT
`
`polymerase described herein. In some cases, components of the reaction mixtures can be liquid, dry,
`
`or a combination thereof.
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`[0022] In other reaction mixtures provided herein, the reaction mixture can comprise: a ligase, a
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`DNA-dependent polymerase that has non-templated activity, wherein the non-templated base can be
`
`N1, and a RT polymerase that has non-templated activity, wherein the non-templated base can be
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`N2, wherein N1 and N2 can be different nucleic acid bases. In one instance, the DNA-dependent
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`polymerase can be selected from an A- and B-family DNA polymerases, a KOD XL, KOD (exo-), a
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`Bst 2.0, a Therminator, a Deep Vent (exo-), a Pfu DNA polymerase, and aTaq. In some cases, a
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`reverse transcriptase used in the mixture can be selected from HIV reverse transcriptase, Moloney
`
`murine leukemia virus, SuperScript IITM (ThermoFisher), and SuperScript 111““.
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`[0023] Provided herein are kits comprising: an adapter, a first ligase that has a preference for a first
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`nucleic acid form, a second ligase that has a preference for a second nucleic acid form, and a buffer.
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`In some cases, a kit can further comprise instructions for use. A kit provided herein can comprise: a
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`ligase, a DNA-dependent polymerase that has non-templated activity, wherein the non-templated
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`base is N1, and a RT polymerase that has non-templated activity, wherein the non-templated base
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`can be N2, wherein N1 and N2 can be different nucleic acid bases. Kits provided herein can further
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`comprise instructions for use. The DNA-dependent polymerase of a kit described herein can be
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`selected from a A- and B-family DNA polymerases, a KOD XL, KOD (exo-), a Bst 2.0, a
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`Therminator, a Deep Vent (exo-), a Pfu DNA polymerase, and aTaq. Whereas a reverse
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`transcriptase can be selected from HIV reverse transcriptase, Moloney murine leukemia virus,
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`SuperScript 11““, and SuperScript 111““. A kit provided herein may further comprise a control.
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`[0024] Provide herein are methods of sequencing for different nucleic acids forms. A method of
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`sequencing can comprise: providing a sample comprising different nucleic acid forms, denaturing
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`the nucleic acid forms in a sample, ligating a first adapter to one end of a first nucleic acid form
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`using a ligase that has a preference of the first nucleic acid form, and ligating a second adapter to
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`one end of a second nucleic acid form using a ligase that has preference of the second nucleic acid
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`form, wherein the first and the second adapter comprise different identifying sequences, and
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`sequencing the ligated nucleic acids, thereby identifying the different nucleic acid forms in the
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`sample. The method can further comprise amplification by a polymerase, wherein the polymerase
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`can be a DNA-dependent polymerase and/or an RT polymerase. In some cases, the sequencing
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`described herein can be by a next generation sequencing, a massively-parallel sequencing, a
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`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
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`sequencing, a Sanger sequencing, a shotgun sequencing, or a Gilbert's sequencing assay.
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`[0025] Also provided herein are methods for concurrent processing different nucleic acid forms in a
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`sample. These methods can comprise: denaturing the nucleic acid forms in a sample, ligating a first
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`adapter to one end a first nucleic acid form and a second nucleic acid form using a ligase,
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`amplifying using a DNA-dependent polymerase that has non-templated activity, wherein the non-
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`templated base can be N1, and amplifying using a RT polymerase that has non-templated activity,
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`wherein the non-templated base can be N2, wherein N1 and N2 can be different nucleic acid bases.
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`In some cases, a first nucleic acid form or a second nucleic acid form can be DNA, ssDNA, RNA or
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`ssRNA. In some cases the DNA-dependent polymerase can be selected from A- and B-family DNA
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`polymerases, KOD XL, KOD (exo-), Bst 2.0, Therminator, Deep Vent (exo-), Pfu DNA
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`polymerase, and Taq. Whereas, a reverse transcriptase can be selected from HIV reverse
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`transcriptase, Moloney murine leukemia virus, SuperScript IITM, and SuperScript IIITM.
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`[0026] Also provided herein are a method for processing different nucleic acid forms in a sample
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`comprising:
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`(a) denaturing said different nucleic acid forms in a sample, wherein said different
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`nucleic acid forms comprise a first nucleic acid form and a second nucleic acid form, (b) attaching
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`a first adapter to said first nucleic acid form and a second adapter to said second 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
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`or a first sequence to amplified products of said amplification of said first nucleic acid form, and (d)
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`amplifying said second nucleic acid form using a reverse transciptase polymerase that has non-
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`templated activity, wherein said non-templated activity comprises adding at least one N2 nucloetide
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`or a second sequence to amplified products of said amplification of said second nucleic acid form,
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`wherein said Nl nucleotide and said N2 nucleotide are different nucleotides or said first sequence is
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`different from said second sequence.
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`[0027] In some cases, said first nucleic acid form is a DNA molecule or said second nucleic acid
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`form is RNA a molecule. In some cases, said first nucleic acid form is ssDNA and said second
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`nucleic acid form is ssRNA. In some cases, 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. In some cases, said reverse transcriptase, is selected from HIV
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`reverse transcriptase, Moloney murine leukemia virus, SuperScript II , and SuperScript III . In some
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`cases, the method further comprises distinguishing said first nucleic acid form from said second
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`nucleic acid form based on said non-templated activity of said reverse transcriptase or based on said
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`non-templated activity of said DNA-dependent polymerase. In some cases, the method further
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`comprises distinguishing said first nucleic acid form from said second nucleic acid form based on
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`said Nl or N2 nucleotides or said first or second sequences. In some cases, said attaching of said
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`first adapter or of said second adapter comprises performing a ligation reaction or primer extenstion
`
`reaction. In some cases, the attaching occurs at the 3’ end of the first nucleic acid form or of the
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`3’end of the second nucleic acid form.
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`INCORPORATION BY REFERENCE
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`[0028] All publications, patents, and patent applications mentioned in this specification are herein
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`incorporated by reference in their entireties to the same extent as if each individual publication,
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`patent, or patent application was specifically and individually indicated to be incorporated by
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`reference.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0029] The novel features of the invention are set forth with particularity in the appended claims. A
`
`better understanding of the features and advantages of the present invention will be obtained by
`
`reference to the following detailed description that sets forth illustrative embodiments, in which the
`
`principles of the invention are utilized, and the accompanying drawings of which:
`
`[0030] FIG. 1 shows exemplary approaches for processing DNA and RNA in a sample by adding
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`adapters to single-stranded nucleic acids.
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`[0031] FIG. 2 depicts exemplary techniques to detect various nucleic acid forms in a sample using
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`polymerases with non-template activity.
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`[0032] FIG. 3 depicts
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`ligation/primer extension approaches using polymerases having non-
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`templated activity to detect various nucleic acid forms in a sample.
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`[0033] FIG. 4 depicts exemplary approaches to detect various nucleic acid forms in a sample,
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`including approaches using a second adapter that contains both double-stranded and single-stranded
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`regions.
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`[0034] FIG. 5 depicts exemplary non-templated approaches to detect various nucleic acid forms in
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`a sample, including an approach using a strand-displacing polymerase.
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`[0035] FIG. 6 depicts a approaches for detecting cell-free nucleic acids, or other low-quality forms,
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`in a sample.
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`[0036] FIG. 7 depicts exemplary primer extension-non-templated approaches using a successive
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`mode.
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`[0037] FIG. 8 shows exemplary primer extension-non-templated approaches using a concurrent
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`mode.
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`[0038] FIG. 9 exemplary approaches for distinguishing different structural forms of the nucleic
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`acids in a sample.
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`[0039] FIG. 10 shows an electrophoric gel illustrating the efficiency of different DNA and RNA
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`ligases in a single reaction mixture provided by the disclosure. Lane A1 of the gel shows the
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`molecular ladder (L), Lanes B2 and C2 is the product produced using a CircLigase II. Lanes D2 and
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`E2 is the product produced using a thermostable App-DNA/RNA ligase. Lanes F2 and G2 is the
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`product produced using a T4 RNA ligase 1.
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`[0040] FIG. 11A and FIG. 11B show bar graphs comparing the recovery of the input DNA and
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`RNA of the starting sample with the final output DNA and RNA detected after conducting the
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`methods of the disclosure. 11A shows recovery of DNA and RNA product with a SMARTer
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`Reverse Transcriptase. 11B shows recovery of the DNA and RNA product with a Bst 2.0
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`Polymerase.
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`[0041] FIG. 12 shows an electrophoric gel illustrating nucleic acid products detected using the
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`methods of the disclosure.
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`[0042] FIG. 13 depicts a primer extension reaction using various reverse transcriptase enzymes.
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`[0043] FIG. 14 depicts a bar graph comparing the performance of an embodiment of the ligation
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`method with a commercial kit by NuGEN. The white bars indicate the number of nucleic acid
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`products detected by the ligation method. The hatched bars indicate the number of nucleic acid
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`products detected NuGEN method. The X-aXis shows the name of the selected pathogens for the
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`study.
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`[0044] FIG. 15 depicts a bar graph comparing the performance of an embodiment of the ligation
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`method with a commercial kit by NuGEN. The white bars indicate the number of nucleic acid
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`products detected by the ligation method. The hatched bars indicate the number of nucleic acid
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`products detected NuGEN method. The X-aXis shows the name of the selected pathogens for the
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`study.
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`[0045] FIG. 16 depicts a plot of the quantity versus fragment length for both human chr21 and
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`pathogen cell-free DNA detected using the methods provided herein.
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`[0046] FIG. 17 illustrates the activity of polymerases having non-template activity. The non-
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`templated nucleotides are indicated by “NNNNN”, where N could be any nucleotide and any
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`number of Ns can be used. In this illustration, the non-templated nucleotides are added to the 3’ end
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`of the nascent growing strand.
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`[0047] FIG. 18 depicts the non-template activity of a polymerase. The y-aXis shows the number of
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`reads detected and the X-aXis shows the number of non-templated bases added at the 3’end by the
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`polymerase.
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`[0048] FIG. 19 depicts a computer control system that is programmed or otherwise configured to
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`implement the methods and systems provided herein.
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`[0049] FIG. 20 depicts
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`splint ligase approaches to detect various nucleic acid forms in a sample.
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`DETAILED DESCRIPTION
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`[0050] The following passages describe different aspects of the invention in greater detail. Each
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`aspect, embodiment, or feature of the invention may be combined with any other aspect,
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`embodiment, or feature the invention unless clearly indicated to the contrary.
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`I.
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`Definitions
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`[0051] Unless defined otherwise, all technical and scientific terms used herein have the meaning
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`commonly understood by a person skilled in the art to which this invention belongs.
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`[0052] “ Detect,” as used herein can refer to quantitative or qualitative detection, including,
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`without limitation, detection by identifying the presence, absence, quantity, frequency,
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`concentration, sequence, form, structure, origin, or amount of an analyte.
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`[0053] “Nucleic acid” as used herein, can refer to a polymer of nucleotides and is generally
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`synonymous with the term “polynucleotide.” The nucleotides may comprise a deoxyribonucleotide,
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`a ribonucleotide, a deoxyribonucleotide analog, ribonucleotide analog, or any combination thereof.
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`The term “nucleic acid” may also include nucleic acids with modified backbones. Nucleic acid can
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`be of any length. Nucleic acid may perform any function, known or unknown. The following are
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`non-limiting examples of nucleic acids: coding or non-coding regions of a gene or gene fragment,
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`loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA
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`(tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA),
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`micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,
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`plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid
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`probes, primers, mitochondrial DNA, cell-free nucleic acids, viral nucleic acid, bacterial nucleic
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`acid, and genomic DNA. A nucleic acid may comprise one or more modified nucleotides, such as
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`methylated nucleotides or methylated nucleotide analogs. If present, modifications to the nucleotide
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`structure may be imparted before or after assembly of the polymer. The sequence of nucleotides
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`may be interrupted by non-nucleotide components. A nucleic acid may be further modified after
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`polymerization, such as by conjugation with a labeling component. A nucleic acid may be single-
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`stranded, double-stranded or have higher numbers strands (e.g., triple-stranded).
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`[0054] “A”, “an”, and “the”, as used herein, can include plural referents unless expressly and
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`unequivocally limited to one referent.
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`[0062] As used herein, the term “or” is used to refer to a nonexclusive “or”, as such, “A or B”
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`includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.
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`[0055] “Identifying sequence element” can refer to an index, a code, a barcode, a random sequence,
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`an adaptor, an overhang of non-templated nucleic acids, a tag comprising one or more non-
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`templated nucleotides