Method of processing nucleic acid sample

Abstract

The present disclosure provides methods and systems for amplifying and analyzing nucleic acid samples. The present disclosure provides methods for preparing cDNA and / or DNA molecules and cDNA and / or DNA libraries using modified reverse transcriptase enzymes.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201780083440.X, application date November 10, 2017, and invention name "Method for Processing Nucleic Acid Samples".

[0002] Cross Reference

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 421,028, filed on November 11, 2016, and U.S. Provisional Application No. 62 / 477,211, filed on March 27, 2017, which are each incorporated herein by reference in their entirety. Background Art

[0004] The common technique for studying gene expression in living cells is to produce complementary deoxyribonucleic acid (cDNA) from ribonucleic acid (RNA) molecules. This technology provides a way to study RNA by living cells, which avoids directly analyzing the unstable RNA itself. As the first step in cDNA synthesis, the RNA molecules from organisms are separated from organism cells or tissue extracts. After messenger RNA (mRNA) separation, the affinity chromatography method such as oligo dT (short sequence of deoxythymidine nucleotides) is used to anneal the oligonucleotide sequence to the separated mRNA molecules, and RNA / DNA primers can be used as templates to produce cDNA copies of RNA sequences using enzymes with reverse transcriptase activity. Therefore, the reverse transcription of mRNA is a key step in various forms of gene expression analysis. Usually, mRNA is reversely transcribed into cDNA for subsequent analysis by primer extension or polymerase chain reaction.

[0005] Reverse transcriptase has both RNA-guided DNA polymerase activity and DNA-guided DNA polymerase activity. The reverse transcription of the RNA template may require a primer sequence that anneals to the RNA template to start DNA synthesis from the 3'OH of the primer. At room temperature, reverse transcriptase can allow the formation of fully matched and mismatched DNA / RNA hybrids. In some cases, reverse transcriptase may produce a large amount of nonspecific cDNA products due to some nonspecific initiation events. Nonspecific reverse transcriptase products can interfere with subsequent cDNA analysis, such as cDNA sequencing, real-time polymerase chain reaction (PCR) and alkaline agarose gel electrophoresis, etc. The nonspecific cDNA templates produced due to nonspecific reverse transcriptase activity may have specific difficulties in applications such as real-time PCR. Specifically, these nonspecific cDNA products may produce erroneous signals that can complicate real-time PCR signals and product analysis. Therefore, reducing nonspecific reverse transcriptase activity can facilitate the higher specificity of cDNA synthesis. At present, there is no reliable and easy-to-use method for improving reverse transcription specificity. The present disclosure meets these and other needs.

[0006] Several methods can be used to obtain transcriptome data from single cells. A pioneer method uses reverse transcriptase and oligo-dT primers, wherein the T7 phage RNA polymerase promoter sequence is connected to the 5' end of the oligo-dT run. The resulting cDNA is transcribed into multiple RNA copies and then converted back to cDNA (Phillips et al., Methods 10 (3): 283-288 (1996)). This often truncates the cDNA molecule, thereby losing the 5' sequence of the original mRNA, especially for relatively long transcripts, and when using lower amount (LQ) cells as starting materials, multiple rounds of processing are required, which further aggravates cDNA truncation. The latest modification (Hashimshony et al., Cell Reports (Cell Rep.) 2 (3): 666-673 (2012)) enables multiple analysis, but this still favors 3' end sequences. Other methods are based on PCR amplification of cDNA (Liu et al., Methods Enzymol. 303:45-55 (1999); Ozsolak et al., Genome Res. 20(4):519-525 (2010)); Gonzalez et al., PLoS ONE. 5(12):el4418 (2010); Kanamori et al., Genome Res. 21(7):1150-1159 (2011); Islam et al., Genome Res. 21(7):1160-1167 (2011); Tang et al., Nat. Methods. 6(5):377-382 (2009); Kurimoto et al., Nucleic Acids Res. Res.) 34(5):e42 (2006); Qiu S et al., Front Genet. 3:124 (2012)).

[0007] However, these methods may result in biased representation of sequences along the mRNA and fail to obtain the complete sequence of the mRNA (eg, long mRNA) due to exclusion of DNA templates (eg, long DNA templates) even when long PCR reactions are used. Summary of the invention

[0008] The present disclosure provides methods for amplifying cDNA from RNA isolated from relatively low amounts of cells and / or single cells. The present disclosure also provides methods for transcriptome analysis, wherein cells are not subjected to stress (eg, high temperature for cDNA analysis), thereby maintaining transcriptome expression profiles.

[0009] In one embodiment, the present disclosure relates to a method for preparing a complementary deoxyribonucleic acid (cDNA) molecule, comprising:

[0010] (a) annealing a primer to a template nucleic acid molecule, thereby producing an annealed template nucleic acid molecule; and

[0011] (b) mixing, in the presence of nucleotides:

[0012] i. the annealed template nucleic acid molecule;

[0013] ii. one or more receptor nucleic acid molecules; and

[0014] iii. A modified reverse transcriptase, wherein the modified reverse transcriptase produces a plurality of contiguous complementary deoxyribonucleic acid molecules by: i) reverse transcribing the sequence of the annealed template nucleic acid molecule; ii) migrating to a receptor nucleic acid molecule; and iii) reverse transcribing the sequence of the receptor nucleic acid molecule at a temperature of about 12°C to about 42°C with an error rate of up to about 5%.

[0015] In some embodiments, the migration is independent of the sequence identity between the template and the receptor nucleic acid molecule. In some embodiments, (a) and (b) are performed in a single container. In some embodiments, the method further includes performing the method on a plurality of heterogeneous template nucleic acid molecules including a plurality of different ribonucleic acid (RNA) molecules. In some embodiments, a plurality of different ribonucleic acid molecules include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (mRNA) and long non-coding RNA (lncRNA). In some embodiments, a plurality of continuous complementary deoxyribonucleic acid molecules are prepared within a maximum of about 2 hours. In some embodiments, the template nucleic acid molecule is selected from the group consisting of: artificial fragmented DNA template, natural fragmented DNA template, artificial fragmented ribonucleic acid (RNA) template, natural fragmented ribonucleic acid (RNA) template or a combination thereof.

[0016] In some embodiments, the modified reverse transcriptase amplifies the annealed template nucleic acid molecule with a processivity of at least about 80% per base. In some embodiments, the modified reverse transcriptase comprises at least one improved enzymatic property relative to a wild-type unmodified reverse transcriptase. In some embodiments, the at least one improved enzymatic property is selected from the group consisting of: higher thermostability, higher specific activity, higher processivity, higher strand displacement, higher end-to-end template jumping, higher affinity, and higher fidelity relative to the wild-type unmodified reverse transcriptase. In some embodiments, the modified reverse transcriptase is an R2 reverse transcriptase. In some embodiments, the modified reverse transcriptase has at least about 90% identity to SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67, and contains at least one substitution modification relative to SEQ ID NO:52.

[0017] In some embodiments, the method further comprises adding a tag to the template nucleic acid molecule after performing (a) and (b), thereby generating a plurality of continuous complementary deoxyribonucleic acid molecules with tags. In some embodiments, the method further comprises sequencing the plurality of continuous complementary deoxyribonucleic acid molecules with tags. In some embodiments, the modified reverse transcriptase further comprises a tag. In some embodiments, the tag is selected from the group consisting of: biotin, azido, acetylene, His tag, calmodulin tag, CBP, CYD, Strep II, FLAG tag, HA tag, Myc tag, S tag, SBP tag, Softag-1, Softag-3, V5 tag, Xpress tag, isopeptag, SpyTag B, HPC peptide tag, GST, MBP, biotin carboxyl carrier protein, glutathione-S-transferase tag, green fluorescent protein tag, maltose binding protein tag, Nus tag, Strep tag and thioredoxin tag.

[0018] In some embodiments, the method further comprises performing a polymerase chain reaction (PCR) amplification reaction to form one or more amplicons. In some embodiments, (a) and (b) and the PCR amplification reaction are performed in the same container. In some embodiments, the PCR amplification is performed at a temperature sufficient to inactivate the reverse transcriptase.

[0019] In some embodiments, one or more receptor nucleic acid molecules include modified nucleotides that stop the reverse transcription by the modified reverse transcriptase. In some embodiments, the method further includes obtaining a sample including one or more template nucleic acid molecules from a subject and annealing one or more primers to the one or more template nucleic acid molecules. In some embodiments, the sample is a tissue sample. In some embodiments, the sample includes one or more free nucleic acids, and the method further includes performing the reactions of (a) and (b) in the same container.

[0020] In some embodiments, the method further comprises depleting at least one ribosomal RNA (rRNA) of a sample comprising one or more template nucleic acid molecules prior to annealing the one or more primers. In some embodiments, depleting the at least one ribosomal RNA (rRNA) comprises hybridizing an oligonucleotide to the rRNA. In some embodiments, depleting the at least one ribosomal RNA (rRNA) comprises oligonucleotide probe-guided endonucleolitic cleavage of the rRNA. In some embodiments, the one or more template nucleic acid molecules comprise at least one transfer RNA (tRNA), and the method further comprises depleting at least one tRNA of the one or more template nucleic acid molecules prior to annealing the one or more primers.

[0021] In some embodiments, the method is performed without purifying one or more annealed template nucleic acid molecules. In some embodiments, the method further comprises purifying a mixture comprising a plurality of continuous complementary deoxyribonucleic acid molecules. In some embodiments, the purification comprises two purification steps. In some embodiments, the two purification steps comprise a nickel affinity purification step and a heparin affinity purification step.

[0022] In some embodiments, the method is performed on about 0.1 nM to about 100 nM of one or more template nucleic acid molecules. In some embodiments, the method further comprises annealing one or more random primers to the template nucleic acid molecules. In some embodiments, the primers hybridize to one or more adapter sequences. In some embodiments, the template nucleic acid molecules are derived from a single cell.

[0023] In one embodiment, the present disclosure relates to a method for preparing a complementary deoxyribonucleic acid molecule, comprising:

[0024] (a) annealing one or more primers to a certain amount of template nucleic acid molecules, thereby generating one or more annealed template nucleic acid molecules; and

[0025] (b) mixing, in the presence of nucleotides:

[0026] i. one or more annealed template nucleic acid molecules;

[0027] ii. one or more receptor nucleic acid molecules; and

[0028] iii. A modified reverse transcriptase, whereby the modified reverse transcriptase produces multiple contiguous complementary deoxyribonucleic acid molecules by reverse transcribing the sequence of an annealed template nucleic acid molecule, migrating to an acceptor nucleic acid molecule, and reverse transcribing the sequence of the acceptor nucleic acid molecule without the need for thermal cycling in a single reaction vessel.

[0029] In some embodiments, the migration is independent of the sequence identity between the template and the receptor nucleic acid molecule. In some embodiments, a plurality of continuous complementary deoxyribonucleic acid molecules are prepared within about 2 hours at most. In some embodiments, the template nucleic acid molecule of a certain amount is selected from the group consisting of: artificial fragmentation DNA template, natural fragmentation DNA template, artificial fragmentation ribonucleic acid (RNA) template, natural fragmentation ribonucleic acid (RNA) template or a combination thereof. In some embodiments, the template nucleic acid molecule of a certain amount includes one or more free nucleic acids. In some embodiments, the modified reverse transcriptase includes at least one improved enzyme property relative to the wild-type unmodified reverse transcriptase. In some embodiments, the improved enzyme property is selected from the group consisting of: higher thermal stability, higher specific activity, higher processivity, higher chain displacement, higher end-to-end template jump, higher affinity and higher fidelity relative to the wild-type unmodified reverse transcriptase. In some embodiments, the modified reverse transcriptase is an R2 reverse transcriptase. In some embodiments, the modified reverse transcriptase has at least 90% identity to SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67, and contains at least one substitution modification relative to SEQ ID NO:52.

[0030] In some embodiments, the method further includes adding a tag to the certain amount of template nucleic acid molecules after performing (a) and (b), thereby generating a plurality of continuous complementary deoxyribonucleic acid molecules with tags. In some embodiments, the method further includes sequencing a plurality of continuous complementary deoxyribonucleic acid molecules with tags. In some embodiments, the modified reverse transcriptase includes a tag. In some embodiments, the tag is selected from the group consisting of: biotin, azido, acetylene, His tag, calmodulin tag, CBP, CYD, Strep II, FLAG tag, HA tag, Myc tag, S tag, SBP tag, Softag-1, Softag-3, V5 tag, Xpress tag, isopeptag, SpyTag B, HPC peptide tag, GST, MBP, biotin carboxyl carrier protein, glutathione-S-transferase tag, green fluorescent protein tag, maltose binding protein tag, Nus tag, Strep tag and thioredoxin tag. In some embodiments, one or more receptor nucleic acid molecules include modified nucleotides that stop reverse transcription by the modified reverse transcriptase.

[0031] In some embodiments, the method further includes obtaining a sample from a subject. In some embodiments, the sample includes one or more free nucleic acids, and the method further includes performing the reactions of (a) and (b) in the same container. In some embodiments, the sample is a tissue sample. In some embodiments, the method further includes exhausting at least one ribosomal RNA (rRNA) of one or more template nucleic acid molecules before annealing one or more primers. In some embodiments, exhausting at least one ribosomal RNA (rRNA) includes hybridizing an oligonucleotide with rRNA. In some embodiments, exhausting the at least one ribosomal RNA (rRNA) includes oligonucleotide probe-guided endonucleolytic cleavage of rRNA. In some embodiments, exhausting at least one transfer RNA (tRNA) of a certain amount of template nucleic acid molecules occurs before annealing one or more primers.

[0032] In some embodiments, the method is performed without purifying one or more annealed template nucleic acid molecules. In some embodiments, the method further comprises purifying a mixture comprising a plurality of continuous complementary deoxyribonucleic acid molecules. In some embodiments, the purification comprises two purification steps. In some embodiments, the two purification steps comprise a nickel affinity purification step and a heparin affinity purification step. In some embodiments, the certain amount of template nucleic acid molecules is one or more template nucleic acid molecules of about 0.1 nM to about 100 nM. In some embodiments, the one or more primers comprise one or more random primers. In some embodiments, the one or more primers hybridize with one or more adapter sequences. In some embodiments, a certain amount of template nucleic acid molecules originates from a single cell.

[0033] In one embodiment, the present disclosure relates to a polypeptide having reverse transcriptase activity and comprising an amino acid sequence having at least 90% identity to SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67 and containing at least one substitution modification relative to SEQ ID NO:52.

[0034] In one embodiment, the disclosure relates to a purified polypeptide comprising one or more domains from an R2 reverse transcriptase, wherein the purified polypeptide produces one or more copies of a complementary deoxyribonucleic acid molecule from a template nucleic acid molecule with an error rate of up to about 5%.

[0035] In some embodiments, the polypeptide reverse transcribes the template nucleic acid molecule with at least about 80% processivity per base. In some embodiments, the polypeptide reverse transcribes the template nucleic acid molecule at a temperature of about 12° C. to about 42° C. In some embodiments, the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67.

[0036] In one embodiment, the disclosure relates to a non-naturally occurring enzyme comprising one or more domains from an R2 reverse transcriptase, wherein the non-naturally occurring enzyme produces a complementary deoxyribonucleic acid product in a period of less than three hours with a performance index greater than 1.0 for at least one enzyme property, the at least one enzyme property selected from the group consisting of: improved stability, specific activity, protein expression, purification, processivity, strand displacement, template jumping, increased DNA / RNA affinity, and fidelity compared to a purified enzyme having SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67.

[0037] In some embodiments, R2 reverse transcriptase and / or non-natural enzyme and / or polypeptide can have at least one mutation and / or modification. In some embodiments, R2 and / or non-natural enzyme and / or polypeptide include C952S, and / or C956S, and / or C952S, C956S (double mutant), and / or C969S, and / or H970Y, and / or R979Q, and / or R976Q, and / or R1071S, and / or R328A, and / or R329A, and / or Q336A, and / or R328A, R329A, Q336A (triple mutant), and / or G426A, and / or D428A, and / or G426A, D428A (double mutant) mutation, and / or any combination thereof.

[0038] In one embodiment, the present disclosure relates to a method comprising:

[0039] (a) adding a nuclease complex to a plurality of double-stranded nucleic acid molecules, wherein the nuclease complex comprises:

[0040] i. Cas9 nuclease or a functional variant thereof; and

[0041] ii. at least one synthetic guide oligonucleotide, wherein the synthetic guide oligonucleotide is complementary to a ribosomal ribonucleic acid (rRNA) region or a transfer ribonucleic acid (tRNA) region in at least one double-stranded nucleic acid molecule in the plurality of double-stranded nucleic acid molecules;

[0042] (b) allowing the complex to cleave the rRNA or tRNA region of at least one double-stranded nucleic acid molecule, thereby providing at least one cleaved double-stranded nucleic acid molecule, and

[0043] (c) performing nucleic acid sequencing on the at least one cleaved double-stranded nucleic acid molecule or a derivative thereof, thereby obtaining a nucleic acid sequence of the at least one double-stranded nucleic acid molecule lacking the rRNA or tRNA region.

[0044] In some embodiments, the synthetic guide oligonucleotide is complementary to ribosomal ribonucleic acid (rRNA). In some embodiments, the synthetic guide oligonucleotide is complementary to transfer ribonucleic acid (tRNA). In some embodiments, the method does not require denaturation of multiple double-stranded molecules including sequences derived from rRNA and tRNA before sequencing. In some embodiments, multiple double-stranded nucleic acid molecules include cDNA.

[0045] In some embodiments, the method does not require denaturation of multiple double-stranded molecules comprising sequences derived from rRNA and / or tRNA. In some embodiments, multiple double-stranded nucleic acid molecules comprise sequences derived from rRNA and / or tRNA. In some embodiments, the double-stranded nucleic acid molecules comprise cDNA. In some embodiments, the predetermined region is the rRNA region. In some embodiments, the predetermined region is the tRNA region.

[0046] In one embodiment, the present disclosure relates to a method for preparing concatemers of nucleic acid molecules, comprising:

[0047] (a) treating the ends of a plurality of double-stranded nucleic acid molecules;

[0048] (b) adding a first plurality of adaptor molecules to the plurality of double-stranded nucleic acid molecules, wherein the first plurality of adaptor molecules comprises one or more overhang sequences, wherein at least two overhang sequences are complementary to each other, thereby providing a first plurality of adaptor-ligated double-stranded nucleic acid molecules;

[0049] (c) adding a polymerase to the first plurality of adaptor-ligated double-stranded nucleic acid molecules in the absence of a primer, whereby the polymerase forms a first set of adaptor-ligated double-stranded nucleic acid concatemers by joining two or more adaptor-ligated double-stranded nucleic acid molecules through the one or more overhang sequences;

[0050] (d) adding a second plurality of adaptor molecules to the first set, wherein the second plurality of adaptor molecules comprises one or more overhang sequences, wherein at least two of the overhang sequences are complementary to each other, thereby providing a second set of adaptor-ligated double-stranded nucleic acid molecules;

[0051] (e) Repeating (a) to (c) for a set of adaptor molecules to obtain concatemers having a predetermined average length.

[0052] In some embodiments, the processing in (a) comprises end repair. In some embodiments, the polymerase is a reverse transcriptase. In some embodiments, the reverse transcriptase is an R2 reverse transcriptase or a functional variant thereof. In some embodiments, the first plurality of adapter molecules, the second plurality of adapter molecules, or both comprise a unique molecular identifier sequence (UMI). In some embodiments, the polymerase joins two or more adapter-ligated double-stranded nucleic acid molecules in a PCR or isothermal amplification reaction. In some embodiments, the first plurality of adapter molecules, the second plurality of adapter molecules, or both comprise at least one modified nucleotide.

[0053] In some embodiments, the adapter comprises a unique molecular identification sequence (UMI). In some embodiments, the amplification is performed by PCR or isothermal amplification. In some embodiments, the adapter comprises at least one modified nucleotide.

[0054] Advantages of the present disclosure include, but are not limited to: simple, efficient and compatible methods for cDNA and nucleic acid library preparation (e.g., single cell and / or bulk library preparation) that are compatible with various sequence technologies; high-quality library preparation that can be used for single-cell nucleic acid (e.g., RNA) sequencing and bulk nucleic acid (e.g., RNA) sequencing; modified reverse transcriptases with improved enzymatic properties; high conversion rates (e.g., efficiency and / or fidelity) of nucleic acid samples (e.g., RNA, and / or mRNA, and / or DNA) to nucleic acid (e.g., cDNA) libraries; low non-specific product yields; and in transcriptome analysis, since nucleic acid synthesis can be performed at ambient temperature (e.g., 30°C), cells are not subjected to stress (e.g., cDNA synthesis does not require high temperatures).

[0055] Advantages of the present disclosure also include the ability to generate nucleic acid (e.g., cDNA) libraries using random priming or multiple priming, and / or the ability to generate libraries from fragmented / degraded nucleic acid molecules even at relatively low amounts (e.g., 500 femtomoles) of fragmented / degraded nucleic acid molecules (e.g., RNA and DNA). Advantages of the present disclosure also include the ability of the disclosed method to amplify fragments (e.g., amplify complete fragments) and generate multiple copies of the complete fragments for sequencing (e.g., by using a modified reverse transcriptase). Currently available methods may cause amplification of fragments at random positions and may only amplify portions of the fragments, so fragment sequencing and identification according to current methods are not available at relatively low amounts. Advantages of the present disclosure also include amplification by a single-step amplification scheme or by a two-step amplification process, thereby improving specificity and efficiency. Current methods include multi-step amplifications that may result in low yields, low efficiency, and / or low specificity. Another advantage of the method of the present disclosure is the ability to use nucleic acid molecule fragments (e.g., RNA) that are not necessarily complementary to the template for priming. Another advantage of the present disclosure is that the modified enzyme is capable of template hopping at room temperature and / or at a temperature as low as about 30°C. Another advantage of the present disclosure is the ability to perform template hopping in less than three steps ( Figure 1 ) and / or prepare a library from a sample in less than 4 hours. This advantage is particularly important for clinical research and testing and the medical field. Another advantage of the present disclosure is that the methods disclosed herein can improve template jumping, processivity, strand displacement properties, enzyme activity and / or fidelity (e.g., by using a modified reverse transcriptase).

[0056] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. It should be appreciated that the present disclosure is capable of other and different embodiments, and that its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Therefore, the drawings and descriptions are to be regarded as illustrative in nature, rather than restrictive.

[0057] Incorporation by Reference

[0058] All publications, patents and patent applications, and NCBI accession numbers mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, patent application or NCBI accession number was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents, patent applications or NCBI accession numbers incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The novel features of the present disclosure are set forth in detail in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description (which sets forth illustrative embodiments using the principles of the present disclosure) and the accompanying drawings (also referred to herein as "figures" and "diagrams"), in which:

[0060] Figure 1 The difference between the traditional method and the method of the present disclosure in preparing a complementary deoxyribonucleic acid (cDNA) library is shown. Figure 1 The difference between the preparation of samples for sequencing from liquid biopsy samples based on the traditional method and the method of the present disclosure is also shown. The traditional method includes a process that requires about 1-2 days and more than 4-5 hours of hands-on operation time, while the method of the present disclosure includes a process that requires less than about 2 hours and less than about 30 minutes of hands-on operation time;

[0061] Figure 2 A workflow for constructing a library based on the method of the present disclosure is shown;

[0062] Figure 3 The workflow for constructing the library is shown;

[0063] Figure 4 Shows the workflow for library construction using random primers;

[0064] Figure 5A A workflow for constructing a library using RNA priming using fragmented or degraded ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) is shown;

[0065] Figure 5B An example of a workflow for constructing a library using RNA priming using fragmented or degraded RNA or DNA, i.e., a method using specific primers, is shown;

[0066] Fig. 6A A workflow for constructing a library using fragmented or degraded RNA or DNA with donor complexes is shown;

[0067] Figure 6B An example of a workflow for constructing a library using fragmented or degraded RNA or DNA using donor complexes, i.e., using specific primers;

[0068] Figure 7 Schematic diagram showing N-terminal and C-terminal truncations of R2 reverse transcriptase;

[0069] Figure 8 Shown are examples of sequence analysis using selected non-long terminal repeat (LTR) retrotransposons and N-terminal truncation sites upstream of two conserved regions (region 1 and region 0);

[0070] Fig. 9An example using sequence analysis of selected non-LTR retrotransposons and C-terminal truncation sites downstream of the two conserved motifs 8* and 9* is shown;

[0071] Fig.10 Shown is a gel showing purified wild-type R2 reverse transcriptase and N-terminally truncated R2 reverse transcriptase;

[0072] Fig.11 Shown is a polyacrylamide gel electrophoresis (PAGE) gel demonstrating the activity and template jumping properties of the R2 enzyme and Moloney murine leukemia virus (MMLV) reverse transcriptase by using synthetic RNA;

[0073] Fig. 12A shows the workflow for sequencing library preparation;

[0074] Fig. 12B Real-time PCR data showing library preparation based on one-pot (e.g., single-vessel) reactions;

[0075] Fig. 12C A gel showing the amplicons is shown;

[0076] Fig.13 One-pot (e.g., single-container) RNA library preparation using different template amounts is shown;

[0077] Fig.14 One-pot (e.g., single-container) RNA library preparation using different template lengths is shown;

[0078] Fig.15 It is shown that the enzyme activity and template hopping depend on the concentration of sodium chloride (NaCl);

[0079] Fig.16A Shown is the enzyme activity after nickel affinity purification and / or heparin affinity purification observed based on a fluorescently labeled primer (fluorescein);

[0080] Fig. 16B Shows the template jumping properties after nickel affinity purification and / or heparin affinity purification as observed based on SYBR Gold staining;

[0081] Fig.17 R2 enzyme activity and template jumping in the presence of DNA template are shown (lane 1: no enzyme control; lane 2: 0.023 μg / μl enzyme in the presence of DNA template);

[0082] Fig.18A The workflow of the method disclosed herein for sequencing library preparation from a liquid biopsy is shown;

[0083] Fig.18BIt shows that the sample DNA fragments are captured by R2 enzyme using RNA priming method and RNA donor method;

[0084] Fig.18C Shown are sample DNA fragments (at various concentrations) captured by the R2 enzyme using the RNA priming method;

[0085] Fig.18D Shown are sample DNA fragments (at various concentrations) captured by the R2 enzyme using the RNA donor method;

[0086] Fig.19 It is shown that sample DNA fragments were captured using the RNA donor method at concentrations as low as 500 femtomolar;

[0087] Fig. 20 It is shown that the method of the present disclosure can be used for liquid biopsy applications. Fig. 20 The methods described herein are disclosed to demonstrate high sensitivity compared to currently available technologies (e.g., the methods disclosed herein can be used for extremely low DNA amounts as low as 0.3 pg, which is about 100-1000 times more sensitive than currently available methods). The data also demonstrates potential for applications involving DNA amounts even lower than 0.3 pg (e.g., several orders of magnitude lower);

[0088] Fig.21 It is shown that the method disclosed in the present invention has the advantage of detecting mutations in RNA (e.g., exosomal RNA) and free DNA (cell-free DNA). This method can be used to detect rare mutations or low-frequency mutations (e.g., some mutant alleles may appear in less than 1 copy per milliliter of plasma) by combining RNA and free DNA to improve detection sensitivity. This method can be used, for example, to test analytes separated from body fluids (e.g., blood). Body fluids (e.g., plasma) may contain different sources of free nucleic acids. These free sources can be circulating free DNA (e.g., double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA)) and RNA (e.g., extracellular RNA (exRNA) and RNA from exosomes). Free DNA may exist in various stages of fragmentation and / or degradation (e.g., different lengths). Extracellular RNA may include, but is not limited to, messenger RNA (mRNA), transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), and long non-coding RNA (lncRNA). Fig.21 It is also shown that the methods of the present disclosure differentially label free DNA and RNA present in the same or different tubes (eg, the same PCR tube). Labeling facilitates post-sequencing analysis by distinguishing sequences derived from DNA and RNA templates. Fig.21Two different analytes (DNA and RNA) are shown to be polytailed in the presence of terminal deoxynucleotidyl transferase (TdT), poly(A) polymerase, and the specific nucleotide substrates dCTP (or alternatively dGTP or dTTP) and ATP. Briefly, reactions containing RNA and DNA (dsDNA and ssDNA) are mixed and incubated with poly(A) polymerase, TdT, dCTP, and ATP. Fig.21 ). Poly(A) polymerase preferentially uses the preferred substrate ATP to extend RNA, while TdT preferentially uses the preferred deoxy substrate dCTP to extend DNA. Generally, the reaction can be performed simultaneously using two enzymes (e.g., poly(A) polymerase and TdT), or alternatively, can be performed sequentially using one enzyme at a time;

[0089] Fig.22A It was shown that streptavidin-immobilized oligonucleotides can be used to improve the efficiency of specific template capture and template hopping capabilities. Fig.22A The streptavidin-immobilized oligonucleotide is shown to bind to a specific DNA and / or RNA template. In this case, the streptavidin-immobilized oligonucleotide is bound to magnetic beads. Once the specific DNA and / or RNA template is bound to the streptavidin-magnetic bead complex, the template can be enriched. The oligonucleotide can be used as a primer and the template can be transcribed in the presence of an enzyme (e.g., R2 enzyme);

[0090] Fig. 22B It is shown that a complex comprising streptavidin bound to both an oligonucleotide primer and an oligonucleotide acceptor is capable of binding to a specific DNA and / or RNA template. Briefly, a specific template is bound to an oligonucleotide primer, which can then be extended in the presence of an enzyme (e.g., R2 enzyme). Primer extension then undergoes template hopping due to the close proximity between the acceptor oligonucleotide and the specific template;

[0091] Fig. 22C It is shown that a complex comprising streptavidin bound to an oligonucleotide primer, an oligonucleotide acceptor and magnetic beads is able to bind to a specific DNA and / or RNA template. In this case, the specific template is first enriched with magnetic beads. The template is then copied in the presence of an enzyme (e.g., R2 enzyme), and the extended sequence may further undergo template hopping;

[0092] Fig.23A and Fig. 23B Template concatemerization is shown. Some sequencing technologies have long sequencing read lengths (about 500 bp to about 50,000 bp), while other technologies have short sequencing read lengths (about 50 bp to about 250 bp). Most of the free DNA and RNA isolated from body fluids are short fragments (about 20 bp to about 200 bp). Fig.23A and Fig. 23B The method shown in is particularly suitable for sequencing technologies with long sequencing read lengths because the method is able to form long template concatemers. Fig.23A A method for concatemerizing several templates separated by a signaling sequence is shown. In this method, short dsDNA fragments are converted into long concatemers separated by a signaling sequence. Fig. 23B The final product of concatemerization is displayed, which contains specific adapters at both ends. The adapter is designed and has a unique molecular identification sequence (UMI), which allows tracking of labeled molecules and helps to reduce errors during data analysis. In short, dsDNA fragments are joined using two or more adapters. Subsequently, primer-free PCR (or isothermal amplification) is used to extend the joined fragments. The length of the concatemer or the number of connected templates can be determined, for example, by tagging the adapter with modified nucleotides (for example, by introducing methylated nucleotides or by inserting dUTP). The length of the concatemer can be adjusted based on the ratio between modified / unmodified adapters. The adapter sequence can serve as a homology priming position (annealing to the homology point ssDNA fragment, serving as a template and primer). The reaction in PCR undergoes a selected number of cycles (the more cycles, the longer the concatemer) or time (isothermal amplification). The reaction is then stopped, and the long dsDNA concatemer is joined by two unique dsDNA adapters.

[0093] See also Example 13;

[0094] Fig.24A The workflow of template concatenation disclosed in the present invention is shown;

[0095] Fig. 24B Shown is a gel demonstrating concatemerization of 200 bp DNA fragments;

[0096] Fig.25 A schematic reaction is shown in the presence of a reverse transcriptase and a second enzyme or enzymatic activity (i.e., a companion enzyme) such as a ssDNA 3' to 5' exonuclease or a polymerase with editing activity (e.g., a 3' to 5' exonuclease). Examples of companion enzymes include, but are not limited to, T4 DNA polymerase, exonuclease I, and exonuclease T. One function or use of a companion enzyme is to remove excess unused free extension primers. Free primers can produce unwanted products (e.g., free primers can act as jump acceptors or can be used as non-specific primers). Fig.25A reaction flow is shown, which begins with annealing a primer to an RNA template (the primer can be annealed to a specific sequence, or a poly(A) tail, or a poly(A) tail product at the 3' end). The reactants are then mixed with an enzyme (e.g., an R2 enzyme), a polymerase with editing activity (e.g., a 3' to 5' exonuclease), and an acceptor template (e.g., an acceptor template with a protected 3' end). The acceptor template can include a base at the 3' end to protect it from exonuclease hydrolysis. Examples of nucleotides that can be used to protect the acceptor template include, but are not limited to, ribonucleotides, thiophosphates, and nucleotide bases with or without modifications. Alternatively, if, for example, an appropriate ratio of primer to exonuclease is used, it can be performed in a single step Fig.25 The reaction shown in Fig.25 As shown in , if the R2 reverse transcriptase dissociates from the DNA / RNA heteroduplex before completely jumping to the acceptor template, the product is overextended (3' overhang). Subsequently, the 3' to 5' exonuclease activity may regenerate the common end structure of the DNA / RNA duplex. In general, jumping to the acceptor template can be accomplished by a multi-switch mechanism, thereby increasing the reaction yield;

[0097] Fig.26 Bioanalyzer tracking data of next generation sequencing (NGS) libraries are shown. Line 1 represents a fragmented RNA seq library from plasma, and line 2 represents a non-fragmented RNA seq library from plasma (see Example 15);

[0098] Fig. 27 and Fig.28 A method for generating a cell-free deoxyribonucleic acid (cfDNA) library is shown;

[0099] Fig.29 Library preparation is shown: ribosomal RNA and / or transfer RNA and / or PCR products are pulled using complementary oligonucleotides attached to magnetic beads or solid supports to maximize sequencing capacity;

[0100] Fig.30 Library preparation is shown: ribosomal RNA and / or transfer RNA and / or PCR products are subjected to oligonucleotide-guided degradation to maximize sequencing capacity.

[0101] Fig.31 The disclosed technology is shown to be capable of capturing / targeting all types of RNA simultaneously. Fig.31The free RNA library obtained according to the method of the present disclosure is shown. The chart corresponds to the Illumina sequencing results of the library prepared from 20ng free RNA (cfRNA). 28357171 reads were analyzed, and 91.9% were plotted. Examples of captured items / analytes include but are not limited to vault RNA, tRNA, srpRNA, sRNA, snRNA, snoRNA, scRNA, scaRNA, rRNA, RNA, long non-coding RNA (lncRNAs), microRNA (miRNA), large lnc RNA, miscellaneous RNA, 3' overlapping ncRNA, DNA, bidirectional promoter incRNA, lincRNA, MT_tRNA, MT_rRNA, ribozymes, LTR, retroposon and SINE. DETAILED DESCRIPTION

[0102] Although various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Numerous variations, changes, and substitutions may be envisioned by those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present application including the definitions shall prevail. In addition, unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. Unless it is specified that only a specific portion of a patent or patent publication is incorporated by reference, all publications, patents and other references mentioned herein are incorporated by reference for all purposes, just as each individual publication or patent application is specifically and individually specified to be incorporated by reference. In order to further define the present disclosure, the following terms, abbreviations and definitions are provided.

[0104] As used herein, the term "about" refers to variations in quantity that may occur, for example, due to typical measurement and liquid handling procedures used to prepare concentrates or solutions in the real world, inadvertent errors in these procedures, differences in the manufacture, source, or purity of the ingredients used to prepare the composition or perform the implementation method, and so on. The term "about" also encompasses amounts that differ due to different equilibrium conditions of the composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalent values ​​of the quantity. In some embodiments, the term "about" means within 10% of the reported value, or within 5% of the reported value, or within 20% of the reported value.

[0105] The indefinite article "a" and "an" before the elements or components of the present disclosure are not intended to limit the number of instances, i.e., the number of occurrences of the elements or components. Therefore, "a" should be understood to include one or at least one, and unless the number obviously represents the singular, the singular form of the elements or components also includes the plural.

[0106] The terms "annealing", "hybridization" or "binding" are used interchangeably herein to refer to the merging of one or more single-stranded polynucleotide sequences, segments or chains and allowing them to form a double-stranded molecule through base pairing. Two complementary sequences (e.g., ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA)) can anneal or hybridize by forming hydrogen bonds using complementary bases, thereby producing a double-stranded polynucleotide or a double-stranded region of a polynucleotide.

[0107] The term "subject" can be any animal that may benefit from the methods of the present disclosure, including, for example, humans and non-human mammals, such as primates, rodents, horses, dogs, and cats. Subjects include, but are not limited to, eukaryotes; mammals, such as primates, such as chimpanzees or humans, cows, dogs, cats; rodents, such as guinea pigs, rats, mice; lagomorphs; or birds; reptiles; or fish. Subjects particularly intended to be treated using the methods described herein include humans. A subject can be an individual or a patient.

[0108] As used herein, the term "primer extension reaction" generally refers to the denaturation of a double-stranded nucleic acid, the binding of a primer to one or both strands of the denatured nucleic acid, and the stretching of the primer.

[0109] As used herein, the term "reaction mixture" generally refers to a composition including reagents necessary to accomplish nucleic acid amplification (e.g., DNA amplification, RNA amplification), wherein non-limiting examples of these reagents include a primer set specific for a target RNA or target DNA, DNA produced by reverse transcription of RNA, DNA polymerase, reverse transcriptase (e.g., for reverse transcription of RNA), a suitable buffer (including a zwitterionic buffer), cofactors (e.g., divalent and monovalent cations), dNTPs, and other enzymes (e.g., uracil-DNA glycosylase (UNG)), etc. In some cases, the reaction mixture may also include one or more reporters.

[0110] As used herein, "reporter agent" generally refers to a composition that generates a detectable signal, the presence or absence of which can be used to detect the presence of an amplified product.

[0111] As used herein, the term "target nucleic acid" generally refers to a nucleic acid molecule having a nucleotide sequence in a starting nucleic acid molecule population, the presence, amount and / or sequence of which, or a change in one or more of these items, is to be determined. The target nucleic acid can be any type of nucleic acid, including DNA, RNA and analogs thereof.

[0112] As used herein, the term "primer" refers to an oligonucleotide naturally occurring or synthetically produced in restriction enzyme hydrolysis, characterized in that it can be extended relative to the template oligonucleotide, so that when all oligonucleotides are placed in a suitable temperature and pH in the presence of nucleotides, the oligonucleotides whose sequence is complementary to the sequence of at least a portion of the template molecule are connected to the primer. However, it is possible to use the primer itself in this way without fully extending relative to the template, and in some embodiments, the primer is only used as a site for adding a few non-templated nucleotides. For example, a primer with a primer hexamer of at least 6 nucleotides in length can be used. In some embodiments, the primer can be fluorescently labeled (e.g., 5'- / 56FAM / TGATGACGAGGCATTTGGC / 3'). In some embodiments, the length of the primer is about 6 to about 100 nucleotides, or in the range of about 10 to about 70 nucleotides in some embodiments. In some embodiments, a larger primer can be used. In some embodiments, a random primer can be used. In some embodiments, a primer can be a random primer. In some embodiments, one or more primers can be one or more random primers.

[0113] The term "one or more primers" can include any number of primers or random primers. For example, "one or more primers" can include at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 primers or random primers. The one or more primers can include about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 30, about 1 to about 35, about 5 to about 15, about 3 to about 10, about 5 to about 20, about 10 to about 50, about 30 to about 100, or more than about 100 primers. The one or more primers can include any number of primers.For example, the one or more primers can include at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 ,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,150,200,250,300,350,400,450,500,550,600,700,800,900,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,3000,4000,5000,6000,7000, 8000、9000、10000、11000、12000、13000、14000、15000、16000、17000、18000、19000、20000、25000、30000、35000、40000、45000、50000、55000、60000、65000、70000、75000、80000、85000、90000、95000、100000、150000、200000、250000、300000、350000、400000、450000、50000 0000, 550000, 600000, 650000, 700000, 750000, 800000, 850000, 900000, 950000, 1000000, 1500000, 2000000, 2500000, 3000000, 3500000, 4000000, 4500000, 5000000, 5500000, 6000000, 6500000, 7000000, 7500000, 8000000, 8500000, 9000000, 9500000 or 100000000 primers.The one or more primers can comprise about 10 to about 100, about 100 to about 1000, about 1000 to about 10,000, about 10,000 to about 100,000, about 100,000 to about 1,000,000, or about 1,000,000 to about 10,000,000 primers.

[0114] As used herein, the term "random primer" refers to a primer containing a random base sequence therein, and is intended to cover a primer composed partially or entirely of a random base sequence.

[0115] In some embodiments, the primer may comprise an adapter sequence. In some embodiments, the 5' tail sequence of the primer includes a sequence that does not hybridize with the target (adapter sequence). The same adapter sequence in a variety of primers with different 3' target binding sequences (i.e., "universal" 5' tail sequences) can be selected. This allows a single reporter probe sequence to be used to detect any desired target sequence, which is an advantage in the synthesis of more complex reporter probes due to labeling. In some embodiments, the primer may comprise an RNA primer. In some embodiments, the primer may comprise a DNA primer. In some embodiments, the primer may comprise an R2RNA primer. In some embodiments, the primer may comprise one or more random primers.

[0116] As used herein, the term "receptor template" is synonymous with "receptor nucleic acid molecule". In some embodiments, the receptor nucleic acid can be modified. In some embodiments, the receptor nucleic acid molecule can be modified at the 3' end, for example, to protect it from being mistaken for an RNA primer. In some embodiments, the modification of the receptor nucleic acid molecule can include a dideoxy 3' end. In some embodiments, the modification can include a phosphorylated 3' end. In some embodiments, the phosphorylated 3' end of a polynucleotide or a phosphorylated 3' end of a receptor nucleic acid molecule that generally has a hydroxyl group on its 3' end can be used as a 3' blocker due to extension by an enzyme disclosed herein, or for example, the extension of a DNA polymerase can be inhibited or the engagement by a ligase can be inhibited. Another non-limiting example of a 3' blocker includes adding a 3'C3 spacer (three-carbon spacer) to the 3' end of a polynucleotide, which can serve as an effective blocker for polymerase extension. Zhou et al., Clin. Chem., 50: 1328-1335 (2004). Thus, the 3' end can be blocked by adding, for example, a C3 spacer, a phosphate, an amino group (NH2), or any other chemical modification that inhibits the formation of a subsequent phosphodiester bond between the 3' end of the polynucleotide and another nucleotide.

[0117] As used herein, "overhang sequence" refers to a single-stranded region of a nucleic acid extending from a double-stranded region.

[0118] As used herein, an "isolated" polynucleotide means a polynucleotide that has been removed from its natural environment, produced using recombinant technology, or chemically or enzymatically synthesized. A polynucleotide may also be purified, i.e., substantially free of any other polynucleotides and associated cellular products or other impurities.

[0119] As used herein, the term "polymerase" may refer to an enzyme that links individual nucleotides together into a chain, using another chain as a template. In some embodiments, the polymerase is a polymerase with editing capabilities. In some embodiments, the polymerase with editing capabilities can be a 3' to 5' exonuclease, T4 DNA polymerase, exonuclease I, Phi29, Pfu, Vent, KOD, exonuclease III, and exonuclease T. Examples of polymerases can include DNA polymerases, RNA polymerases, RNA-guided DNA polymerases, reverse transcriptases, polypeptides having reverse transcriptase activity or any variants thereof, thermostable polymerases, wild-type polymerases, modified polymerases, Escherichia coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase, PHI 29 (phi29) DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tea polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerase, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, polymerases and variants with 3' to 5' exonuclease activity, modified products and derivatives thereof. In some embodiments, the polymerase can be a reverse transcriptase or a modified reverse transcriptase of the present disclosure. In some embodiments, the polymerase is a single subunit polymerase. The polymerase can have high processivity, that is, the ability of the polymerase to continuously incorporate nucleotides into a nucleic acid template without stripping the nucleic acid template.

[0120] The term "reverse transcriptase" or RT refers to an enzyme containing RNA-guided DNA polymerases and DNA-guided DNA polymerases. RT refers to a group of enzymes having reverse transcriptase activity (e.g., catalyzing the synthesis of DNA from an RNA template). In general, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, reverse plasmid reverse transcriptases, reverse transcriptases, bacterial reverse transcriptases, type II intron-derived reverse transcriptases, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-long terminal repeat (LTR) retrotransposon reverse transcriptases, reverse plasmid reverse transcriptases, reverse transcriptases, and type II intron reverse transcriptases. Other bacterial reverse transcriptases are described in Simon D and Zimmerly S (2008), "A diversity of uncharacterized retroelements in bacteria," Nucleic Acids Res 36(22):7219-7229 and Kojima, KK and Kanehisa, M (2008), "Systematic survey for novel types of prokaryotic retroelements based on gene neighborhood and protein architecture," Mol Biol Evol 25:1395-1404, which describe many classes of non-retroviral reverse transcriptases (i.e., retrocons, group II introns, and diversity-generating retroelements, among others). Reverse transcriptase has been primarily used to transcribe RNA into cDNA, which can then be cloned into a vector for further processing or used in various amplification methods, such as polymerase chain reaction, nucleic acid sequence-dependent amplification (NASBA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), various primer extension reactions, 5'RACE, chemical modification detection, or other techniques that require the synthesis of DNA using an RNA template.

[0121] Retroviral reverse transcriptase

[0122] Moloney murine leukemia virus (M-MLV) reverse transcriptase contains a single subunit of 78 kDa that has RNA-dependent DNA polymerase activity and RNase H activity. This enzyme has been cloned and expressed in fully active form in E. coli (reviewed in Prasad, VR, Reverse Transcriptase, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, p. 135 (1993)).

[0123] Human immunodeficiency virus (HIV) reverse transcriptase is a heterodimer of p66 and p51 subunits, wherein the smaller subunit is derived from the larger subunit by proteolytic cleavage. The p66 subunit has an RNA-dependent DNA polymerase domain and an RNase H domain, while the p51 subunit has only one DNA polymerase domain. Active HIV p66 / p51 reverse transcriptase has also been successfully cloned and expressed in a variety of expression hosts, including Escherichia coli (in Le Grice, SFJ, Reverse Transcriptase, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, p. 163 (1993) reviewed). In the HIV p66 / p51 heterodimer, the 51-kD subunit is catalytically inactive and the 66-kD subunit has DNA polymerase activity and RNase H activity (Le Grice, SFJ et al., EMBO Journal 10:3905 (1991); Hostomsky, Z. et al., J. Virol. 66:3179 (1992)).

[0124] Members of the Avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase family are also heterodimers with two subunits, α (approximately 62 kDa) and β (approximately 94 kDa), wherein the α subunit is derived from the β subunit by proteolytic cleavage (reviewed in Prasad, VR, Reverse Transcriptase, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (1993), p. 135). Members of this family include but are not limited to Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) helper virus MCAV reverse transcriptase, Avian Myeloblastosis Virus MC29 helper virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) helper virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 helper virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 helper virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase and Myeloblastosis Associated Virus (MAV) reverse transcriptase, etc.

[0125] ASLV reverse transcriptase can exist in two other catalytically active structural forms, Ad and a (Hizi, A. and Joklik, WK, J. Biol. Chem. 252:2281 (1977)).

[0126] Sedimentation analysis indicates that α / β and β / β exist as dimers, and that the α form exists in equilibrium between monomeric and dimer forms (Grandgenett, DP et al., Proc. Nat. Acad. Sci. USA 70:230 (1973); Hizi, A. and Joklik, WK, J. Biol. Chem. 252:2281 (1977); and Soltis, DA and Skalka, AM, Proc. Nat. Acad. Sci. USA 85:3372 (1988)). ASLV α / β and β / β reverse transcriptases are the only known examples of retroviral reverse transcriptases that contain three distinct activities in the same protein complex: DNA polymerase activity, RNase H activity, and DNA endonuclease (integrase) activity (reviewed in Skalka, AM, Reverse Transcriptase, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (1993), p. 193). The a form lacks the integrase domain and activity.

[0127] Various forms of each subunit of ASLV reverse transcriptase have been cloned and expressed. These forms include: a 98-kDa precursor polypeptide that is usually proteolytically processed into β and a 4 kDa polypeptide stripped from the β carboxyl terminus (Alexander, F. et al., Journal of Virology 61:5 3 4 (1987) and Anderson, D. et al., Focus 17:53 (1995)); and the mature β subunit (Weis, JH and Salstrom, JS, U.S. Pat. No. 4,663,290 (1987); and Soltis, DA and Skalka, AM, Proc. Natl. Acad. Sci. USA 85:3372 (1988)). (See also Wermer S and Wohrl BM, Eur. J. Biochem. 267:4740-4744 (2000); Wermer S and Wohrl BM, J. Virol. 74:3245-3252 (2000); Wermer S and Wohrl BM, J. Biol. Chem. 274:26329-26336 (1999)). Heterodimeric RSV α / β reverse transcriptase has also been purified from E. coli cells expressing a cloned RSV β gene (Chemov, AP et al., Biomed. Sci. 2:49 (1991)).

[0128] Reverse transcriptase from non-retroviral sources

[0129] Reverse transcriptase can also be separated from a large number of movable genetic factors that are not retroviral sources. These movable genetic factors are present in the genome of higher species and play a role in the life cycle of these movable genetic factors. Known movable genetic factors are for reverse transcriptase encoding genes (in Howard M Temin, Reverse Transcription in the Eukaryotic Genome: Retroviruses. Pararetroviruses, Retrotransposons, and Retrotranscripts, Molecular Biology and Evolution (Mol.Biol.Evol.) 2 (6): Reviewed in 455-468). These factors include but are not limited to retrotransposons. Retrotransposons include non-long terminal repeats (LTR) retrotransposons and LTR movable factors (such as TY3, TY5, non-LTR, LINE-L1, R2, R1). (Reviewed by Cordaux and Batzer in Nature Reviews, Vol. 10, pp. 691-703, October 2009).

[0130] As used herein, "non-LTR retrotransposon" refers to a protein encoded by a non-LTR retrotransposon and a polypeptide fragment thereof having reverse transcriptase activity, and a protein or polypeptide derived therefrom contains one or more amino acid substitutions that enhance its reverse transcriptase activity or have no deleterious effect on the activity. A preferred class of non-LTR retrotransposons is an R2 protein or polypeptide. Therefore, as used herein, "R2 protein or R2 enzyme or polypeptide or a functional fragment thereof" refers to a naturally occurring protein encoded by an R2 element and a polypeptide fragment thereof having reverse transcriptase activity, and a protein or polypeptide derived therefrom contains one or more amino acid substitutions that enhance its reverse transcriptase activity or have no deleterious effect on the activity.

[0131] Retroelements (genetic elements encoding RT) are divided into two major families denoted as LTR-containing retroelements and non-LTR-containing retroelements (Xiong Y, Eickbush TH (1990) "Origin and evolution of retroelements based upon their reverse transcriptase sequences" EMBO Journal 9:3353-62). Non-LTR retroelements are a distinct family of RT encoding elements that include retroplasmids, non-LTR retrotransposons, retrotrons, and mobile group II introns.

[0132] As used herein, the term polymerase "active fraction" is defined as the portion of an enzyme that has polymerase activity. For example, a reverse transcriptase active fraction (RT active fraction) is a portion of an enzyme that has reverse transcriptase activity.

[0133] As used herein, the terms "variant," "modified," "non-naturally occurring," and "mutated" are synonymous and refer to polypeptides or enzymes that differ from the specifically enumerated polypeptides or enzymes due to one or more amino acid insertions, deletions, mutations, and substitutions produced using, for example, recombinant DNA techniques (e.g., mutation). Determining which amino acid residues can be substituted, added, or deleted without eliminating the associated activity can be guided by comparing the sequence of a particular polypeptide to homologous polypeptides (e.g., yeast or bacteria) and minimizing the number of amino acid sequence changes produced in regions of high homology (conserved regions) or by replacing amino acids with common sequences. In some embodiments, the terms "derived," "variant," "modified," "non-naturally occurring," and "mutated" are used interchangeably.

[0134] The mutants disclosed herein can be produced according to any suitable method, including but not limited to the methods described and exemplified herein. For example, mutations of substitution, insertion, deletion and / or side chain modification can be introduced into the nucleotide and amino acid sequences of the relevant gene using any suitable technique (including site-directed mutagenesis) (Meth. Enzymol. 217, Academic Press (1993) compiled by Wu). The λ Red recombinase method can be used to "knock out" genes (Datsenko et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS USA) 97: 6640-6645 (2000)). Multiple gene disruptions without markers that last can be produced. Non-naturally occurring nucleotides and amino acids can also be used.

[0135] As used herein, "homolog" refers to an enzyme that is functionally equivalent to an enzyme having an amino acid sequence with a designated sequence identification number (i.e., has the same enzymatic activity), but may have a limited number of amino acid substitutions, deletions, insertions or additions in the amino acid sequence. In order to maintain the function of the protein, the substitutions may be conservative substitutions, replacing one amino acid with an amino acid having similar properties.

[0136] In some embodiments, a homolog refers to a protein that is at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to an amino acid sequence having a SEQ ID NO corresponding to the protein. Algorithms for determining sequence identity include, for example, BLAST, which is available through the National Center for Biotechnology Information (NCBI). The degree of sequence similarity can be determined, which indicates homology and, therefore, similar or identical function.

[0137] Polynucleotides encoding homologs of various enzymes can be obtained by appropriately introducing substitutions, deletions, insertions and / or additions into the DNA of the enzyme consisting of the nucleotide sequence disclosed herein using methods such as random mutagenesis and site-directed mutagenesis (Nucleic Acid Res. 10, p6487 (1982); Methods in Enzymol. 100, p448 (1983); Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory Press (1989); PCR: A Practical Approach, IRL Press, p200 (1991)). Polynucleotides encoding homologs of various enzymes can be introduced and expressed in a host to obtain homologs.

[0138] The term "heterologous" refers to a molecule or activity derived from a source other than the reference species, while "homologous" refers to a molecule or activity derived from a host microorganism. Accordingly, exogenous expression of encoding nucleic acids of the present disclosure can use either or both heterologous or homologous encoding nucleic acids.

[0139] In some embodiments, the host cell can be selected from, for example, bacteria, yeast, fungi, or any of a variety of other organisms useful as host organisms, and the modified or non-naturally occurring enzyme is produced by such organisms.

[0140] In some embodiments, the host is not clearly defined, and one or more enzyme activities can be incorporated into any suitable host organism using methods such as described herein. In some embodiments, the host is selected from bacteria, yeast, algae, cyanobacteria, fungi or plant cells or any combination thereof. Escherichia coli and Saccharomyces cerevisiae are particularly suitable for host organisms because they are well-characterized microorganisms suitable for genetic engineering.

[0141] As used herein, "enzyme" includes proteins produced by cells that can catalyze biochemical reactions. In addition, unless otherwise specified in the context, as used herein, "enzyme" includes protein fragments that retain relevant catalytic activity and can include artificial enzymes that are synthesized and retain relevant catalytic activity.

[0142] Each enzyme described herein can be linked to other amino acid sequences, as long as the activity is functionally equivalent to the activity of the enzyme. As mentioned above, it should be understood that various enzymes or their homologues can be (multiple) peptide fragments, as long as the activity is functionally equivalent to the activity of the enzyme.

[0143] In some embodiments, the enzymes used in the compositions, methods and kits of the present disclosure include any enzyme having reverse transcriptase activity. These enzymes include, but are not limited to, non-retroviral reverse transcriptases, retroviral reverse transcriptases, retrotransposon reverse transcriptases, non-LTR retrotransposons, R2 reverse transcriptases, LTR retrotransposons, hepatitis B reverse transcriptases, cauliflower mosaic virus reverse transcriptases, bacterial reverse transcriptases, Tth DNA polymerases, TaqDNA polymerases (Saiki, RK et al., Science 239: 487-491 (1988); U.S. Patents 4,889,818 and 4,965,188), Tne DNA polymerases (PCT Publication No. WO 96 / 10640), Tma DNA polymerases (U.S. Patent No. 5,374,553), and mutants, fragments, variants or derivatives thereof. In certain embodiments, the reverse transcriptase used in the present disclosure comprises a retroviral reverse transcriptase, such as an M-MLV reverse transcriptase, an AMV reverse transcriptase, an RSV reverse transcriptase, an RAV reverse transcriptase, an MAV reverse transcriptase, and an ASLV reverse transcriptase in general. Mutant reverse transcriptases can be obtained, for example, by site-directed or random mutations that mutate one or more genes encoding the relevant reverse transcriptase. These mutations can comprise point mutations, deletion mutations, insertion mutations, and truncations. For example, one or more point mutations (e.g., replacing one or more amino acids with one or more different amino acids) can be used to construct mutant reverse transcriptases for use in the present disclosure.

[0144] In some embodiments, the enzyme is selected and / or engineered to exhibit high fidelity and low error rate. The fidelity of nucleotide polymerases is usually measured in the form of error rate, i.e., the frequency of incorporating nucleotides in a manner that violates the well-known Watson-Crick base pairing rules. The fidelity or error rate of a polymerase (e.g., DNA polymerase) can be measured using any suitable analysis. See, e.g., Lundburg et al., 1991 Gene, 108: 1-6. The term "fidelity" can be used to refer to polymerization accuracy, or the ability of a polymerase to distinguish between a correct substrate and an incorrect substrate (e.g., nucleotide) when synthesizing a nucleic acid molecule (e.g., RNA or DNA) complementary to a template. The higher the fidelity of the enzyme, the fewer nucleotides that the enzyme misincorporates in the growing chain during nucleic acid synthesis; that is, increasing or improving fidelity produces a more faithful polymerase with a reduced error rate (reduced misincorporation rate). In some embodiments, the misincorporation error rate is at most about 10-2, 10-4, 10-6, or 10-8.

[0145] In some embodiments, the non-naturally occurring or modified enzyme (e.g., a non-naturally occurring or modified reverse transcriptase, a non-naturally occurring or modified non-LTR retrotransposon, a non-naturally occurring or modified R2 reverse transcriptase) or a modified polypeptide having reverse transcriptase activity exhibits a misincorporation error rate of about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 10% or less, about 15% or less, about 20% or less, about 2 ... 15%, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4%, about 3% or less, about 2%, about 1% or less, about 0.01%, about 0.001%, about 0.0001%, about 0.00001%, about 0.000001%, or about 0.0000001%.

[0146] In some embodiments, the non-naturally occurring or modified enzyme (e.g., a non-naturally occurring or modified reverse transcriptase, a non-naturally occurring or modified non-LTR retrotransposon, a non-naturally occurring or modified R2 reverse transcriptase) or a modified polypeptide having reverse transcriptase activity produces one or more nucleic acid (e.g., cDNA) molecules complementary to a template with an error rate that is at least about 10,000 times lower, at least about 1,500 times lower, at least about 1,000 times lower, at least about 500 times lower, at least about 100 times lower, or at least about 150 times lower than an unmodified or naturally occurring enzyme or an unmodified polypeptide having reverse transcriptase activity. At least about 95 times lower, at least about 90 times lower, at least about 85 times lower, at least about 80 times lower, at least about 75 times lower, at least about 70 times lower, at least about 65 times lower, at least about 60 times lower, at least about 55 times lower, at least about 50 times lower, at least about 45 times lower, at least about 40 times lower, at least about 35 times lower, at least about 30 times lower, at least about 25 times lower, at least about 20 times lower, at least about 15 times lower, at least about 10 times lower, at least about 9 times lower, at least about 8 times lower, at least about 7 times lower, at least about 6 times lower, at least about 5 times lower, at least about 4 times lower, at least about 3 times lower, at least about 2 times lower, or at least about 1 times lower.

[0147] In some embodiments, the sequencing error rate will be equal to or less than about 1 / 100,000 bases. In some embodiments, the error rate of nucleotide sequence determination is equal to or less than about 1 / 10 bases, 1 / 20 bases, 3 / 100 bases, 1 / 100 bases, 1 / 1000 bases, and 1 / 10,000 bases.

[0148] The terms "polynucleotide", "nucleic acid", "nucleotide" and "oligonucleotide" are used interchangeably. These terms refer to polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides) of any length, fragments thereof or analogs thereof. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (one or more) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, transfer-messenger RNA, ribosomal RNA, antisense RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), small interfering RNA (siRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after polymer assembly. Nucleotide sequences can be interspersed with non-nucleotide components. Polynucleotides can be further modified after polymerization, for example by binding to a labeling component. The nucleic acids described herein may contain phosphodiester bonds. In some embodiments, the nucleic acid may be DNA (including, for example, genomic DNA, mitochondrial DNA, and cDNA), RNA (including, for example, mRNA and rRNA), or a hybrid, wherein the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine (xathanine), hypoxathanine (hypoxathanine), isocytosine, isoguanine, etc. Polynucleotides are intended to cover single nucleic acids and multiple nucleic acids. Polynucleotides may be composed of any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides may be composed of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules including DNA and RNA (which may be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions).

[0149] Ribosomal RNA can constitute up to 80% or more of the total RNA in a sample. It is often necessary to separate rRNA from mRNA because rRNA may adversely affect the quantitative analysis of mRNA. One method of separating rRNA from mRNA is to deplete the rRNA of a sample. An example is the use of oligonucleotides (e.g., oligonucleotides homologous to 17SrRNA, 18S rRNA, or 28S rRNA in the case of eukaryotic rRNA or to 16S rRNA or 23S rRNA in the case of bacterial rRNA) to hybridize rRNA molecules. Oligonucleotides are designed so that they can be "captured" and hybridization products removed from the sample. For example, oligonucleotides can be fixed on the surface of, for example, a chromatographic column or a bead. MICROB Express (registered trademark) and MICROBEnrich (registered trademark) (Ambion, Austin, Texas) are examples of commercially available kits for rRNA depletion. Methods and compositions for the depletion of rRNA in a sample are described in U.S. Application No. 10 / 029,397, which is incorporated by reference. The poly (A) tail at the 3' end of most eukaryotic mRNAs can be used to separate these molecules from rRNA and other non-mRNA species lacking such poly (A) tails. In some embodiments, the disclosed methods include, for example, exhausting ribosomal RNA by hybridizing an oligonucleotide with rRNA. In some embodiments, the disclosed methods include exhausting rRNA and / or tRNA by oligonucleotide probe-guided endonucleolytic cleavage of at least one rRNA and / or tRNA sequence. In some embodiments, the deletion may be a partial or complete deletion. In some embodiments, the deletion includes reducing the number of rRNA and / or tRNA of a sample. In some embodiments, the disclosure relates to methods for exhausting rRNA and / or transfer RNA (tRNA). In some embodiments, the disclosure relates to exhausting at least one transfer RNA (tRNA) of one or more template nucleic acid molecules. In some embodiments, exhausting rRNA and / or tRNA occurs before annealing a primer (e.g., one or more primers) to a template.

[0150] In one embodiment, the present disclosure relates to a method for depleting ribosomal RNA and / or transfer RNA of a sample for library sequencing. In some embodiments, the method includes providing a sample including RNA. In some embodiments, the RNA includes ribosomal RNA (rRNA) and / or transfer RNA (tRNA). In some embodiments, the method includes performing a polymerase chain reaction (PCR) to convert rRNA and / or tRNA into double-stranded DNA (dsDNA).

[0151] In some embodiments, the method includes partial or complete (complete) amplification. In some embodiments, the method further includes introducing a complex comprising a nuclease and / or a polynucleotide encoding a nuclease and at least one specially designed guide oligonucleotide. In some embodiments, at least one guide oligonucleotide includes a sequence complementary to at least one rRNA and / or at least one tRNA. In some embodiments, at least one guide oligonucleotide includes at least one sequence complementary to at least one dsDNA. In some embodiments, a nuclease or a polynucleotide encoding a nuclease cleaves at least one chain of dsDNA. In some embodiments, a nuclease or a polynucleotide encoding a nuclease cleaves rRNA and / or tRNA and / or dsDNA, thereby depleting the rRNA and / or tRNA of the sample. In some embodiments, the nuclease is Cas9, or a polynucleotide encoding Cas9 or a functional variant thereof. In some embodiments, the method includes denaturing dsDNA into a single-stranded DNA (ssDNA) chain. In some embodiments, the method further includes introducing at least one oligonucleotide (e.g., a specially designed oligonucleotide) comprising a binding molecule and at least one sequence complementary to at least one ssDNA chain, thereby forming a hybrid complex of an oligonucleotide and at least one ssDNA chain. In some embodiments, the method includes fixing the hybrid complex to at least one solid support. In some embodiments, the hybridization complex is fixed so that the hybridization complex is removed from the sample. In some embodiments, the solid support includes streptavidin. In some embodiments, the method includes introducing at least one oligonucleotide (e.g., a specially designed oligonucleotide) including a binding molecule and at least one sequence complementary to at least one rRNA and / or tRNA, thereby forming a complex including the oligonucleotide and at least one rRNA and / or tRNA. In some embodiments, the method further includes fixing the complex to at least one solid support. In some embodiments, the complex is fixed so that the complex is removed from the sample. In some embodiments, the solid support includes streptavidin. In some embodiments, the binding molecule is biotin.

[0152] In some embodiments, any method of the present disclosure includes a nuclease or a polynucleotide encoding a nuclease. In some embodiments, a nuclease or a polynucleotide encoding a nuclease cleaves at least one chain of a dsDNA. In some embodiments, dsDNA cleavage is an intermediate product of library preparation. In some embodiments, dsDNA cleavage comprises rRNA and / or tRNA (coding sequence). In some embodiments, oligonucleotide-guided nuclear decomposition cleavage may not require or require dsDNA denaturation. In some embodiments, dsDNA denaturation is not required as a significant improvement of the present disclosure. In some embodiments, a nuclease or a polynucleotide encoding a nuclease is Cas9 or a polynucleotide encoding Cas9, or a functional variant thereof.

[0153] In one embodiment, the present disclosure relates to a method comprising adding a nuclease complex to a plurality of double-stranded nucleic acid molecules. In some embodiments, the nuclease complex comprises a Cas9 nuclease or a functional variant thereof and at least one synthetic guide oligonucleotide. In some embodiments, the synthetic guide oligonucleotide is complementary to a ribosomal ribonucleic acid (rRNA) and / or a transfer ribonucleic acid (tRNA) region. In some embodiments, the region is in at least one double-stranded nucleic acid molecule. In some embodiments, the method comprises allowing the complex to cleave rRNA and / or tRNA regions. In some embodiments, the region is present in at least one double-stranded nucleic acid molecule. In some embodiments, the method provides at least one cleaved double-stranded nucleic acid molecule. In some embodiments, the method comprises sequencing (e.g., nucleic acid sequencing) at least one cleaved double-stranded nucleic acid molecule or a derivative thereof. In some embodiments, the method comprises sequencing the nucleic acid sequence of at least one double-stranded nucleic acid molecule lacking an rRNA region and / or a tRNA region. In some embodiments, the method comprises sequencing the nucleic acid sequence of at least one double-stranded nucleic acid molecule including an rRNA region and / or a tRNA region. In some embodiments, the method comprises sequencing a mixture of nucleic acid sequences, wherein the mixture comprises at least one double-stranded nucleic acid molecule including an rRNA region and / or a tRNA region and at least one double-stranded nucleic acid molecule lacking an rRNA region and / or a tRNA region.

[0154] In one embodiment, the present disclosure relates to a method for generating a cell-free deoxyribonucleic acid (cfDNA) library, comprising: providing a sample comprising cfDNA; denaturing the cfDNA to generate a single-stranded DNA (ssDNA) sample; introducing a complex comprising a template, a primer, and a reverse transcriptase to the ssDNA sample in the presence of nucleotides and / or catalytic metals, wherein the reverse transcriptase extends the primer on the template, and then the template jumps to the ssDNA sample, thereby generating a double-stranded DNA (dsDNA) sample, and wherein the dsDNA comprises at least one gap between the template and the ssDNA; introducing a polymerase comprising 3' to 5' exonuclease activity to generate dsDNA having a blunt end and / or a 3' overhang; introducing an asymmetric adaptor comprising a nucleic acid duplex having a single-stranded overhang at the 5' end, wherein the asymmetric adaptor is joined to the 5' end of the dsDNA, and wherein the single-stranded overhang comprises a sequence complementary to at least one polymerase chain reaction (PCR) amplification primer; and performing a PCR reaction to amplify only one strand of the dsDNA.

[0155] In one embodiment, the present disclosure relates to a method for producing a free deoxyribonucleic acid (cfDNA) library. In some embodiments, the method includes providing a sample including cfDNA. In some embodiments, the method includes denaturing cfDNA to produce a single-stranded DNA (ssDNA) sample. In some embodiments, the method includes introducing terminal deoxynucleotidyl transferase (TdT) and deoxyadenosine triphosphate (dATP) into the ssDNA sample to produce a poly (A) and / or poly (C) tail. In some embodiments, the method includes introducing non-extendable nucleotides. In some embodiments, the method includes annealing a complex including a primer and a first adapter to the tail end of the ssDNA sample. In some embodiments, the complex includes a sequence complementary to the tail end. In some embodiments, the method includes introducing a reverse transcriptase (e.g., a modified reverse transcriptase) and a complex including an acceptor and a second adapter in the presence of nucleotides and / or catalytic metals, thereby producing a double-stranded DNA (dsDNA) sample. In some embodiments, the nucleotides include degradable nucleotides. In some embodiments, the reverse transcriptase extends the primer, and then the template jumps to the complex to continue extending. In some embodiments, the complex includes a nucleotide blocker to prevent the reverse transcriptase from reaching the end of the complex and jumping to another complex. In some embodiments, the dsDNA includes an original strand and a copy strand. In some embodiments, the original strand includes at least one gap between the complex and the ssDNA. In some embodiments, the copy strand includes at least one degradable nucleotide. In some embodiments, the method further includes introducing a polymerase including 3' to 5' exonuclease activity to produce a dsDNA with a blunt end or a 3' overhang, and / or introducing a DNA ligase that engages at least one gap. In some embodiments, the method further includes introducing at least one uracil-DNA glycosylase to degrade at least one degradable nucleotide. In some embodiments, the method further includes performing a polymerase chain reaction (PCR) including primers (e.g., at least a first primer and / or at least a first and a second primer) to amplify the original strand. In some embodiments, the primer (e.g., a first primer) includes a sequence complementary to the first adapter, and the second primer includes a sequence complementary to the second adapter.

[0156] In one embodiment, the present disclosure relates to a method for producing a library for sequencing. In some embodiments, the method includes providing a sample including free ribonucleic acid (cfRNA). In some embodiments, the method includes subjecting the sample to high temperature. In some embodiments, the high temperature is sufficient to cause RNA (e.g., cfRNA) to undergo transphosphorylation. In some embodiments, the method further includes introducing a phosphatase. In some embodiments, the phosphatase can convert the phosphate moiety of the RNA into a 3'-hydroxyl. In some embodiments, the method further includes introducing adenosine triphosphate and a polymerase to produce a poly (A) tail on the 3'-hydroxyl of the RNA. In some embodiments, the method further includes introducing a primer, an acceptor, and a reverse transcriptase in the presence of nucleotides. In some embodiments, the primer includes a sequence complementary to the poly (A) tail, thereby annealing to the poly (A) tail. In some embodiments, the reverse transcriptase extends the primer, and then the template jumps to the acceptor to continue extending. In some embodiments, the method further includes introducing at least one solid support to fix excess primers and non-specific primer products to at least one solid support, thereby removing excess primers and non-specific primer products from the sample. In some embodiments, the method further includes performing a polymerase chain reaction (PCR) reaction to amplify RNA. In some embodiments, the method further comprises using an isothermal amplification reaction.

[0157] In one embodiment, the present disclosure relates to a method for preparing a nucleic acid library for sequencing. In some embodiments, the method includes obtaining a plurality of nucleic acid molecules. In some embodiments, the method includes inducing a non-enzymatic intramolecular phosphatase transfer of at least one nucleic acid molecule (e.g., at least one of a plurality of nucleic acid molecules). In some embodiments, the non-enzymatic intramolecular phosphatase transfer can occur by increasing the temperature (e.g., increasing the temperature of a plurality of nucleic acid molecules). In some embodiments, the non-enzymatic intramolecular phosphatase transfer and / or the temperature increase produce nucleic acid molecules with free 5'-phosphate moieties (e.g., a plurality of nucleic acid molecules can have free 5'-phosphate moieties). In some embodiments, the method includes adding a phosphatase to a nucleic acid molecule with a free 5'-phosphate moiety. In some embodiments, the phosphatase converts one or more of the free 5'-phosphate moieties into hydroxyls. In some embodiments, this causes a plurality of nucleic acid molecules to have free hydroxyls. In some embodiments, the method includes mixing a plurality of nucleic acid molecules with a polymerase (e.g., in the presence of a certain amount of adenosine triphosphate). In some embodiments, the polymerase produces a poly (A) tail on the free hydroxyl. In some embodiments, the method comprises mixing (i) one or more primers comprising a sequence complementary to the poly(A) tail, (ii) one or more receptor nucleic acid molecules, and (iii) a modified reverse transcriptase in the presence of nucleotides. In some embodiments, the modified reverse transcriptase produces a plurality of continuous complementary deoxyribonucleic acid molecules by reverse transcribing the sequence of the annealed template nucleic acid molecule, migrating to the receptor nucleic acid molecule, and reverse transcribing the sequence of the receptor nucleic acid molecule. In some embodiments, the method comprises adding at least one solid support. In some embodiments, the solid support immobilizes an excess of one or more primers comprising a sequence complementary to the poly(A) tail. In some embodiments, the method comprises performing a polymerase chain reaction (PCR) reaction. In some embodiments, the method comprises performing isothermal amplification.

[0158] In one embodiment, the present disclosure relates to a method for producing a library for sequencing. In some embodiments, the method includes providing a sample including at least one nucleic acid molecule (e.g., ribonucleic acid (e.g., cfRNA)). In some embodiments, the method includes subjecting the sample (or nucleic acid molecule) to a high temperature sufficient to cause the nucleic acid molecule (e.g., RNA) to undergo phosphate transfer. In some embodiments, the method further includes adding a catalytic metal (e.g., magnesium) and / or a polyamine. In some embodiments, the method includes introducing a phosphatase to convert the phosphate moiety of the nucleic acid molecule (e.g., RNA) into a 3'-hydroxyl. In some embodiments, the method includes introducing adenosine triphosphate and a polymerase to produce a poly (A) tail. In some embodiments, the poly (A) tail is produced on the 3'-hydroxyl of the nucleic acid molecule (e.g., RNA). In some embodiments, the method includes introducing a primer, an acceptor, and a reverse transcriptase to the sample or nucleic acid molecule in the presence of nucleotides. In some embodiments, the primer includes a sequence complementary to the poly (A) tail, thereby annealing to the poly (A) tail. In some embodiments, the reverse transcriptase extends the primer, and then the template jumps to the acceptor to continue extending. In some embodiments, the method includes introducing at least one solid support. In some embodiments, the solid support fixes excess primers and non-specific primer products. In some embodiments, excess primers and non-specific primer products are removed from the sample / mixture. In some embodiments, the method further includes performing a polymerase chain reaction (PCR) reaction to amplify nucleic acid molecules (e.g., RNA). In some embodiments, the method further includes an isothermal amplification reaction.

[0159] In one embodiment, the present disclosure relates to a method of depleting rRNA and / or tRNA, and / or to a method comprising reverse transcribing at least one nucleic acid molecule by performing an amplification reaction. In some embodiments, at least one nucleic acid molecule is rRNA and / or tRNA. In some embodiments, the amplification reaction provides a plurality of double-stranded nucleic acid molecules (e.g., cDNA). In some embodiments, the method comprises adding a nuclease or a polypeptide comprising a nuclease and / or a guide oligonucleotide. In some embodiments, the method comprises adding a complex comprising a nuclease or a polypeptide comprising a nuclease and a guide oligonucleotide. In some embodiments, the nuclease or a polypeptide comprising a nuclease is Cas9 or a polypeptide comprising Cas9. In some embodiments, the guide oligonucleotide is complementary to a region in a nucleic acid molecule. In some embodiments, the guide oligonucleotide is complementary to a predetermined region in at least one nucleic acid molecule (e.g., a double-stranded nucleic acid molecule). In some embodiments, the oligonucleotide guides the nuclease (e.g., Cas9) to a cleavage site. In some embodiments, a complex comprising a nuclease and a guide oligonucleotide cleaves a region or a predetermined region of a nucleic acid molecule. In some embodiments, the cleavage is located at a gene. In some embodiments, the method comprises sequencing the cleaved nucleic acid molecule (e.g., a double-stranded nucleic acid molecule) (e.g., sequencing a library of cleaved nucleic acid molecules). In some embodiments, the double-stranded nucleic acid molecule includes a sequence derived from rRNA, tRNA or both rRNA and tRNA. In some embodiments, the predetermined region of at least one double-stranded nucleic acid molecule is a region of rRNA, tRNA or both (e.g., a combination). In some embodiments, the predetermined region is an rRNA region. In some embodiments, the predetermined region is a tRNA region. In some embodiments, the method does not require denaturation of nucleic acid molecules. In some embodiments, the method does not require denaturation of multiple double-stranded nucleic acid molecules. In some embodiments, the double-stranded nucleic acid molecule includes a sequence derived from rRNA and / or tRNA before sequencing. In some embodiments, the double-stranded nucleic acid molecule includes cDNA.

[0160] In one embodiment, the present disclosure relates to a method for preparing a concatemer of nucleic acid molecules. In some embodiments, the method includes processing the ends of a plurality of double-stranded nucleic acid molecules. In some embodiments, the method includes adding a first plurality of adapter molecules to a plurality of double-stranded nucleic acid molecules. In some embodiments, the first plurality of adapter molecules include one or more overhang sequences. In some embodiments, at least two of the one or more overhang sequences are complementary to each other. In some embodiments, the method provides a first plurality of adapters to connect double-stranded nucleic acid molecules. In some embodiments, the method includes adding a polymerase (e.g., adding a polymerase to the first plurality of adapters to connect double-stranded nucleic acid molecules). In some embodiments, the polymerase is added in the absence of a primer. In some embodiments, the method does not include adding a primer. In some embodiments, the polymerase forms a first group of adapters to connect double-stranded nucleic acid concatemers. In some embodiments, two or more adapters are connected to double-stranded nucleic acid molecules by joining two or more than two adapters to form a first group of adapters to connect double-stranded nucleic acid concatemers by one or more overhang sequences. In some embodiments, the method includes adding a second plurality of adapter molecules to the first group (e.g., the first adapter molecule). In some embodiments, the second plurality of adapter molecules include one or more overhang sequences. In some embodiments, at least two of the one or more overhang sequences are complementary to each other. In some embodiments, the method provides a second group of adapters to connect double-stranded nucleic acid molecules. In some embodiments, any of the previous embodiments can be repeated utilizing a set of adaptor molecules, resulting in concatemers comprising a predetermined average length.

[0161] In one embodiment, the present disclosure relates to a method for preparing a concatemer of nucleic acid molecules. In some embodiments, the method includes performing end repair on at least one nucleic acid molecule and / or multiple double-stranded nucleic acid molecules. In some embodiments, the method includes adding at least one or more adapter molecules to at least one nucleic acid molecule and / or multiple double-stranded nucleic acid molecules. In some embodiments, adding at least one or (first) multiple adapter molecules to at least one nucleic acid molecule and / or multiple double-stranded nucleic acid molecules includes joining. In some embodiments, adding at least one or (first) multiple adapter molecules to at least one nucleic acid molecule and / or multiple double-stranded nucleic acid molecules includes reverse transcriptase (e.g., R2 reverse transcriptase or modified reverse transcriptase). In some embodiments, at least one or more adapter molecules include one or more overhang sequences. In some embodiments, at least two overhang sequences are complementary to each other (e.g., thereby providing (first) multiple adapters to connect double-stranded nucleic acid molecules). In some embodiments, at least one or more adapter molecules include a sequence (e.g., overhang sequence) connected / joined to the 3' end of the nucleic acid molecule and / or a sequence (e.g., overhang sequence) connected / joined to the 5' end of the nucleic acid molecule. In some embodiments, the nucleic acid molecule includes adapters on the 3' end and the 5' end. In some embodiments, the adapter bound to the 3' end is complementary to the adapter bound to the 5' end. In some embodiments, the adapter sequence is unknown. In some embodiments, the adapter sequence is predetermined. In some embodiments, the adapter acts as a template and / or primer. In some embodiments, the adapter bound to the 3' end of a nucleic acid molecule can be bound to the adapter on the 5' end of another nucleic acid molecule. In some embodiments, the method further includes adding a polymerase to the adapter connected to the nucleic acid molecule. In some embodiments, the method further includes adding a polymerase to the (first) plurality of adapters to connect double-stranded nucleic acid molecules. In some embodiments, a polymerase is added in the absence of a primer. In some embodiments, the polymerase connects two or more than two adapters to form a first group of adapters to connect double-stranded nucleic acid concatemers by one or more overhang sequences. In some embodiments, the polymerase allows the adapters connected to the nucleic acid molecules to form concatemers. In some embodiments, the method includes adding a second plurality of adapter molecules to the first group. In some embodiments, the second plurality of adapter molecules include one or more overhang sequences. In some embodiments, at least two overhang sequences are complementary to each other. In some embodiments, a second group of adapters are formed to connect double-stranded nucleic acid molecules. In some embodiments, the concatemer length or the number of ligated templates can be determined, for example, by tagging the adaptors with modified nucleotides (e.g., by introducing methylated nucleotides or by inserting dUTP). In some embodiments, the concatemer length can be adjusted based on the ratio between modified / unmodified adaptors.In some embodiments, the adapter sequence can act as a homology priming position (annealing to the homology point ssDNA fragment, acting as a template and primer). In some embodiments, the method includes amplifying the concatemer by PCR or an isothermal reaction. In some embodiments, the PCR reaction undergoes a selected number of cycles (the more cycles, the longer the concatemer) or a selected time (isothermal amplification). In some embodiments, the reaction is stopped and the (long) dsDNA concatemer is joined with two unique dsDNA adapters. In some embodiments, the length of the concatemer can be manipulated. In some embodiments, the length of the concatemer can be determined based on at least the number of PCR cycles and / or the amount of time (e.g., time in isothermal amplification) and / or based on the modified nucleotides present in the adapter. In some embodiments, the adapter includes a unique molecular recognition sequence (UMI). In some embodiments, the polymerase connects two or more adapters to double-stranded nucleic acid molecules in a PCR or isothermal amplification reaction. In some embodiments, the adapter includes at least one modified nucleotide.

[0162] In some embodiments, amplify nucleic acids from biological samples obtained from subjects. In some cases, biological samples are obtained directly from subjects. In some embodiments, biological samples obtained directly from subjects refer to biological samples that have been further processed after being obtained from subjects. In some embodiments, biological samples obtained directly from subjects refer to biological samples that have not been further processed after being obtained from subjects, but any method can be used to collect biological samples from subjects for further processing. For example, blood is obtained directly from subjects by accessing the circulatory system of the subject, removing blood from the subject (e.g., via a needle) and injecting the removed blood into a container. The container may include reagents (e.g., anticoagulants) so that the blood sample is suitable for further analysis. In another example, a swab can be used to obtain epithelial cells on the surface of the oropharynx of a subject. After obtaining a biological sample from a subject, a swab containing a biological sample can be contacted with a fluid (e.g., a buffer) to collect biological fluids from the swab.

[0163] In some embodiments, the biological sample has been purified. In some embodiments, the biological sample has not been purified. In some embodiments, when the biological sample is provided to the test tube, the nucleic acid of the biological sample has not yet been extracted. For example, when the biological sample is provided to the test tube, the RNA or DNA in the biological sample may not have been extracted from the biological sample. In some embodiments, before the biological sample is provided to the reaction vessel (e.g., a test tube), the target nucleic acid (e.g., target RNA or target DNA) present in the biological sample may not be concentrated. Any suitable biological sample including nucleic acid can be obtained from the subject.

[0164] The present disclosure relates to a non-naturally occurring or modified enzyme (e.g., a non-naturally occurring or modified reverse transcriptase, a modified reverse transcriptase) or a modified polypeptide having reverse transcriptase activity, which has improved enzymatic properties compared to a naturally occurring or wild-type or unmodified enzyme (e.g., a wild-type reverse transcriptase) or an unmodified polypeptide having reverse transcriptase activity. In some embodiments, the non-naturally occurring or modified enzyme is an enzyme having reverse transcriptase activity. In some embodiments, the non-naturally occurring or modified enzyme is a modified reverse transcriptase. In some embodiments, the non-naturally occurring or modified enzyme is a modified non-retroviral reverse transcriptase. In some embodiments, the non-naturally occurring or modified enzyme is a modified non-LTR retrotransposon. In some embodiments, the non-naturally occurring or modified enzyme is a modified R2 reverse transcriptase. In some embodiments, the non-naturally occurring or modified enzyme or modified polypeptide having reverse transcriptase activity can amplify the template nucleic acid molecule with a processivity of at least about 80% per base, at least about 85% per base, at least about 88% per base, at least about 89% per base, at least about 90% per base, at least about 91% per base, at least about 92% per base, at least about 93% per base, at least about 94% per base, at least about 95% per base, at least about 96% per base, at least about 97% per base, at least about 98% per base, at least about 99% per base, at least about 99.5% per base, or at least about 100% per base.

[0165] In some embodiments, the non-naturally occurring or modified enzyme or modified polypeptide having reverse transcriptase activity can or is capable of amplifying a template nucleic acid molecule with processivity measured at a temperature between about 12° C. and about 40° C. In some embodiments, the temperature is between about 10° C. and about 35° C., between about 12° C. and about 30° C., between about 25° C. and about 40° C., or between about 12° C. and about 42° C. In some embodiments, the temperature is between about 8° C. and about 50° C., between about 2° C. and about 60° C., between about 8° C. and about 42° C., between about 6° C. and about 32° C., or between about 7° C. and about 35° C.

[0166] In some embodiments, the non-naturally occurring or modified enzyme or modified polypeptide having reverse transcriptase activity can amplify or is capable of amplifying a template nucleic acid molecule with a processivity of at least about 80% per base at a temperature of about or up to about 4°C, about or up to about 8°C, about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, or about or up to about 42°C; or at least about 85% per base at a temperature of about 35°C, about or up to about 40°C, about or up to about 42°C, about or up to about 45°C, about or up to about 50°C; at least about 89% per base at a temperature of about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, or about or up to about 42°C; at least about 89% per base at a temperature of about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, or about or up to about 42°C; at least about 90% of each base at a temperature of about or up to about 4°C, about or up to about 45°C, about or up to about 50°C; at least about 91% of each base at a temperature of about or up to about 4°C, about or up to about 8°C, about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, or about or up to about 42°C; at least about 91% of each base at a temperature of about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, about or up to about 42°C at least about 85% of each base at a temperature of about 50°C; at least about 95% of each base at a temperature of about or up to about 4°C, about or up to about 8°C, about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, or about or up to about 42°C; at least about 85% of each base at a temperature of about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, about or up to about 42°C, about or up to about 45°C, about or up to about 50°C;At least about 99% of each base at a temperature of about or up to about 4°C, about or up to about 8°C, about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, or about or up to about 42°C; at least about 99% of each base at a temperature of about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, or about or up to about 42°C; at least about 85% per base at a temperature of about or up to about 40°C, about or up to about 42°C, about or up to about 45°C, about or up to about 50°C; at least about 99.5% per base at a temperature of about or up to about 4°C, about or up to about 8°C, about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, or about or up to about 42°C; At least about 85% of each base at a temperature of about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, about or up to about 42°C, about or up to about 45°C, about or up to about 50°C; or at least about 85% of each base at a temperature of about or up to about 4°C, about or up to about 8°C, about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C , at least about 100% per base at a temperature of about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, or about or up to about 42°C; at least about 85% per base at a temperature of about or up to about 12°C, about or up to about 15°C, about or up to about 20°C, about or up to about 25°C, about or up to about 30°C, about or up to about 35°C, about or up to about 40°C, about or up to about 42°C, about or up to about 45°C, or about or up to about 50°C. ;

[0167] In some embodiments, the non-naturally occurring or modified enzyme or modified polypeptide having reverse transcriptase activity can amplify or is capable of amplifying a template nucleic acid molecule with a processivity of at least about 80% per base at a temperature up to about 35°C; at least about 85% per base at a temperature up to about 40°C; at least about 88% per base at a temperature up to about 35°C; at least about 89% per base at a temperature up to about 40°C; at least about 90% per base at a temperature up to about 35°C; at least about 91% per base at a temperature up to about 40°C; at least about 92% per base at a temperature up to about 35°C; at least about 93% per base at a temperature up to about 40°C; at least about 94% per base at a temperature up to about 40°C; at least about 95% per base at a temperature up to about 35°C; at least about 96% per base at a temperature up to about 40°C; at least about 97% per base at a temperature up to about 35°C; at least about 98% per base at a temperature up to about 40°C; at least about 99% per base at a temperature up to about 40°C; at least about 99.5% per base at a temperature up to about 40°C; or at least about 100% per base at a temperature up to about 40°C.

[0168] In some embodiments, the improved enzyme property is selected from at least one of the following: improved stability (e.g., improved thermostability), improved specific activity, improved protein expression, improved purification, improved processivity, improved strand displacement, improved template hopping, improved DNA / RNA affinity, and improved fidelity. In some embodiments, the non-naturally occurring enzyme or modified enzyme or modified polypeptide having reverse transcriptase activity amplifies the template nucleic acid molecule. In some embodiments, the non-naturally occurring enzyme or modified enzyme or modified polypeptide having reverse transcriptase activity that amplifies the template nucleic acid molecule has a performance index greater than about 1, greater than about 2, greater than about 3, greater than about 4, greater than about 5, greater than about 6, greater than about 7, greater than about 8, greater than about 9, greater than about 10, greater than about 15, greater than about 20, greater than about 25, greater than about 30, greater than about 35, greater than about 40, greater than about 45, greater than about 50, greater than about 60, greater than about 70, greater than about 80, greater than about 90, or greater than about 100 for at least one enzyme property. In some embodiments, the enzyme properties and / or performance indicators are achieved at temperatures of at least about 50°C, at least about 42°C, at least about 40°C, at least about 39°C, at least about 38°C, at least about 37°C, at least about 36°C, at least about 35°C, at least about 34°C, at least about 33°C, at least about 32°C, at least about 31°C, at least about 32°C, at least about 33°C, at least about 34°C, at least about 35°C, at least about 36°C, at least about 37 ... ℃, equal to or lower than or up to about 30℃, equal to or lower than or up to about 29℃, equal to or lower than or up to about 28℃, equal to or lower than or up to about 27℃, equal to or lower than or up to about 26℃, equal to or lower than or up to about 25℃, equal to or lower than or up to about 23℃, equal to or lower than or up to about 20℃, equal to or lower than or up to about 15℃, equal to or lower than or up to about 13℃, equal to or lower than or up to about 12℃, equal to or lower than or up to about 10℃, equal to or lower than or up to about 8℃, equal to or lower than or up to about 4℃.In some embodiments, the non-naturally occurring enzyme or modified enzyme (e.g., a modified reverse transcriptase) or modified polypeptide having reverse transcriptase activity exhibits a processivity for a given nucleotide substrate that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 37.5%, at least about 40%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 56%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 10 ... In some embodiments, the non-naturally occurring or modified enzyme is a non-naturally occurring reverse transcriptase. In some embodiments, the modified enzyme is a modified reverse transcriptase.

[0169] The present disclosure relates to processes and / or methods that require significantly less hands-on time, for example, the execution of the process is much simpler and the duration required is much shorter than other methods for RNA sequencing and / or liquid biopsy. In some embodiments, the methods and processes of the present disclosure include a process less than about 2 hours and / or a hands-on time less than about 30 minutes. In some embodiments, the process is less than about 20 hours, less than about 15 hours, less than about 12 hours, less than about 11 hours, less than about 10 hours, less than about 9 hours, less than about 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour or less than about 30 minutes. In some embodiments, the hands-on time is less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, or less than about 15 minutes.

[0170] In some embodiments, the method for preparing a nucleic acid library and / or a complementary cDNA library comprises preparing the library in at most about 1 hour, at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 7 hours, at most about 10 hours, at most about 15 hours, or at most about 20 hours.

[0171] In one embodiment, the present disclosure relates to methods and processes that enable discovery of novel markers and mutations for cancer, and / or provide methods for precision medicine. In some embodiments, the methods and processes disclosed herein provide a higher sensitivity of <0.1% for capturing minor alleles in ctDNA (current methods available have a sensitivity of >1%). In some embodiments, the methods and / or processes of the present disclosure comprise a one-pot (e.g., single container) method, a 1-step process, and prepare a library from a sample in an amount of time equal to or less than about 2 hours. See Figure 2 .

[0172] The present disclosure relates to a method for preparing a modified reverse transcriptase, the method comprising at least one of the following steps: (a) random or rational mutation of a nucleic acid sequence encoding a reverse transcriptase; (b) amino acid truncation of a nucleic acid sequence encoding a reverse transcriptase; (c) variation of a nucleic acid sequence encoding a reverse transcriptase, including insertion, deletion or substitution of amino acid residues; and (d) fusion of a nucleic acid sequence encoding a reverse transcriptase with a protein or domain. In some embodiments, the nucleic acid sequence obtained in any one of steps (a) to (d) is expressed in a host cell. In some embodiments, the modified reverse transcriptase comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some embodiments, the nucleic acid sequence is a combination of DNA, RNA, or RNA and DNA. In some embodiments, the method comprises screening a host cell expressing a modified reverse transcriptase. In some embodiments, the method comprises preparing a modified reverse transcriptase expressed by a host cell. In some embodiments, the method may comprise purifying a modified reverse transcriptase according to any method comprising any method disclosed herein. In some embodiments, the method may include determining reverse transcriptase activity, estimating reverse transcriptase activity fractions, and / or testing the stability and / or robustness of a modified reverse transcriptase. In some embodiments, reverse transcriptase activity is determined, estimated, and / or tested using a reverse transcriptase activity assay, or tested using a reverse transcriptase activity assay. In some embodiments, reverse transcriptase activity, reverse transcriptase activity fractions, and / or the stability and / or robustness of a modified reverse transcriptase are increased / improved compared to an unmodified or naturally occurring reverse transcriptase.

[0173] The present disclosure relates to a method for preparing complementary deoxyribonucleic acid (cDNA) molecules. In some embodiments, the method includes annealing a primer to a template nucleic acid molecule, thereby producing an annealed template nucleic acid molecule. In some embodiments, the method further includes mixing the annealed template nucleic acid molecule, one or more receptor nucleic acid molecules and a modified reverse transcriptase in the presence of nucleotides. In some embodiments, the modified reverse transcriptase produces a plurality of continuous complementary deoxyribonucleic acid molecules. In some embodiments, a plurality of continuous complementary deoxyribonucleic acid molecules are prepared within a maximum of about 2 hours. In some embodiments, a plurality of continuous complementary deoxyribonucleic acid molecules are produced by reversely transcribing the sequence of the annealed template nucleic acid molecule with a modified reverse transcriptase. In some embodiments, the modified reverse transcriptase then migrates to a receptor nucleic acid molecule (e.g., one or more receptor nucleic acid molecules). In some embodiments, the reverse transcriptase (e.g., a modified reverse transcriptase) is capable of reverse transcribing the sequence of the template and / or receptor nucleic acid molecule at a temperature of about 12° C. to about 42° C. In some embodiments, the reverse transcriptase (e.g., a modified reverse transcriptase) is capable of reverse transcribing the sequence of a template and / or a receptor nucleic acid molecule at a temperature of about 8°C to about 50°C (e.g., about 8°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 48°C). In some embodiments, the reverse transcriptase (e.g., a modified reverse transcriptase) is capable of reverse transcribing the sequence of a template and / or a receptor nucleic acid molecule at a temperature of up to about 4°C, up to about 8°C, up to about 15°C, up to about 20°C, up to about 25°C, up to about 30°C, up to about 35°C, up to about 40°C, up to about 45°C, or up to about 48°C. In some embodiments, reverse transcription occurs at an error rate of up to about 5%. In some embodiments, the reverse transcriptase (e.g., a modified reverse transcriptase) is capable of reverse transcribing the template and / or the receptor nucleic acid molecule with an error rate of up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, or up to about 1%. In some embodiments, the reverse transcriptase (e.g., a modified reverse transcriptase) can migrate from the template to the receptor nucleic acid molecule regardless of the sequence identity between the template and the receptor nucleic acid molecule. In some embodiments, the method is prepared in a single container. In some embodiments, the template nucleic acid molecule is a fragmented DNA template, a fragmented RNA template, a non-fragmented DNA template, a non-fragmented RNA template, or a combination thereof. In some embodiments, the method further includes adding a tag to the template nucleic acid molecule, thereby generating a plurality of continuous complementary deoxyribonucleic acid molecules with tags. In some embodiments, the method further includes performing a polymerase chain reaction amplification reaction, thereby forming one or more amplicons.

[0174] The present disclosure relates to methods for preparing complementary deoxyribonucleic acid (cDNA) molecules using modified reverse transcriptases. In some embodiments, the methods for preparing cDNA molecules are performed by template jumping. In some embodiments, the modified reverse transcriptase has improved enzymatic properties compared to a naturally occurring or unmodified or wild-type enzyme (e.g., a wild-type reverse transcriptase). In some embodiments, the method for preparing cDNA molecules comprises: (a) annealing a primer to a template; and (b) mixing the template annealed to the primer with the modified reverse transcriptase with a receptor nucleic acid molecule (e.g., a receptor RNA, DNA, or a combination thereof) in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce a cDNA molecule complementary to the template and / or a receptor nucleic acid molecule. Figure 3 ). In some embodiments, the enzyme (e.g., a modified reverse transcriptase) generates continuous cDNA molecules by migrating from the template to the receptor nucleic acid molecule. In some embodiments, template jumping is independent of the sequence identity between the template and the receptor nucleic acid molecule. In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, step (a) includes step (b) (e.g., step (a) and step (b) are combined into one step). In some embodiments, at least one of steps (a) and / or step (b) further includes adding a hot start thermostable polymerase. In some embodiments, the method of the present disclosure is performed in a single test tube. In some embodiments, the method of the present disclosure further includes a polymerase chain reaction (PCR) amplification reaction. In some embodiments, the PCR amplification reaction is performed in a single test tube (e.g., the same test tube from steps (a) and (b)). In some embodiments, all steps of the method of the present disclosure are performed in a single test tube.

[0175] The present disclosure relates to a method for preparing a concatemer of nucleic acid molecules for sequencing. In some embodiments, the method includes joining a nucleic acid molecule with a first adapter. In some embodiments, the method further includes forming a concatemer by amplifying the joined nucleic acid molecule by performing a nucleic acid amplification reaction. In some embodiments, an amplification reaction is performed in the absence of a primer. In some embodiments, the method further includes joining a concatemer with a second adapter. In some embodiments, the adapter (first and / or second adapter) is designed to allow recombination of molecules (e.g., nucleic acid molecules, and / or templates, and / or primers, and / or receptors) or annealing and extension based on homology. In some embodiments, the nucleic acid amplification reaction is a polymerase chain reaction (PCR) or isothermal amplification. In some embodiments, the first adapter includes a unique molecular identification (UMI) sequence. In some embodiments, the first adapter acts as a primer. In some embodiments, the first adapter includes a single-stranded nucleic acid. In some embodiments, the single-stranded nucleic acid includes single-stranded DNA (ssDNA). In some embodiments, the second adapter includes a double-stranded nucleic acid. In some embodiments, the double-stranded nucleic acid includes double-stranded DNA (dsDNA). In some embodiments, the first adapter is different from the second adapter. In some embodiments, the first adapter includes two or more adapters. In some embodiments, the second adaptor comprises two or more adaptors. In some embodiments, both ends of the nucleic acid molecule comprise adaptors. In some embodiments, only one end of the nucleic acid molecule comprises an adaptor. In some embodiments, both the 3' end and the 5' end of the nucleic acid molecule comprise adaptors.

[0176] The present disclosure relates to methods for preparing complementary deoxyribonucleic acid (cDNA) molecules using modified reverse transcriptases. In some embodiments, the method for preparing cDNA molecules is performed by template jumping. In some embodiments, the modified reverse transcriptase has improved enzyme properties compared to naturally occurring or unmodified or wild-type enzymes (e.g., wild-type reverse transcriptases). In some embodiments, the method for preparing cDNA molecules includes mixing primers, templates, modified reverse transcriptases, and receptor nucleic acid molecules (e.g., receptor RNA, DNA, or a combination thereof) under conditions sufficient to produce cDNA molecules complementary to templates and / or receptor nucleic acid molecules in the presence of nucleotides (e.g., dNTPs). In some embodiments, the method includes adding a hot start thermostable polymerase (e.g., to a mixing step). In some embodiments, the method of the present disclosure is performed in a single test tube. In some embodiments, the method of the present disclosure further includes a polymerase chain reaction (PCR) amplification reaction. In some embodiments, the PCR amplification reaction is performed in a single test tube (e.g., a test tube identical to the mixing step). In some embodiments, all steps of the method of the present disclosure are performed in a single test tube (single container).

[0177] In some embodiments, the method for preparing a cDNA molecule comprises: (a) annealing one or more primers to a template; and (b) mixing the template annealed to the one or more primers with a modified reverse transcriptase with a receptor nucleic acid molecule (e.g., receptor RNA, DNA, or a combination thereof) in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce a cDNA molecule complementary to the template and / or the receptor nucleic acid molecule. Figure 4 ). In some embodiments, the method for preparing a cDNA molecule is performed by template jumping. In some embodiments, step (a) and step (b) are performed simultaneously. In some embodiments, step (a) includes step (b) (e.g., step (a) and step (b) are combined into one step). In some embodiments, at least one of step (a) and / or step (b) further includes adding a hot start thermostable polymerase. In some embodiments, the method of the present disclosure is performed in a single test tube. In some embodiments, the method of the present disclosure further includes a polymerase chain reaction (PCR) amplification reaction. In some embodiments, the PCR amplification reaction is performed in a single test tube (e.g., the same test tube used in steps (a) and (b) or from steps (a) and (b)). In some embodiments, all steps of the disclosed method are performed in a single test tube.

[0178] In some embodiments, the method for preparing cDNA molecules includes mixing one or more primers, templates, modified reverse transcriptases and receptor nucleic acid molecules (e.g., receptor RNA, DNA, or a combination thereof) under conditions sufficient to produce cDNA molecules complementary to templates and / or receptor nucleic acid molecules in the presence of nucleotides (e.g., dNTPs). In some embodiments, the method for preparing cDNA molecules is performed by template jump. In some embodiments, the method includes adding a hot start thermostable polymerase (e.g., to a mixing step). In some embodiments, the method of the present disclosure is performed in a single test tube. In some embodiments, the method of the present disclosure further includes a polymerase chain reaction (PCR) amplification reaction. In some embodiments, the PCR amplification reaction is performed in a single test tube (e.g., a test tube identical to the mixing step). In some embodiments, all steps of the disclosed method are performed in a single test tube.

[0179] The present disclosure relates to a method for preparing a nucleic acid molecule, comprising: mixing a fragment or degraded template (e.g., a nucleic acid fragment), a primer, a modified reverse transcriptase, and a receptor nucleic acid molecule in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce the nucleic acid molecule. Figure 5A and Figure 5B). In some embodiments, the receptor nucleic acid molecule comprises a modified nucleotide. In some embodiments, primer extension stops at a modified nucleotide. In some embodiments, the modified reverse transcriptase comprises at least one improved enzymatic property relative to a wild-type, naturally occurring or unmodified reverse transcriptase. In some embodiments, the primer is an RNA primer. In some embodiments, the primer is an engineered primer (e.g., an engineered RNA primer). In some embodiments, the primer has been optimized. In some embodiments, the primer is an optimized and / or engineered primer (e.g., an optimized and / or engineered RNA primer). In some embodiments, the primer is an RNA R2 primer. In some embodiments, the method for preparing a nucleic acid molecule is performed by template jumping. In some embodiments, the mixing step of the disclosed method further comprises adding a hot start thermostable polymerase. In some embodiments, the disclosed method is performed in a single test tube. In some embodiments, the disclosed method further comprises a polymerase chain reaction (PCR) amplification reaction. In some embodiments, the PCR amplification reaction is performed in the same single test tube. In some embodiments, all steps of the disclosed method are performed in a single test tube.

[0180] The present disclosure relates to a method for preparing a nucleic acid molecule, comprising: mixing a fragment or degraded template (e.g., a nucleic acid fragment), a donor complex, a modified reverse transcriptase, and an acceptor nucleic acid molecule in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce the nucleic acid molecule. Fig. 6A and Figure 6B ). In some embodiments, the recipient nucleic acid molecule comprises a modified nucleotide. In some embodiments, primer extension stops at the modified nucleotide. In some embodiments, the modified reverse transcriptase comprises at least one improved enzymatic property relative to a wild-type or naturally occurring or unmodified reverse transcriptase. In some embodiments, the donor complex comprises a template and a primer. In some embodiments, the donor complex is a donor R2 complex. In some embodiments, the donor R2 complex comprises an RNA R2 primer. In some embodiments, the method for preparing a nucleic acid molecule is performed by template jumping. In some embodiments, the mixing step of the disclosed method further comprises adding a hot start thermostable polymerase. In some embodiments, the disclosed method is performed in a single test tube. In some embodiments, the disclosed method further comprises a polymerase chain reaction (PCR) amplification reaction. In some embodiments, the PCR amplification reaction is performed in the same single test tube (e.g., the same single test tube used to prepare the nucleic acid molecule). In some embodiments, all steps of the disclosed method are performed in a single test tube.

[0181] The present disclosure relates to methods for preparing complementary deoxyribonucleic acid (cDNA) libraries using modified reverse transcriptases. In some embodiments, the methods for preparing cDNA libraries use template jumping. In some embodiments, the modified reverse transcriptase has improved enzymatic properties compared to naturally occurring or wild-type or unmodified enzymes (e.g., wild-type reverse transcriptases). In some embodiments, the methods for preparing cDNA libraries include: (a) annealing a primer or one or more primers to a template; and (b) mixing a template annealed to a primer or a template annealed to one or more primers with a receptor nucleic acid molecule (e.g., receptor RNA, DNA, or a combination thereof) in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce cDNA molecules complementary to the template and / or receptor nucleic acid molecules. In some embodiments, the methods for preparing cDNA libraries include mixing a primer or one or more primers, a template, a modified reverse transcriptase, and a receptor nucleic acid molecule (e.g., receptor RNA, DNA, or a combination thereof) in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce cDNA molecules complementary to the template and / or receptor nucleic acid molecules. In some embodiments, the enzyme (e.g., a modified reverse transcriptase) generates continuous cDNA molecules by migrating from a template to a receptor nucleic acid molecule. In some embodiments, template jump is independent of the sequence identity between the template and the receptor nucleic acid molecule. In some embodiments, the method further includes amplifying cDNA molecules to produce a cDNA library. In some embodiments, step (a) and step (b) are performed simultaneously. In some embodiments, step (a) includes step (b) (e.g., step (a) and step (b) are combined into one step). In some embodiments, the mixing step or at least one of step (a) and / or step (b) further includes adding a hot start thermostable polymerase. In some embodiments, the method of the present disclosure is performed in a single test tube. In some embodiments, the method of the present disclosure further includes a polymerase chain reaction (PCR) amplification reaction. In some embodiments, the PCR amplification reaction is performed in a single test tube (e.g., for a mixing step or from a mixing step, or in steps (a) and (b) or from the same test tube of steps (a) and (b)). In some embodiments, all steps of the disclosed method are performed in a single test tube.

[0182] The present disclosure relates to a method for preparing a cDNA and / or DNA library, comprising mixing a fragment or degraded template (e.g., a nucleic acid fragment), a primer, a modified reverse transcriptase, and a receptor nucleic acid molecule under conditions sufficient to produce a nucleic acid (e.g., cDNA and / or DNA) molecule in the presence of nucleotides (e.g., dNTPs). In some embodiments, the receptor nucleic acid molecule comprises a modified nucleotide. In some embodiments, primer extension stops at a modified nucleotide. In some embodiments, the modified reverse transcriptase comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some embodiments, the primer is an RNA R2 primer. In some embodiments, the method further comprises amplifying a nucleic acid (e.g., cDNA and / or DNA) molecule to produce a cDNA library. In some embodiments, the method for preparing cDNA and / or DNA and / or nucleic acid molecules is performed by template jumping.

[0183] The present disclosure relates to a method for preparing a cDNA and / or DNA library, comprising: mixing a fragment or degraded template (e.g., a nucleic acid fragment), a donor complex, a modified reverse transcriptase, and an acceptor nucleic acid molecule in the presence of nucleotides (e.g., dNTPs) under conditions sufficient to produce nucleic acid (e.g., cDNA and / or DNA) molecules. In some embodiments, the acceptor nucleic acid molecule comprises a modified nucleotide. In some embodiments, primer extension stops at a modified nucleotide. In some embodiments, the modified reverse transcriptase comprises at least one improved enzymatic property relative to a wild-type or unmodified reverse transcriptase. In some embodiments, the donor complex comprises a template and a primer. In some embodiments, the donor complex is a donor R2 complex. In some embodiments, the donor R2 complex comprises an RNA R2 primer. In some embodiments, the method further comprises amplifying nucleic acid (e.g., cDNA and / or DNA) molecules to produce a cDNA and / or DNA library. In some embodiments, the method for preparing cDNA and / or DNA and / or nucleic acid molecules is performed by template jumping.

[0184] In some embodiments, the methods of the present disclosure may include a donor complex. In some embodiments, the donor complex includes a template and a primer. In some embodiments, the methods of the present disclosure may include a template. In some embodiments, the template is a fragmented and / or degraded template. In some embodiments, the template is not fragmented. In some embodiments, the template is RNA, DNA, or a combination of DNA and RNA. In some embodiments, the RNA is mRNA. In some embodiments, the template is mRNA.

[0185] The present disclosure relates to a method for preparing a library for sequencing, comprising: (a) obtaining a sample with free nucleic acid from a subject; and (b) adding a modified reverse transcriptase, a template (e.g., a nucleic acid template), nucleotides, a receptor nucleic acid molecule, and one or more primers to the nucleic acid. In some embodiments, the method further comprises performing an amplification reaction on the free nucleic acid (cf nucleic acid) derived from the sample to produce a plurality of amplicons. In some embodiments, the amplification reaction comprises 35 or fewer amplification cycles. In some embodiments, the method comprises generating a library for sequencing. In some embodiments, the library comprises a plurality of amplicons. In some embodiments, the modified reverse transcriptase is capable of template hopping and / or comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some embodiments, the nucleic acid is a combination of DNA, RNA, or RNA and DNA.

[0186] The present disclosure relates to a method for preparing complementary deoxyribonucleic acid (cDNA) molecules using template jumping, which includes mixing a primer or one or more primers, a messenger RNA (mRNA) template, nucleotides, a modified reverse transcriptase, a receptor nucleic acid molecule and a catalytic metal in a single test tube under conditions sufficient to produce continuous cDNA molecules. In some embodiments, the continuous cDNA molecules are complementary to the mRNA template and / or the receptor nucleic acid molecule. In some embodiments, the modified reverse transcriptase includes at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some embodiments, continuous cDNA molecules are produced. In some embodiments, the modified reverse transcriptase undergoes migration from the template to the receptor nucleic acid molecule.

[0187] The present disclosure relates to a method for preparing a library for sequencing, comprising mixing free nucleic acid, a modified reverse transcriptase, a template, nucleotides, an acceptor nucleic acid molecule, a catalytic metal, and one or more primers in a single test tube under conditions sufficient to produce a library. In some embodiments, the modified reverse transcriptase comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase.

[0188] In some embodiments, the nucleic acid molecule includes an unknown nucleic acid sequence. In some embodiments, the template includes an unknown nucleic acid sequence. In some embodiments, the migration of the template to the receptor nucleic acid molecule is independent of the sequence identity between the template and the receptor nucleic acid molecule. In some embodiments, the receptor nucleic acid molecule includes modified nucleotides that can stop primer extension. In some embodiments, the free nucleic acid is free DNA (cfDNA), circulating tumor DNA (ctDNA) and / or formalin-fixed paraffin-embedded DNA (FFPE DNA) or a combination thereof.

[0189] In some embodiments, the disclosed method can add a hot start thermostable polymerase at or before any step of the method and / or while performing a mixing step. For example, a hot start thermostable polymerase can be added while a modified reverse transcriptase is added to the reaction. A hot start thermostable polymerase can be added while a receptor nucleic acid molecule is added, and / or while a template and / or primer and / or reverse transcriptase and / or nucleotides are added to a reaction tube. In some embodiments, a hot start thermostable polymerase is added before the start of the PCR reaction. In some embodiments, a hot start thermostable polymerase is added before or while the RT reaction. In some embodiments, the hot start thermostable polymerase is a hot start Taq polymerase. Amplification of the target nucleic acid can occur on beads. In some embodiments, amplification does not occur on beads. Amplification can be performed by isothermal amplification, such as isothermal linear amplification. In some embodiments, a hot start PCR can be performed, wherein the reaction is heated to 95°C before adding the polymerase, for example for two minutes, or the polymerase can be kept inactive until the first heating step in the first cycle. Hot start PCR can be used to minimize nonspecific amplification.

[0190] In some embodiments, the disclosed method is performed in a single test tube. In some embodiments, all steps of the disclosed method are performed in a single test tube. In some embodiments, the method (from start to finish) is performed in a single test tube. In some embodiments, the same test tube used for RT reaction is used for PCR amplification reaction.

[0191] In some embodiments, PCR amplification is performed at a temperature sufficient to inactivate the reverse transcriptase. In some embodiments, PCR amplification is performed at a temperature sufficient to activate a hot-start thermostable polymerase.

[0192] The present disclosure relates to methods for amplifying free nucleic acid molecules of a sample. In some embodiments, the sample is a biological sample. In some embodiments, nucleic acid amplification including reverse transcriptase (e.g., modified reverse transcriptase) is performed on the free nucleic acid molecules. In some embodiments, nucleic acid amplification including reverse transcriptase (e.g., modified reverse transcriptase) is performed on the free nucleic acid molecules under conditions of amplifying nucleic acid molecules with a specified continuous synthesis capacity. In some embodiments, the processivity is at least about 80% per base, at least about 81% per base, at least about 82% per base, at least about 83% per base, at least about 84% per base, at least about 85% per base, at least about 86% per base, at least about 87% per base, at least about 88% per base, at least about 89% per base, at least about 90% per base, at least about 91% per base, at least about 92% per base, at least about 93% per base, at least about 94% per base, at least about 95% per base, at least about 96% per base, at least about 97% per base, at least about 98% per base, at least about 99% per base, or at least about 100% per base. In some embodiments, processivity is performed at a temperature of about or up to about or down to about 12°C, about or up to about or down to about 13°C, about or up to about or down to about 14°C, about or up to about or down to about 15°C, about or up to about or down to about 16°C, about or up to about or down to about 17°C, about or up to about or down to about 18°C, about or up to about or down to about 19°C, about or up to about or down to about 20°C, about or up to about or down to about 21°C, about or up to about or down to about 22°C, about or up to about or down to about 23°C, about or up to about or down to about 24°C, about or up to about or down to about 25°C, about or up to about or down to about 26°C, about or up to about or down to about 27°C, about or up to about or down to about 28°C, about or up to about or down to about 29°C, about or up to about or down to about 30°C, about or up to about or down to about 31°C, about or up to about or down to about 32°C, about or up to about or down to about 33°C, about or up to about or down to about 34°C, about or up to about or down to about 35°C, about or up to about or down to about 36°C, about or up to about or down to about 37°C, about or up to about or down to about 38°C, about or up to about about or at most about or at least about 39°C, about or at most about 40°C, about or at most about 45°C, about or at most about or at least about 50°C, about or at most about or at least about 60°C, about or at most about or at least about 70°C, about or at most about or at least about 80°C, about or at most about or at least about 90°C, about or at most about or at least about 100°C, about or at most about or at least about 110°C, about or at most about or at least about 120°C, about or at most about or at least about 130°C, about or at most about or at least about 140°C, about or at most about or at least about 150°C, about or at most about or at least about 160°C, about or at most about or at least about 170°C, about or at most about or at least about 180°C, about or at most about or at least about 190°C.In some embodiments, the processivity is at least about 80% per base, at least about 81% per base, at least about 82% per base, at least about 83% per base, at least about 84% per base, at least about 85% per base, at least about 86% per base, at least about 87% per base, at least about 88% per base, at least about 89% per base, at least about 90% per base, at least about 91% per base, at least about 92% per base, at least about 93% per base, at least about 94% per base, at least about 95% per base, at least about 96% per base, at least about 97% per base, at least about 98% per base, at least about 99% per base, or at least about 100% per base at a temperature of about or up to about or down to about 30°C, about or up to about or down to about 12°C, about or up to about or down to about 45°C, about or up to about or down to about 35°C. In some embodiments, the reverse transcriptase is a non-LTR retrotransposon or a modified non-LTR retrotransposon. In some embodiments, the reverse transcriptase is an R2 reverse transcriptase or a modified R2 reverse transcriptase. In some embodiments, the reverse transcriptase is an R2 non-LTR retrotransposon or a modified R2 non-LTR retrotransposon.

[0193] The present disclosure relates to a method for preparing a complementary deoxyribonucleic acid (cDNA) library and / or a DNA library from a plurality of single cells. In some embodiments, the method comprises the following steps: stripping nucleic acid from each single cell to provide a plurality of individual nucleic acid samples. In some embodiments, the nucleic acid in each individual nucleic acid sample is from a single cell. In some embodiments, the method further comprises annealing the nucleic acid template to one or more primers. In some embodiments, the method further comprises mixing a nucleic acid template annealed to one or more primers using an acceptor template (or acceptor nucleic acid molecule) and a modified reverse transcriptase under conditions that effectively produce cDNA and / or DNA molecules in the presence of nucleotides. In some embodiments, the modified reverse transcriptase is capable of template hopping and / or comprises at least one enzyme property that is improved relative to a wild-type or unmodified reverse transcriptase. In some embodiments, the method further comprises amplifying cDNA molecules and / or DNA molecules to produce cDNA and / or DNA libraries.

[0194] The present disclosure relates to methods for detecting nucleic acid molecules. In some embodiments, the method includes mixing a sample including nucleic acid molecules with an acceptor template (or acceptor nucleic acid molecule), a modified reverse transcriptase, a primer, and nucleotides under conditions effective to produce nucleic acid molecules. In some embodiments, the modified reverse transcriptase includes at least one improved enzymatic property relative to a wild-type or unmodified reverse transcriptase. In some embodiments, the acceptor template (or acceptor nucleic acid molecule) includes at least one modified nucleotide. In some embodiments, the modified nucleotide can stop primer extension. In some embodiments, the method further includes amplifying the nucleic acid molecule.

[0195] The present disclosure relates to a method for detecting, diagnosing and / or prognosing a disease (e.g., cancer) in a subject, comprising: (a) obtaining sequence information of a nucleic acid sample (e.g., a free nucleic acid sample) derived from a subject, and (b) detecting circulating tumor nucleic acids in the sample using the sequence information derived from step (a). In some embodiments, obtaining sequence information according to step (a) includes using one or more adapters. In some embodiments, one or more adapters include a molecular barcode. The adapter may include one or more terminal modifications. The adapter may include a 5' phosphate. The adapter may include two 5' phosphates. The adapter may include a 3' hydroxyl. The adapter may include two 3' hydroxyls. The adapter may lack a 3' hydroxyl.

[0196] In some embodiments, the molecular barcode comprises a random sequence. In some embodiments, the method is capable of detecting free nucleic acid that is less than or equal to about 0.75%, 0.50%, 0.25%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.0 ... 0.0005%, or 0.00001%, 1%, 1.75%, 1.5%, 1.25%, 2%, 3%, 4%, 5%, 6%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. In some embodiments, the methods are capable of detecting circulating tumor nucleic acids that are less than or equal to about 0.75%, 0.50%, 0.25%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0005%, or 0.006% of the total circulating nucleic acids. 0.00001%, 1%, 1.75%, 1.5%, 1.25%, 2%, 3%, 4%, 5%, 6%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. In some embodiments, the method is capable of detecting a percentage of circulating tumor nucleic acid (ct nucleic acid) that is less than or equal to 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.50%, 0.25%, 0.1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0005% or 0.00001% of total free nucleic acid.In some embodiments, the sequence information includes information about at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 70, 80, 100, 200, or 300 genomic regions. In some embodiments, the sequence information includes information about less than all, nearly all, or all of the genome sequenced. In some embodiments, concentrations as low as 50 ng cfDNA can provide complete genome sequencing.

[0197] In some embodiments, the methods of the present disclosure can be used to determine the presence of a subject's disease (e.g., cancer). In some embodiments, determining the presence of a subject's cancer includes obtaining a sample from the subject according to any of the methods described herein and detecting nucleic acid molecules (e.g., nucleic acid fragments) in the sample. In some embodiments, determining the presence of a subject's disease (e.g., cancer) includes amplifying and / or sequencing nucleic acid molecules. In some embodiments, the presence of nucleic acid molecules indicates cancer. In some embodiments, the presence of nucleic acid molecules indicates a prenatal state. In some embodiments, the nucleic acid molecules and / or templates include unknown sequences. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample includes circulating tumor DNA. In some embodiments, the biological sample includes a tissue sample.

[0198] In some embodiments, the methods of the present disclosure include detecting an amplicon produced by an amplification primer, wherein the presence of the amplicon determines the presence of a modified reverse transcriptase in the sample.

[0199] In some embodiments, the methods of the present disclosure include providing a prenatal diagnosis based on the presence or absence of a nucleic acid molecule (eg, a cDNA molecule).

[0200] The present disclosure relates to a kit for producing nucleic acid molecules (e.g., cDNA molecules), comprising: one or more primers, nucleotides, at least one modified reverse transcriptase, a template, and instructions for performing any of the methods disclosed in the present disclosure. In some embodiments, the kit can be used to detect nucleic acids, the kit comprising a nucleic acid template (e.g., a DNA template), at least one modified reverse transcriptase, nucleotides, and instructions for performing any of the methods disclosed in the present disclosure. In some embodiments, the modified reverse transcriptase present in the kit or to be used in the kit is active and / or capable of template jumping at a temperature equal to or below about or above about 4°C, 8°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 52°C, 55°C or 60°C. In some embodiments, nucleic acid and / or template (such as nucleic acid template, DNA or RNA) exist with following concentration: as low as about 50 femtomoles, as low as about 60 femtomoles, as low as about 70 femtomoles, as low as about 75 femtomoles, as low as about 80 femtomoles, as low as about 90 femtomoles, as low as about 100 femtomoles, as low as about 120 femtomoles, as low as about 150 femtomoles, as low as about 200 femtomoles, as low as about 250 femtomoles, as low as about 300 femtomoles, as low as about 350 femtomoles, as low as about 400 femtomoles, as low as about 500 femtomoles, as low as about 550 femtomoles, as low as about 600 femtomoles, as low as about 700 femtomoles or as low as about 800 femtomoles. In some embodiments, the test kit may include one or more primers and / or templates annealed to primers. The present disclosure also relates to a test kit for producing modified enzymes, modified reverse transcriptases or modified polypeptides. In some embodiments, the test kit comprises a PCR step and / or a component for PCR.

[0201] In some embodiments, the present disclosure relates to a kit for detecting nucleic acids, comprising a template, at least one modified reverse transcriptase, nucleotides, and instructions for performing the disclosed methods. In some embodiments, the nucleic acid is present at a concentration of at least about 50 femtomolar, at least about 20 femtomolar, at least about 100 femtomolar, or greater than about 1000 femtomolar.

[0202] The present disclosure relates to any method disclosed herein, wherein the method may further comprise detecting at least one amplicon produced by the amplification primer. In some embodiments, the presence of at least one amplicon indicates the presence of at least one modified reverse transcriptase in the sample.

[0203] In some embodiments, any of the methods of the present disclosure does not include a purification step. In some embodiments, any of the methods of the present disclosure includes at least one purification step. In some embodiments, any of the methods of the present disclosure includes at least two purification steps. In some embodiments, any of the methods of the present disclosure includes at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, or at least twenty purification steps.

[0204] The present disclosure relates to a method for preparing a library for sequencing.

[0205] In some embodiments, the modified reverse transcriptase is a modified non-retroviral reverse transcriptase. In some embodiments, the modified reverse transcriptase is a modified non-LTR retrotransposon. In some embodiments, the modified reverse transcriptase is a modified R2 reverse transcriptase.

[0206] In some embodiments, variants or modified enzymes, or non-natural enzymes, or modified polypeptides have improved enzyme properties compared to unmodified, wild-type or naturally occurring enzymes or polypeptides. In some embodiments, improved enzyme properties are selected from at least one of the following: increased stability (e.g., increased thermal stability), increased specific activity, increased protein expression, improved purification, improved processivity, improved strand displacement, increased template jump and improved fidelity. In some embodiments, the term stability may include but is not limited to thermal stability, storage stability and pH stability. In some embodiments, specific activity is a measure of the enzymatic activity (units) of a protein or enzyme relative to the total amount of the protein or enzyme used in the reaction. In some embodiments, specific activity is measured based on the ability of an enzyme to produce cDNA molecules. In some embodiments, specific activity is measured with U / mg protein determined based on a primer extension reaction. In some embodiments, the characteristics of a changed or improved property can be characterized by a performance index (PI), wherein PI is a performance ratio of a variant, a modified enzyme or a non-naturally occurring enzyme compared to a wild-type or compared to a naturally occurring enzyme or protein. The term "performance index (PI)" may refer to the performance ratio of a variant polypeptide to a parent polypeptide, or a modified enzyme to an unmodified enzyme (e.g., a reverse transcriptase), or a non-naturally occurring enzyme to a naturally occurring enzyme for a specified performance characteristic. In some embodiments, the specified performance or enzyme property characteristic may include, but is not limited to, stability (e.g., thermostability), specific activity, protein expression, purification, processivity, end-to-end template hopping, and / or fidelity. In some embodiments, the PI is greater than about 0.5, while in other embodiments, the PI is about 1 or greater than about 1. In some embodiments, the variant polypeptide, modified enzyme (e.g., a modified reverse transcriptase), or non-naturally occurring enzyme includes modifications at one or more amino acid positions. In some embodiments, the modified enzyme or non-naturally occurring enzyme has a performance index (PI) of about 0.1 or greater, about 0.2 or greater, about 0.3 or greater, about 0.4 or greater, about 0.5 or greater, about 0.6 or greater, about 0.7 or greater, about 0.8 or greater, about 0.9 or greater, about 1 or greater, about 1.2 or greater, about 1.5 or greater, about 2 or greater, etc. about 2.5 or more, about 3 or more, about 3.5 or more, about 4 or more, about 4.5 or more, about 5 or more, about 5.5 or more, about 6 or more, about 6.5 or more, about 7 or more, about 8 or more, about 9 or more, about 10 or more, about 50 or more, about 75 or more, about 100 or more, about 500 or more, about 1000 or more.In some embodiments, the variant or modified enzyme has a performance index (PI) of about 0.1 to about 1, about 0.5 to about 1, about 0.1 to about 2, about 1 to about 2, about 0.5 to about 2, about 0.5 to about 10, about 1 to about 10, about 0.1 to about 10, about 1 to about 5, about 0.5 to about 5, about 0.5 to about 20, about 0.3 to about 20, about 5 to about 10, about 1.5 to about 10, about 1.5 to about 50, about 1 to about 50, about 1.5 to about 100, about 1.5 to about 75, about 4 to about 10, 3 to about 10, about 3 to about 25, about 3 to about 50, about 2 to about 20, about 2 to about 100, about 2 to about 1000, about 1 to about 1000. In some embodiments, the performance index is determined for protein expression. In some embodiments, the performance index is determined for at least one characteristic that improves the properties of the enzyme. In some embodiments, the performance index is determined for purification. In some embodiments, the performance index is determined for stability (e.g., thermostability). In some embodiments, the performance index is determined for specific activity. In some embodiments, the performance index is determined for processivity. In some embodiments, the performance index is determined for strand displacement. In some embodiments, the performance index is determined for template jump. In some embodiments, the performance index is determined for fidelity. In some embodiments, the characteristics of the improved enzyme properties are selected from the group consisting of increased thermostability, increased specific activity, and increased protein expression. In some embodiments, the performance index is determined at 30°C. In some embodiments, the enzyme properties are analyzed at 30°C. In some embodiments, enzyme properties, stability (e.g., thermostability), specific activity, protein expression, purification, processivity, strand displacement, template jump, and / or fidelity are determined at 30°C. In some embodiments, the performance index for measuring enzyme properties is determined at a specific temperature. In some embodiments, the temperature is about 25°C to about 42°C. In some embodiments, the temperature is about 8°C to about 50°C. In some embodiments, performance indicators for measuring enzyme properties can be determined at temperatures ranging from about 8°C to about 50°C, about 12°C to about 42°C, about 25°C to about 42°C, about 25°C to about 40°C, about 28°C to about 38°C, about 30°C to about 38°C, about 35°C to about 37°C, about 27°C to about 38°C, about 27°C to about 37°C, about 26°C to about 42°C, about 25°C to about 38°C, about 27°C to about 38°C, about 29°C to about 38°C, about 29°C to about 32°C.In some embodiments, the performance index for measuring enzyme properties can be determined at a temperature of about 8° C. or less, about 12° C. or less, about 20° C. or less, about 4° C. or less, about 55° C. or less, about 37° C. or less, about 25° C. or less, about 28° C. or less, about 30° C. or less, about 32° C. or less, about 34° C. or less, about 35° C. or less, about 36° C. or less, about 33° C. or less, about 31° C. or less, about 60° C. or less, about 38° C. or less, about 39° C. or less, about 40° C. or less, about 41° C. or less, about 42° C. or less, about 50° C. or less. In some embodiments, the temperature can be in the range of about 25° C. to about 80° C.

[0207] In some embodiments, the modified enzyme has a specific activity of about 5 U / mg to about 140,000 U / mg, about 5 U / mg to about 125,000 units / mg, about 50 U / mg to about 100,000 U / mg, about 100 U / mg to about 100,000 U / mg, about 250 U / mg to about 100,000 U / mg, about 500 U / mg to about 100,000 U / mg, about 1000 U / mg to about 100,000 U / mg, about 5000 U / mg to about 100,000 U / mg, about 10,000 U / mg to about 100,000 U / mg, about 25,000 U / mg to about 75,000 U / mg. In some embodiments, the range of specific activity includes a specific activity of about 20,000 U / mg to about 140,000 U / mg, a specific activity of about 20,000 U / mg to about 130,000 U / mg, a specific activity of about 20,000 U / mg to about 120,000 U / mg, a specific activity of about 20,000 U / mg to about 110,000 U / mg, a specific activity of about 20,000 U / mg to about 100,000 U / mg, a specific activity of about 20,000 U / mg to about 90,000 U / mg. / mg, a specific activity of about 25,000 U / mg to about 140,000 U / mg, a specific activity of about 25,000 U / mg to about 130,000 U / mg, a specific activity of about 25,000 U / mg to about 120,000 U / mg, a specific activity of about 25,000 U / mg to about 110,000 U / mg, a specific activity of about 25,000 U / mg to about 100,000 U / mg, and a specific activity of about 25,000 U / mg to about 90,000 U / mg. In some embodiments, the lower limit of the specific activity range can vary within the following ranges: 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, and 80,000 U / mg. In some embodiments, the upper limit of the range can vary within the following ranges: 150,000, 140,000, 130,000, 120,000, 110,000, 100,000, and 90,000 U / mg.

[0208] In some embodiments, the sample is a biological sample. In some embodiments, the biological sample includes circulating tumor DNA. In some embodiments, the biological sample includes a tissue sample. In some embodiments, the nucleic acid is from the sample. In some embodiments, the sample is a liquid biopsy sample. In some embodiments, the sample can be an RNA sample. In some embodiments, the RNA sample can be used for various purposes, including but not limited to PCR, conjugation, transcriptome analysis, microarray analysis, Northern blot analysis, and cDNA library construction. In some embodiments, the present disclosure relates to a method for amplifying a cDNA library from a relatively low amount of cells and / or single cells in an amount and quality suitable for transcriptome analysis or microarray analysis by, for example, sequencing.

[0209] In some embodiments, nucleic acid and / or template have unknown sequence. In some embodiments, nucleic acid and / or template are RNA, DNA or a combination of RNA and DNA. In some embodiments, RNA is mRNA. In some embodiments, mRNA includes internal initiation. In some embodiments, nucleic acid can be fragmented nucleic acid and / or degraded nucleic acid. In some embodiments, template can be fragmented template and / or degraded template. In some embodiments, nucleic acid can be non-fragmented nucleic acid and / or non-degraded nucleic acid. In some embodiments, template can be non-fragmented template and / or non-degraded template. In some embodiments, nucleic acid and / or template indicate disease. In some embodiments, nucleic acid and / or template indicate cancer. In some embodiments, nucleic acid is equal to or less than about 0.01 micromolar. In some embodiments, nucleic acid is between about 0.1nM and about 100nM. In some embodiments, nucleic acid is equal to or less than about 500 femtomolar.

[0210] In some embodiments, RNA is obtained from a source selected from the group consisting of single cells, cultured cells, tissues, RNA based on RNA transcription amplification (e.g., TTR amplified RNA or other DNA dependent RNA polymerase transcribed RNA), RNA-promoter-driven transcribed RNA, aRNA, aRNA amplified RNA, single cell mRNA library, isolated mRNA, RNA contained in cells, and combinations of RNA sources. In some embodiments, RNA is prepared from a plurality of fixed cells, wherein the fixed cells are protected from RNA degradation and permeabilized for enzyme penetration. In some embodiments, fixed cells are obtained from fixative treated cultured cells, frozen fresh tissue, fixative treated fresh tissue, or paraffin embedded tissue on a slide.

[0211] In some embodiments, RNA molecules can be products of in vitro synthesis, or can be isolated from cells or tissues (Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, Wiley, 1995). Cells and tissues suitable for obtaining RNA suitable for practicing the present disclosure can include animal cells and plant cells. In some embodiments, cells include mammalian cells and insect cells. RNA can also be isolated from prokaryotic cells such as bacteria.

[0212] In some embodiments, the template is RNA, DNA, or a combination of RNA and DNA. In some embodiments, the template may be a fragmented template and / or a degraded template. In some embodiments, the template is not degraded and / or fragmented. In some embodiments, RNA is mRNA. In some embodiments, the template is an RNA template. In some embodiments, the template is a DNA template. In some embodiments, the template is a DNA and / or RNA template. In some embodiments, the template is a mixture of DNA and RNA. In some embodiments, RNA includes any type of RNA (e.g., one or more of rRNA, tRNA, mRNA, and / or snRNA). In some embodiments, RNA includes a mixture of at least one type of RNA. In some embodiments, DNA may include a mixture or at least one of the following: genomic DNA or nuclear DNA, mitochondrial DNA, Y-line DNA, autosomal DNA, ribosomal DNA, or a combination thereof. In some embodiments, the template is a polymer of any length. In some embodiments, the template is about 20 bases to about 100 bases, about 30 bases to about 500 bases, about 30 bases to about 1000 bases, about 50 bases to about 300 bases, about 100 bases to about 600 bases, about 200 bases to about 800 bases, about 200 bases to about 600 bases, about 100 bases to about 2000 bases, about 100 bases to about 2500 bases, about 200 bases to about 5000 bases, about 200 bases to about 1000 bases, about 200 to about 10000 bases.In some embodiments, the template is at least about 10 bases, at least about 20 bases, at least about 30 bases, at least about 40 bases, at least about 50 bases, at least about 60 bases, at least about 70 bases, at least about 80 bases, at least about 90 bases, at least about 100 bases, at least about 150 bases, at least about 200 bases, at least about 250 bases, at least about 300 bases, at least about 350 bases, at least about 400 bases, at least about 450 bases, at least about 500 bases, at least about 550 bases, at least about 600 bases, at least about 650 bases, at least about 700 bases, at least about 750 bases, at least about bases, at least about 800 bases, at least about 850 bases, at least about 900 bases, at least about 950 bases, at least about 1000 bases, at least about 1100 bases, at least about 1200 bases, at least about 1300 bases, at least about 1400 bases, at least about 1500 bases, at least about 1700 bases, at least about 2000 bases, at least about 2200 bases, at least about 2500 bases, at least about 2700 bases, at least about 3000, at least about 3500 bases, at least about 4000 bases, at least about 4500 bases, at least about 5000 bases, at least about 10,000 bases or at least about 50,000 bases.In some embodiments, the template is about or at least about or at most about 10 bases, about or at least about or at most about 20 bases, about or at least about or at most about 30 bases, about or at least about or at most about 40 bases, about or at least about or at most about 50 bases, about or at least about or at most about 60 bases, about or at least about or at most about 70 bases, about or at least about or at most about 80 bases, about or at least about or at most about 90 bases, about or at least about or at most about 100 bases, about or at least about or at most about 150 bases, about or at least about or at most about 160 bases, about or at least about or at most about 170 bases, about or at least about or at most about 180 bases, about or at least about or at most about 190 bases, about or at least about or at most about 200 bases, about or at least about or at most about 210 bases, about or at least about or at most about 220 bases, about or at least about or at most about 230 bases, about or at least about or at most about 240 bases, about or at least about or at most about 250 bases, about or at least about about or at least about or at most about 200 bases, about or at least about or at most about 250 bases, about or at least about or at most about 300 bases, about or at least about or at most about 350 bases, about or at least about or at most about 400 bases, about or at least about or at most about 450 bases, about or at least about or at most about 500 bases, about or at least about or at most about 550 bases, about or at least about or at most about 600 bases, about or at least about or at most about 650 bases, about or at least about or at most about 700 bases, about or at least about or at most about 750 bases, about or at least about or at most about 800 bases, about or at least about or at most about 850 bases, about or at least about or at most about 900 bases, about or at least about or at most about 950 bases, about or at least about or at most about 1000 bases, about or at least about or at most about 1100 bases, about or at least about or at most about 1200 bases, about or at least about or at most about 1300 bases, about or at least about or at most about 1400 bases, about or at least about or at most about 1500 bases, about or at least about or at most about 1700 bases, about or at least about At least about or at most about 2000 bases, about or at least about or at most about 2200 bases, about or at least about or at most about 2500 bases, about or at least about or at most about 2700 bases, about or at least about or at most about 3000, about or at least about or at most about 3500 bases, about or at least about or at most about 4000 bases, about or at least about or at most about 4500 bases, about or at least about or at most about 5000 bases, about or at least about or at most about 10,000 bases or about or at least about or at most about 50,000 bases. In some embodiments, the template DNA can be a double-stranded DNA template (dsDNA template) or a single-stranded DNA template (ssDNA template). In some embodiments, the template RNA can be a double-stranded RNA template (dsRNA template) or a single-stranded RNA template (ssRNA template).

[0213] In some embodiments, the template is from a single cell. In some embodiments, the template is from a plurality of cells. In some embodiments, the template includes a low copy number DNA or RNA or a combination of DNA and / or RNA. In some embodiments, low copy number refers to a sample containing equal to or less than about 250 picograms (e.g., 100 picograms) of, for example, a mixture of template and / or DNA and / or RNA and / or DNA and RNA. In some embodiments, RNA may include at least one of the following: messenger RNA (mRNA), transfer RNA, transfer-messenger RNA, ribosomal RNA, antisense RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), small interfering RNA (siRNA) or any combination thereof. In some embodiments, the template is from a sample. In some embodiments, the total amount of template is the total amount of template in the sample. In some embodiments, the total amount of template is the total amount of template in the reaction mixture. In some embodiments, the total amount of template is the total amount of template in a pot (e.g., a single container). In some embodiments, the total amount of template is from about 1 femtomolar (fM) to about 100 micromolar, from about 40 femtomolar to about 0.01 micromolar, from about 50 femtomolar to about 500 femtomolar, from about 50 femtomolar to about 0.01 micromolar, from about 50 femtomolar to about 0.1 micromolar, from about 50 femtomolar to about 500 picomolar, from about 50 femtomolar to about 500 nanomolar, from about 50 femtomolar to about 500 micromolar, from about 50 femtomolar to about 1 picomolar, from about 40 femtomolar to about 1 nanomolar, from about 1 femtomolar to about 1 picomolar, from about 0.0001 micromolar to about 0.01 micromolar, from about 0.0001 micromolar to about 0.1 micromolar, or from about 0.1 nM to about 100 nM.In some embodiments, the total amount of template is equal to or is at least about or less than about 1000 micromolar, equal to or is at least about or less than about 500 micromolar, equal to or is at least about or less than about 250 micromolar, equal to or is at least about or less than about 100 micromolar, equal to or is at least about or less than about 50 micromolar, equal to or is at least about or less than about 25 micromolar, equal to or is at least about or less than about 10 micromolar, equal to or is at least about or less than about 1 micromolar, equal to or is at least about or less than about 0.1 micromolar, equal to or is at least about or less than about 0.01 micromolar, equal to or is at least about or less than about 0.001 micromolar, equal to or is at least about or less than about 0.0001 micromolar, equal to or at least about or less than about 2000 nanomolar, equal to or at least about or less than about 500 nanomolar, equal to or at least about or less than about 250 nanomolar, equal to or at least about or less than about 200 nanomolar, equal to or at least about or less than about 50 nanomolar, equal to or at least about or less than about 25 nanomolar, equal to or at least about or less than about 20 nanomolar, equal to or at least about or less than about 2 nanomolar, equal to or at least about or less than about 0.2 nanomolar, equal to or at least about or less than about 0.01 nanomolar, equal to or at least about or less than about 0.001 ... about or less than about 0.0001 nanomoles, equal to or at least about or less than about 3000 picomoles, equal to or at least about or less than about 500 picomoles, equal to or at least about or less than about 250 picomoles, equal to or at least about or less than about 300 picomoles, equal to or at least about or less than about 50 picomoles, equal to or at least about or less than about 25 picomoles, equal to or at least about or less than about 30 picomoles, equal to or at least about or less than about 3 picomoles, equal to or at least about or less than about 0.3 picomoles, equal to or at least about or less than about 0.01 picomoles, equal to or at least about or less than about 0.001 picomoles, equal to or At least about or less than about 0.0001 picomoles, equal to or at least about or less than about 5000 femtomoles, equal to or at least about or less than about 500 femtomoles, equal to or at least about or less than about 250 femtomoles, equal to or at least about or less than about 50 femtomoles, equal to or at least about or less than about 25 femtomoles, equal to or at least about or less than about 10 femtomoles, equal to or at least about or less than about 1 femtomoles, equal to or at least about or less than about 0.1 femtomoles, equal to or at least about or less than about 0.01 femtomoles, equal to or at least about or less than about 0.001 femtomoles, equal to or at least about or less than about 0.0001 femtomoles.

[0214] In some embodiments, the template may be present in any relevant nucleic acid sample, including but not limited to nucleic acid samples isolated from a single cell, multiple cells (e.g., cultured cells), tissues, organs, or organisms (e.g., bacteria, yeast, etc.). In some embodiments, nucleic acid samples are cells, tissues, organs, etc. isolated from mammals (e.g., humans, rodents (e.g., mice), or any other mammals). In some embodiments, nucleic acid samples are isolated from sources other than mammals, such as bacteria, yeast, insects (e.g., fruit flies), amphibians (e.g., frogs (e.g., Xenopus)), viruses, plants, or any other non-mammalian nucleic acid sample sources.

[0215] In some embodiments, the template is optimized. In some embodiments, the receptor template or receptor nucleic acid molecule includes at least one modified nucleotide. In some embodiments, the receptor template or receptor nucleic acid molecule is engineered to improve template jump and / or conversion efficiency. In some embodiments, the receptor template or receptor nucleic acid molecule is optimized at the 3' end. In some embodiments, optimization prevents secondary structure formation and / or nucleotide synthesis.

[0216] In some embodiments, the methods disclosed in the present disclosure may further include optimization of the template (e.g., donor template). In some embodiments, optimization of the template includes contacting the template (e.g., RNA) with a reagent capable of removing the 5' cap structure of the template (e.g., mRNA). In some embodiments, the cap structure is removed under conditions that allow the cap structure to be removed by the reagent. In some embodiments, the methods disclosed in the present disclosure further include, for example, dephosphorylation of the decapping template. In some embodiments, the method further includes adding a dephosphorylating agent to the decapping template under conditions that allow dephosphorylation.

[0217] In some embodiments, any method of the present disclosure may further include the optimization of template. In some embodiments, the optimization of template includes: under the condition that the cap structure is allowed to be removed by reagent, the sample including the template is contacted with the reagent of the 5' cap structure of the removal template. In some embodiments, the optimization of template may further include adding a dephosphorylating agent under the condition that the decapping template is allowed to be dephosphorylated by reagent. In some embodiments, the template (such as RNA molecule) is dephosphorylated after synthesis or separation. In some embodiments, dephosphorylation is realized by treating nucleic acid (such as RNA) molecule with alkaline phosphatase. In some embodiments, the separated donor template (such as RNA or mRNA) is decapped and dephosphorylated after separation. Nucleic acid (such as RNA) decapping method comprises enzymatic method (such as by using pyrophosphatase, such as tobacco pyrophosphatase) and chemical method (such as periodate oxidation and β elimination). The dephosphorylation method of nucleic acid (such as RNA) can use alkaline phosphatase. In some embodiments, mRNA has been separated through decapping (such as using tobacco acid pyrophosphatase) and dephosphorylation (such as by using alkaline phosphatase). In some embodiments, the RNA cap structure is removed by enzymatic treatment of the mRNA with pyrophosphatase or by chemical decapping (eg, by periodate oxidation and beta elimination). In some embodiments, the mRNA is modified with a tag.

[0218] In some embodiments, any of the methods of the present disclosure is performed in one pot (e.g., a single container). In some embodiments, the reaction is performed in one pot (e.g., a single container). In some embodiments, the reaction is a one-pot (e.g., a single container) reaction.

[0219] In some embodiments, template hopping is dependent on the concentration of the acceptor nucleic acid molecule.

[0220] In some embodiments, a modified enzyme (e.g., a modified reverse transcriptase), a modified reverse transcriptase, a non-naturally occurring enzyme, a modified polypeptide having reverse transcriptase activity comprises at least one modification relative to a wild-type, unmodified counterpart, or a naturally occurring enzyme. In some embodiments, a modified non-LTR retrotransposon comprises at least one modification of a wild-type or unmodified non-LTR retrotransposon. In some embodiments, a modified R2 reverse transcriptase comprises at least one modification of a wild-type or unmodified R2 reverse transcriptase. In some embodiments, a modified reverse transcriptase comprises at least one modification of a wild-type or unmodified reverse transcriptase. In some embodiments, a modified polypeptide having reverse transcriptase activity comprises at least one modification of a wild-type or unmodified polypeptide having reverse transcriptase activity. In some embodiments, the modification comprises at least one truncation (e.g., N-terminal truncation, C-terminal truncation, and / or N-terminal and C-terminal truncation). In some embodiments, the modification comprises site-directed incorporation and / or addition and / or deletion and / or substitution of amino acids at relevant positions. In some embodiments, the modification enhances the biological properties of the modified enzyme or modified polypeptide relative to the wild-type or unmodified enzyme or polypeptide. In some embodiments, modification improves at least one enzyme property of the modified enzyme or polypeptide relative to the wild-type or unmodified enzyme or polypeptide. In some embodiments, modification serves as, for example, a connection point for a label and a protein half-life extender and a connection point for attaching a variant to a solid support surface. In some embodiments, the disclosure relates to methods for producing cells capable of producing a modified enzyme (e.g., a modified reverse transcriptase) or a modified polypeptide, and methods for producing a vector containing a DNA or RNA encoding a modified enzyme (e.g., a modified reverse transcriptase) or a modified polypeptide. In some embodiments, truncation is based on a two-step method. In some embodiments, the first step for selecting truncation includes analyzing the domain and motif structure and function of a class of enzymes or proteins or polypeptides. In some embodiments, the enzyme or protein or polypeptide is a non-LTR retrotransposon, a reverse transcriptase, an R2 reverse transcriptase, an LTR retrotransposon, an R2 non-LTR retrotransposon, or any combination thereof. In some embodiments, the enzyme or protein or polypeptide is from a different organism. In some embodiments, all domains of the enzyme or protein or polypeptide are present. In some embodiments, all domains are present to ensure reverse transcriptase activity. In some embodiments, all domains are present to ensure the unique properties necessary for the disclosure. In some embodiments, the domain responsible for reverse transcriptase activity is not modified. In some embodiments, the R2 domain does not include modification. In some embodiments, the R2 domain may include modification. In some embodiments, the truncated variants display expression levels. In some embodiments, the truncated variants displaying promising expression levels are further subjected to minor adjustments in sequence (step two). In some embodiments, the minor adjustments in sequence include deletions, insertions and / or substitutions of amino acids.In some embodiments, the deletion, insertion and / or substitution of amino acids may comprise one or several amino acids. In some embodiments, the deletion, insertion and / or substitution of amino acids further optimizes expression and / or stability (e.g., thermal stability).

[0221] In some embodiments, the modified enzyme (e.g., a modified reverse transcriptase), the modified reverse transcriptase, or the modified polypeptide has an N-terminal truncation, a C-terminal truncation, or both relative to a wild-type or unmodified enzyme (e.g., a wild-type reverse transcriptase) or a wild-type or unmodified polypeptide. In some embodiments, the polymerase comprises an N-terminal truncation, a C-terminal truncation, or both. In some embodiments, the modified reverse transcriptase comprises an N-terminal truncation, a C-terminal truncation, or a combination of N-terminal and C-terminal truncations. In some embodiments, the modified enzyme comprises an N-terminal truncation, a C-terminal truncation, or a combination of N-terminal and C-terminal truncations. In some embodiments, the modified polypeptide comprises an N-terminal truncation, a C-terminal truncation, or a combination of N-terminal and C-terminal truncations. In some embodiments, the truncation comprises the sequence MAHHHHHHVGTVGTGGGSGGASTAL. In some embodiments, the modified reverse transcriptase, the modified enzyme, the modified polypeptide, the modified non-LTR retrotransposon, or the modified R2 reverse transcriptase comprises a truncation of less than about 100 amino acid residues. In some embodiments, the modified reverse transcriptase, modified enzyme, modified polypeptide, modified non-retrotransposon, or modified R2 reverse transcriptase comprises at least one of: (a) an amino-terminal truncation of less than about 400 amino acid residues and (b) a carboxyl-terminal truncation of less than about 400 amino acid residues. In some embodiments, the modified reverse transcriptase, modified enzyme, modified polypeptide, modified non-LTR retrotransposon, or modified R2 reverse transcriptase lacks up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 50, about 75, about 100, about 120, about 150, about 175, about 200, about 220, about 250, about 275, about 280, about 290, about 300, about 325, about 350, about 375, about 380, about 390, about 400, or about 450 amino acids at the N-terminus, the C-terminus, or both.In some embodiments, the modified reverse transcriptase, modified enzyme, modified polypeptide, modified non-LTR retrotransposon, or modified R2 reverse transcriptase may alternatively or additionally have one or more internal deletions of up to: about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acids, about 30, about 50, about 75, about 100, about 120, about 150, about 175, about 200, about 220, about 250, about 275, about 280, about 290, about 300, about 325, about 350, about 375, about 380, about 390, or a total of about 450 amino acids. In some embodiments, the N-terminal truncation, the C-terminal truncation, or both may comprise a deletion of about 1 to about 50 amino acids, about 1 to about 25, about 1 to about 70, about 10 to about 50, about 20 to about 30, about 15 to about 100, about 1 to about 150, about 15 to about 60, about 15 to about 40, about 1 to about 10, about 10 to 35, about 50 to about 100, about 20 to about 150, about 200 to about 350, about 25 to about 350, about 150 to about 400, about 50 to about 400, about 50 to about 450, about 200 to about 400, or about 50 to about 350, or about 50 to about 400 amino acids. In some embodiments, the N-terminal truncation removes at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 90, at least about 95, at least about 100, at least about 120, at least about 130, at least about 140, at least about 150, at least about 175, at least about 200, at least about 220, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 375, or at least about 400 amino acids. In some embodiments, the C-terminal truncation removes at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 90, at least about 95, at least about 100, at least about 120, at least about 130, at least about 140, at least about 150, at least about 175, at least about 200, at least about 220, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 375, or at least about 400 amino acids.In some embodiments, the N-terminal truncation lacks about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 65, about 70, about 75, about 80, about 90, about 95, about 100, about 120, about 130, about 140, about 150, about 175, about 200, about 220, about 250, about 275, about 300, about 325, about 350, about 375, or about 400 amino acids. In some embodiments, the C-terminal truncation lacks about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 65, about 70, about 75, about 80, about 90, about 95, about 100, about 120, about 130, about 140, about 150, about 175, about 200, about 220, about 250, about 275, about 300, about 325, about 350, about 375, or about 400 amino acids. In some embodiments, the N-terminal truncation lacks no more than about 5, no more than about 10, no more than about 15, no more than about 20, no more than about 25, no more than about 30, no more than about 35, no more than about 40, no more than about 50, no more than about 60, no more than about 65, no more than about 70, no more than about 75, no more than about 80, no more than about 90, no more than about 95, no more than about 100, no more than about 120, no more than about 130, no more than about 140, no more than about 150, no more than about 175, no more than about 200, no more than about 220, no more than about 250, no more than about 275, no more than about 300, no more than about 325, no more than about 350, no more than about 375, or no more than about 400 amino acids. In some embodiments, the C-terminal truncation lacks no more than about 5, no more than about 10, no more than about 15, no more than about 20, no more than about 25, no more than about 30, no more than about 35, no more than about 40, no more than about 50, no more than about 60, no more than about 65, no more than about 70, no more than about 75, no more than about 80, no more than about 90, no more than about 95, no more than about 100, no more than about 120, no more than about 130, no more than about 140, no more than about 150, no more than about 175, no more than about 200, no more than about 220, no more than about 250, no more than about 275, no more than about 300, no more than about 325, no more than about 350, no more than about 375, or no more than about 400 amino acids. In some embodiments, the truncation comprises an N-terminal truncation that removes at least about, at most about, or about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 amino acids.In some embodiments, the truncation comprises a C-terminal truncation that removes at least about, at most about, or about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 amino acids. In some embodiments, the N-terminal truncation, the C-terminal truncation, or both can be greater than about 500 amino acids, greater than about 1000 amino acids, greater than about 1500 amino acids, greater than about 2000 amino acids, greater than about 5000 amino acids, greater than about 10000 amino acids, greater than about 100000 amino acids, greater than about 1000000 amino acids.

[0222] In some embodiments, truncations in regions that affect the functional activity of a protein or enzyme can be engineered. In some embodiments, truncations in regions that do not affect the functional activity of a protein or enzyme can be engineered. Truncations can include truncations of less than about 5, less than about 10, less than about 15, less than about 20, less than about 25, less than about 30, less than about 35, less than about 40, less than about 45, less than about 50, less than about 60, less than about 70, less than about 80, less than about 90, less than about 100, less than about 125, less than about 150, less than about 200, less than about 250, less than about 300, less than about 350, less than about 400 or more amino acids. Truncations can include truncations of greater than about 5, greater than about 10, greater than about 15, greater than about 20, greater than about 25, greater than about 30, greater than about 35, greater than about 40, greater than about 45, greater than about 50, greater than about 60, greater than about 70, greater than about 80, greater than about 90, greater than about 100, greater than about 125, greater than about 150, greater than about 200, greater than about 250, greater than about 300, greater than about 350, greater than about 400, or more amino acids. Truncations can include about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 87%, about 90%, about 92%, about 95%, or about 100% truncation of the polypeptide or enzyme.

[0223] In some embodiments, the variant or modified enzyme or modified protein may include one or more modifications at an amino acid position. In some embodiments, the variant, mutant or modified polypeptide or enzyme of the present disclosure may have an increased activity compared to a corresponding unmutated or unmodified or wild-type polymerase or compared to one or more polymerases (e.g., RNA-dependent DNA polymerase or reverse transcriptase), such as increased RNA-dependent DNA polymerase activity or DNA-dependent DNA polymerase activity. In some embodiments, the polymerase or reverse transcriptase with increased activity can be a modified polymerase or modified reverse transcriptase having at least about 5% increase, at least about 10% increase, at least about 25% increase, at least about 30% increase, at least about 50% increase, at least about 100% increase, at least about 150% increase, at least about 200% increase, at least about 300% increase, at least about 500% increase, at least about 1,000% increase, at least about 2,500% increase, or at least about 5,000% increase compared to (1) the corresponding non-mutated or wild-type enzyme; or (2) a specific polymerase (e.g., RNA-dependent DNA polymerase, reverse transcriptase) or a specific reverse transcriptase or a group of polymerases or a group of reverse transcriptases. In some embodiments, the modified polymerase or modified reverse transcriptase of the present disclosure may have an increase in activity of about 5% to about 5,000%, about 5% to about 2,500%, about 5% to about 1000%, about 5% to about 500%, about 5% to about 250%, about 5% to about 100%, about 5% to about 50%, about 5% to about 25%, about 25% to about 5,000%, about 25% to about 2,500%, about 25% to about 1,000%, about 25% to about 500%, about 25% to about 250%, about 25% to about 100%, about 100% to about 5,000%, about 100% to about 2,500%, about 100% to about 1000%, about 100% to about 500%, or about 100% to about 250%. The increase in RNA-dependent DNA polymerase activity and / or DNA-dependent DNA polymerase activity of a modified polymerase or modified reverse transcriptase of the present disclosure can also be measured based on the relative activity compared to (1) the corresponding unmodified or wild-type enzyme; or (2) a specific polymerase (e.g., RNA-dependent DNA polymerase, reverse transcriptase) or a specific reverse transcriptase or a group of polymerases or a group of reverse transcriptases.In some embodiments, the increase in relative activity is at least about 1.1, 1.2, 1.5, 2, 5, 10, 25, 50, 75, 100, 150, 200, 300, 500, 1,000, 2,500, 5,000, 10,000, or 25,000-fold when the activity of a modified polymerase or modified reverse transcriptase of the present disclosure is compared to (1) a corresponding non-mutated or wild-type enzyme; or (2) a specific polymerase (e.g., an RNA-dependent DNA polymerase, a reverse transcriptase) or a specific reverse transcriptase or a group of polymerases or a group of reverse transcriptases. Thus, the modified polymerase or modified reverse transcriptase of the present disclosure may have an increased RNA-dependent DNA polymerase and / or increased DNA-dependent DNA polymerase activity of about 1.1 to about 25,000 times, about 1.1 to about 10,000 times, about 1.1 to about 5,000 times, about 1.1 to about 2,500 times, about 1.1 to about 1,000 times, about 1.1 to about 50 0 times, about 1.1 times to about 250 times, about 1.1 times to about 50 times, about 1.1 times to about 25 times, about 1.1 times to about 10 times, about 1.1 times to about 5 times, about 5 times to about 25,000 times, about 5 times to about 5,000 times, about 5 times to about 1,000 times, about 5 times to about 500 times, about 5 times to about 100 times, about 5 times to about 50 times, about 5 times to about 25 times, about 50 times to about 25,000 times, about 50 times to about 5,000 times, about 50 times to about 1,000 times, about 50 times to about 500 times, about 50 times to about 100 times, about 100 times to about 25,000 times, about 1,000 times to about 25,000 times, about 4,000 times to about 25,000 times, about 10,000 times to about 25,000 times, about 15,000 times to about 25,000 times, about 1,000 times to about 10,000 times, about 2, 500-fold to about 10,000-fold, about 5,000-fold to about 10,000-fold, about 7,500-fold to about 10,000-fold, about 1,000-fold to about 15,000-fold, about 2,500-fold to about 15,000-fold, about 5,000-fold to about 15,000-fold, about 7,500-fold to about 15,000-fold, about 10,000-fold to about 15,000-fold, or about 12,500-fold to about 15,000-fold.

[0224] In some embodiments, the modified enzyme, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme exhibits a change (e.g., increased or decreased) processivity for a given nucleotide substrate relative to an unmodified or naturally occurring counterpart. In some embodiments, the processivity exhibited by the modified enzyme, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme for a given nucleotide substrate is at least about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000%, or 10,000% of the processivity of the reference enzyme for the same nucleotide substrate. In some embodiments, the reference enzyme is the unmodified counterpart of the modified enzyme. In some embodiments, the reference enzyme is a reverse transcriptase. In some embodiments, the reference enzyme is a non-LTR retrotransposon. In some embodiments, the reference enzyme is an LTR retrotransposon. In some embodiments, the reference enzyme is an R2 reverse transcriptase. In some embodiments, the reference enzyme or polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 and / or SEQ ID NO:48.In some embodiments, R2 comprises at least one mutation and / or modification. In some embodiments, R2 comprises the following amino acid sequence: SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67. In some embodiments, R2 and / or the modified enzyme and / or the modified reverse transcriptase include mutations at one or more amino acids selected from, but not limited to, C952S, and / or C956S, and / or C952S, C956S (double mutant), and / or C969S, and / or H970Y, and / or R979Q, and / or R976Q, and / or R1071S, and / or R328A, and / or R329A, and / or Q336A, and / or R328A, R329A, Q336A (triple mutant), and / or G426A, and / or D428A, and / or G426A, D428A (double mutant), and / or any combination thereof. In some embodiments, the amino acid position is based on SEQ ID NO: 52. In some embodiments, the amino acid position is based on wild-type reverse transcriptase. In some embodiments, the amino acid position is based on wild-type R2. In some embodiments, cysteine ​​is mutated. In some embodiments, cysteine ​​is mutated to serine. In some embodiments, arginine is mutated. In some embodiments, arginine is mutated to serine or glutamine or alanine. In some embodiments, amino acids are mutated to alanine. In some embodiments, glutamine is mutated to alanine. In some embodiments, aspartic acid is mutated to alanine. In some embodiments, glycine is mutated to alanine. In some embodiments, histidine is mutated to tyrosine.

[0225] In some embodiments, a variant or modified enzyme (eg, a modified reverse transcriptase) or modified polypeptide having reverse transcriptase activity of the present disclosure includes at least one altered property that improves the properties of the enzyme. In some embodiments, the variants or modified enzymes (e.g., modified reverse transcriptases) or modified polypeptides having reverse transcriptase activity of the present disclosure comprise at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% sequence identity to the amino acid sequences disclosed in the present disclosure. In some embodiments, the variant or modified enzyme or modified polypeptide having reverse transcriptase activity comprises at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% and / or at least about 99% sequence identity to the amino acid sequence corresponding to the GenBank number in Table 1.In some embodiments, the variant or modified enzyme or modified polypeptide having reverse transcriptase activity comprises at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and / or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 and / or SEQ ID NO:48.In some embodiments, the variant or modified enzyme or modified polypeptide having reverse transcriptase activity comprises at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and / or at least about 99% sequence identity to the amino acid sequence set forth in the following sequences: SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 and / or SEQ ID NO:67. See Table 2. SEQ ID NO:49 is an example of R2 RT C truncation, SEQ ID NO:50 is an example of R2 RT N truncation, SEQ ID NO:51 is an example of R2 RT N and C truncation, and SEQ ID NO:52 is an example of R2 wild type. SEQ ID NO:53-67 are examples of R2 with single, double or triple mutations (Table 2). In some embodiments, the modified reverse transcriptase is derived from an arthropod. In some embodiments, the arthropod is Bombyx mori.

[0226] Table 1

[0227]

[0228]

[0229]

[0230] Table 2

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] In some embodiments, polypeptides, proteins, enzymes, modified enzymes (e.g., modified reverse transcriptases), modified polypeptides, non-naturally occurring enzymes or variants include, but are not limited to, fusions with proteins, domains, fusion partners, carrier proteins, target sequences, antigenic determinants, or any combination thereof. In some embodiments, reverse transcriptases or modified reverse transcriptases are fused to proteins, domains, fusion partners, target sequences, antigenic determinants, or any combination thereof. In some embodiments, non-LTR retrotransposons or modified non-LTR retrotransposons are fused to proteins, domains, fusion partners, target sequences, antigenic determinants, or any combination thereof. In some embodiments, modified LTR retrotransposons are fused to proteins, domains, fusion partners, target sequences, antigenic determinants, or any combination thereof. In some embodiments, modified R2 non-LTR retrotransposons are fused to proteins, domains, fusion partners, target sequences, antigenic determinants, or any combination thereof. In some embodiments, modified R2 reverse transcriptases are fused to proteins, domains, fusion partners, target sequences, antigenic determinants, or any combination thereof. In some embodiments, the modified reverse transcriptase is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof. In some embodiments, the variant is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof. In some embodiments, a polypeptide having reverse transcriptase activity is fused to a protein, a domain, a fusion partner, a target sequence, an antigenic determinant, or any combination thereof.

[0248] In some embodiments, the fused polypeptide, protein, enzyme, modified enzyme (e.g., modified reverse transcriptase), modified polypeptide, non-naturally occurring enzyme, or variant thereof increases stability (e.g., increases thermal stability), increases shelf life, increases activity fraction, and / or improves purification compared to a wild-type counterpart, a naturally occurring enzyme, or an unfused polypeptide, protein, enzyme, or variant thereof. In some embodiments, the modified reverse transcriptase comprises a fusion partner or carrier protein. In some embodiments, the selection of a fusion protein, domain, fusion partner, target sequence, antigenic determinant, or any combination thereof is based on a mechanism that produces reduced or increased stability (e.g., increased thermal stability), reduced or increased shelf life, and / or reduced or increased expression levels (Costa et al., "Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system" Front Microbiol. 2014 Feb 19; 5:63). In some embodiments, a fusion tag increases the solubility of its partner protein. In some embodiments, the fusion protein forms a micelle-like structure. In some embodiments, the micelle-like structure is a misfolded or unfolded protein that is shielded and protected from the influence of solvents and / or the soluble protein domain is outward. In some embodiments, the fusion partner attracts a chaperone. In some embodiments, the fusion tag forces its partner protein to enter a chaperone-mediated folding path. In some embodiments, MBP and / or N-utilizing substance (NusA) are two fusion tags that present this mechanism. In some embodiments, the fusion partner has an inherent chaperone-like activity. In some embodiments, the hydrophobic small piece of the fusion tag interacts with a partially folded chaperone protein (passenger protein), thereby preventing self-aggregation and promoting proper folding. In some embodiments, the solubility enhancement partner may play a passive role in the folding of its target protein, thereby reducing the chance of protein aggregation. In some embodiments, the fusion partner captures charge. In some embodiments, the strongly acidic fusion partner inhibits protein aggregation.In some embodiments, the fusion is with, but is not limited to, Fh8, MBP, NusA, Trx, SUMO, GST, SET, GB1, ZZ, HaloTag, SNUT, Skp, T7PK, EspA, Mocr, Ecotin, CaBP, ArsC, IF2 domain I, an expression tag, an expression tag that is part of IF2 domain I, RpoA, SlyD, Tsf, RpoS, PotD, Crr, msyB, yjgD, rpoD, His6, or any combination thereof. In some embodiments, the fusion improves protein solubility and / or purification. In some embodiments, Fh8 can be used as an effective solubility enhancement partner and / or stabilizes purification. In some embodiments, the Fh8 fusion tag has an amino acid sequence comprising MPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGK LDLKELVSILSS. In some embodiments, the codon optimized sequence comprises ATGCCGTCTGTTCAGGAAGTTGAAAAACTGCTGCACGTTCTGGACCGTAACGGTGACGGTAAAGTTTCTGCGGAAGAACTGAAAGCGTTCGCGGACGACTCTAAATGCCCGCTGGACTCTAACAAAATCAAAGCGTTCATCAAAGAACACGACAAAAACAAAGACGGTAAACTGGACCTGAAAGAACTGGTTTCTATCCTGTCTTCTTAG. In some embodiments, the enzyme, or modified enzyme (e.g., modified reverse transcriptase), or protein (e.g., modified protein), or polypeptide (e.g., modified polypeptide), or variant, or product, or nucleic acid molecule, or cDNA molecule, or template, or receptor nucleic acid molecule, or primer, or RNA, or DNA, or fragment nucleic acid, or degraded nucleic acid may include one or more tags. In some embodiments, fragmented or degraded RNA or DNA or variants thereof may include one or more tags. In some embodiments, R2 reverse transcriptase or variants thereof may include one or more tags. In some embodiments, the non-LTR retrotransposon protein or the polypeptide with reverse transcriptase activity or its variant may include one or more tags. In some embodiments, the cDNA molecule may include one or more tags. In some embodiments, the tag may be captured on a solid support, thereby facilitating the separation of the enzyme or protein or polypeptide or variant or product of the present disclosure. In some embodiments, the tag may be biotin that can be recognized by avidin. The affinity tag may include multiple biotin residues for increasing binding to multiple avidin molecules.In some embodiments, the tag may comprise a functional group (e.g., an azido or ethynyl group) that enables capture by copper (I)-mediated click chemistry (see HC Kolb and KB Sharpless, Drug Discovery Today, 2003, 8 (24), 1128-1137). In some embodiments, the tag may comprise an antigen that can be captured by an antibody bound to a solid support. In some embodiments, the tag may include but is not limited to a His tag, a His6 tag, a calmodulin tag, CBP, a CYD (covalent but detachable NorpD peptide), a Strep II, a FLAG tag, a HA tag, a Myc tag, an S tag, a SBP tag, a Softag-1, a Softag-3, a V5 tag, an Xpress tag, an Isopeptag, a SpyTag, a B, a HPC (protein C heavy chain) peptide tag, a GST, a MBP, a biotin, a biotin carboxyl carrier protein, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose binding protein tag, a Nus tag, a Strep tag, a thioredoxin tag, and a combination thereof. In some embodiments, the tagged molecules may be sequenced.

[0249] In some embodiments, the molecular barcode can be connected to any region of the molecule. For example, the molecular barcode can be connected to the 5' end or 3' end of a polynucleotide (e.g., DNA, RNA). For example, the target-specific region of the molecular barcode includes a sequence complementary to the sequence in the 5' region of the molecule. The target-specific region of the molecular barcode may also include a sequence complementary to the sequence in the 3' region of the molecule. In some cases, the molecular barcode connects to a region within a gene or gene product. For example, genomic DNA is fragmented, and a sample tag or molecular identification marker is connected to the fragmented DNA. In other cases, the RNA molecule is subjected to alternative splicing, and the molecular barcode is connected to a variant of the alternative splicing. In another example, the polynucleotide is hydrolyzed, and the molecular barcode is connected to the hydrolyzed polynucleotide. In another example, the target-specific region of the molecular barcode includes a sequence complementary to the sequence within the molecule.

[0250] In some embodiments, the methods of the present disclosure include introducing a biotin moiety or another affinity purification moiety into, for example, a nucleic acid molecule, such as DNA, RNA, or a combination of DNA and RNA. In some embodiments, the method further includes immobilizing the nucleic acid molecule with the affinity purification tag on a solid support. In some embodiments, the solid support is an agarose resin or magnetic beads to which an affinity purification substance, such as avidin, streptavidin, chitin, glutathione, etc., is bound.

[0251] In some embodiments, the enzyme or protein or polypeptide or variant or product of the present disclosure can be bound to a solid support. In some embodiments, fragmented or degraded nucleic acids (e.g., RNA or DNA) or variants thereof can be bound to a solid support. In some embodiments, R2 reverse transcriptase or variants thereof can be bound to a solid support. In some embodiments, non-LTR retrotransposon proteins or polypeptides with reverse transcriptase activity or variants thereof can be bound to a solid support. In some embodiments, cDNA molecules can be bound to a solid support. In some embodiments, the solid support can be glass, plastic, porcelain, resin, agarose, silica or other materials. In some embodiments, the solid support can be a substantially flat substrate, gel, microbeads, magnetic beads, membrane or other suitable shape and size plate. In some embodiments, microbeads may have a diameter between 10nm and several millimeters. In some embodiments, the solid support can be non-porous or porous, with holes of various densities and sizes. In some embodiments, DNA and / or RNA fragments can be captured on a solid support, and unwanted DNA and / or RNA can be washed off. In some embodiments, DNA and / or RNA fragments can be stripped from a solid support, for example, by using a restriction enzyme.

[0252] In some embodiments, the solid support may include a target nucleic acid binding region, wherein the target nucleic acid binding region includes a sequence selected from the group consisting of a gene-specific sequence, an oligo-dT sequence, a random polymer, and any combination thereof. In some embodiments, the solid support further includes a target nucleic acid or its complement. In some embodiments, the solid support includes a plurality of target nucleic acids or their complements, including about 0.01% to about 100% of the transcripts of the transcriptome of an organism or its complement, or about 0.01% to about 100% of the genes of the genome of an organism or its complement. In some embodiments, the cell markers of a plurality of oligonucleotides include a first random sequence connected to a second random sequence by a first marker connection sequence; and the molecular markers of a plurality of oligonucleotides include random sequences. In some embodiments, the solid support is selected from the group consisting of: a polydimethylsiloxane (PDMS) solid support, a polystyrene solid support, a glass solid support, a polypropylene solid support, an agarose solid support, a gelatin solid support, a magnetic solid support, a Pluronic solid support, and any combination thereof. In some embodiments, a plurality of oligonucleotides include a linker, the linker includes a linker functional group, and the solid support includes a solid support functional group; wherein the solid support functional group and the linker functional group are connected to each other. In some embodiments, the linker functional group and the solid support functional group are individually selected from the group consisting of: C6, biotin, streptavidin, primary amine, aldehyde, ketone and any combination thereof. In some embodiments, the molecular labeling of the plurality of oligonucleotides includes at least 15 nucleotides.

[0253] In some embodiments, the fusion partner can be removed from its target protein by enzymatic cleavage, chemical cleavage and / or by using an in vivo cleavage strategy. In some embodiments, a protease can be used for tag removal. In some embodiments, the protease can be an endoprotease, a serine protease, a factor Xa, an enterokinase, an α-thrombin, a viral protease, a tobacco etch virus (TEV), a human rhinovirus 3C protease, a SUMO protease, an exoprotease, a metallocarboxypeptidase or an aminopeptidase. In some embodiments, the fusion tag can be removed by a purification step. In some embodiments, the initial affinity purification step comprises (e.g., by a histidine tag at the N-terminus of the fusion protein) cleaving the purified fusion protein mixed into a solution with an endoprotease (e.g., a his-tagged protease) to separate from the tag. The cleaved target protein can be recovered in a flow sample after a second affinity purification step, wherein the cleaved fusion tag and the added protease are collected in the sample after elution.

[0254] In some embodiments, the modified enzymes, modified reverse transcriptases, non-naturally occurring enzymes, or modified polypeptides having reverse transcriptase activity of the present disclosure exhibit activity in the absence of thermal cycling, are capable of template jumping, and / or produce nucleic acid molecules (e.g., cDNA molecules). In some embodiments, the modified reverse transcriptase, modified enzyme, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity capable of template jumping and / or generating nucleic acid cDNA molecules at a temperature in the range of about 25°C to about 42°C, about 12°C to about 42°C, about 8°C to about 50°C, about 4°C to about 60°C, about 27°C to about 35°C, about 28°C to about 33°C, about 29°C to about 32°C, about 30°C to about 37°C, about 26°C to about 38°C, about 30°C to about 37°C, about 25°C to about 32°C, about 29°C to about 31°C, about 27°C to about 38°C, about 29°C to about 38°C. In some embodiments, the non-naturally occurring enzyme, modified reverse transcriptase, modified enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity at about 30°C, or at about 35°C, or at about 25°C, capable of template jumping and / or producing nucleic acid molecules (e.g., cDNA molecules). In some embodiments, the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity at temperatures of about 38°C or less, about 42°C or less, about 50°C or less, about 60°C or less, about 35°C or less, about 30°C or less, about 28°C or less, about 25°C or less, about 20°C or less, about 12°C or less, about 8°C or less, or about 4°C or less. In some embodiments, the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity at a temperature of about 36° C. or less, is capable of template hopping, and / or produces nucleic acid molecules (e.g., cDNA molecules). In some embodiments, the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity at room temperature, is capable of template hopping, and / or produces nucleic acid molecules (e.g., cDNA molecules).In some embodiments, the modified enzymes, modified reverse transcriptases, non-naturally occurring enzymes, or modified polypeptides having reverse transcriptase activity of the present disclosure exhibit activity, are capable of template jumping, and / or produce nucleic acid molecules (e.g., cDNA molecules) at temperatures of about or up to about 8°C, about or up to about 12°C, about or up to about 20°C, about or up to about 25°C, about or up to about 28°C, about or up to about 30°C, about or up to about 31°C, about or up to about 32°C, about or up to about 33°C, about or up to about 34°C, about or up to about 35°C, about or up to about 36°C, about or up to about 39°C, about or up to about 40°C, about or up to about 41°C, about or up to about 42°C, about or up to about 50°C, about or up to about 55°C, about or up to about 60°C. In some embodiments, the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity of the present disclosure exhibits activity, is capable of template jumping, and / or produces nucleic acid molecules (e.g., cDNA molecules) at a temperature equal to or less than about any temperature between about 42°C and about 80°C, or between about 35°C and about 80°C, or between about 30°C and about 50°C, or between about 8°C and about 50°C, or between about 12°C and about 42°C.

[0255] In some embodiments, the modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme of the present disclosure has at least one altered property relative to an unmodified or naturally occurring enzyme. In some embodiments, the altered property enables the modified enzyme, modified reverse transcriptase, non-naturally occurring enzyme, or modified polypeptide having reverse transcriptase activity to generate nucleic acid molecules and / or complementary deoxyribonucleic acid (cDNA) molecules from a template nucleic acid molecule without thermal cycling. In some embodiments, the modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme of the present disclosure is capable of producing one or more copies of a nucleic acid molecule or cDNA molecule with an error rate of at most about 0.5%, at most about 1%, at most about 1.5%, at most about 2%, at most about 2.5%, at most about 3%, at most about 3.5%, at most about 4%, at most about 4.5%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 40%, at most about 45%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, or at most about 80%. In some embodiments, the modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme of the present disclosure is a variant of any of the sequences disclosed herein. In some embodiments, the modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme of the present disclosure is a variant of any of the sequences corresponding to the accession numbers provided in Table 1. In some embodiments, the modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme of the present disclosure is a variant of any of the sequences provided in SEQ ID NO: 1-67. In some embodiments, the modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or non-naturally occurring enzyme of the present disclosure has at least one altered characteristic that improves an enzyme property relative to an unmodified or naturally occurring enzyme. In some embodiments, the altered characteristic of at least one improved enzyme property comprises at least one of: increased / improved stability (e.g., increased / improved thermostability), increased / improved specific activity, increased / improved protein expression, increased / improved purification, increased / improved processivity, increased / improved strand displacement, increased / improved template jumping, and increased / improved fidelity.

[0256] In one embodiment, the present disclosure relates to a non-naturally occurring enzyme that reverse transcribes a template nucleic acid molecule to produce a complementary deoxyribonucleic acid (cDNA) product and amplifies the cDNA product with a processivity of at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% per base measured at about 12°C, about 15°C, about 20°C, about 25°C, about 30°C, about 32°C, about 35°C, about 40°C.

[0257] In some embodiments, the non-naturally occurring enzyme has a performance index greater than about 1.0 for at least one enzyme property. In some embodiments, the enzyme property is at least one of the group consisting of improved stability (e.g., improved thermostability), specific activity, protein expression, purification, processivity, strand displacement, template hopping, increased DNA / RNA affinity, and fidelity.

[0258] In one embodiment, the present disclosure relates to a non-naturally occurring enzyme that reverse transcribes a template nucleic acid molecule to produce a complementary deoxyribonucleic acid (cDNA) product, a nucleic acid product, and amplifies a cDNA product within a period of about 3 hours or less and / or with a performance index greater than about 1.0 for at least one enzyme property selected from the group consisting of: improved stability (e.g., improved thermostability), specific activity, protein expression, purification, processivity, strand displacement, template jumping, increased DNA / RNA affinity, and fidelity. In some embodiments, the temperature is about 25°C to about 40°C (e.g., about 28°C, about 30°C, about 32°C, about 35°C, or about 37°C). In some embodiments, the temperature is about 8°C to about 50°C (e.g., about 8°C, about 20°C, about 42°C, about 45°C, or about 50°C).

[0259] In one embodiment, the present disclosure relates to a non-naturally occurring enzyme that reverse transcribes a template nucleic acid molecule to produce a complementary deoxyribonucleic acid (cDNA) product and amplifies the cDNA product within a period of 3 hours or less (e.g., 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, or 30 minutes or less) and / or for a given nucleotide substrate with a processivity that is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 95%, or at least about 98% higher than the processivity of a reference enzyme for the same nucleotide substrate.

[0260] In one embodiment, the present disclosure relates to a non-naturally occurring enzyme that reverse transcribes a template nucleic acid molecule to produce a nucleic acid product and amplifies the nucleic acid product within a period of 3 hours or less (e.g., 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, or 30 minutes or less) and / or for a given nucleotide substrate with a processivity that is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 95%, or at least about 98% higher than the processivity of a reference enzyme for the same nucleotide substrate.

[0261] In one embodiment, the present disclosure provides a method for amplifying a nucleic acid molecule, comprising performing nucleic acid amplification on a nucleic acid molecule using a modified reverse transcriptase. In some embodiments, the reverse transcriptase is capable of amplifying a nucleic acid molecule with a processivity of at least about 80%, at least about 88%, at least about 90%, at least about 95%, or at least about 98% per base at about 4°C, about 8°C, about 12°C, about 30°C, about 28°C, about 29°C, about 32°C, about 35°C, about 37°C, about 42°C, about 50°C, or above about 42°C.

[0262] In some embodiments, the method of the present disclosure further comprises reverse transcribing the template nucleic acid molecule using a modified reverse transcriptase to produce a nucleic acid molecule. In some embodiments, the nucleic acid molecule is a free nucleic acid molecule. In some embodiments, the template nucleic acid molecule is a free nucleic acid molecule.

[0263] In some embodiments, amplification products are generated using primer extension or stretching reactions. Primer extension / stretching reactions can include cycles of incubating the reaction mixture at a denaturing temperature for a denaturing duration and incubating the reaction mixture at a stretching temperature for a stretching duration.

[0264] Any type of nucleic acid amplification reaction can be used to amplify target nucleic acid and produce amplified products. In addition, the amplification of nucleic acid can be linear, exponential or a combination thereof. Amplification can be based on emulsion or can be not based on emulsion. Non-limiting examples of nucleic acid amplification methods include reverse transcription, primer extension, polymerase chain reaction, ligase chain reaction, helicase-dependent amplification, asymmetric amplification, rolling circle amplification and multiple displacement amplification (MDA). In certain embodiments, amplified products may be DNA. In the case of amplified target RNA, DNA can be obtained by reverse transcription RNA, and the subsequent amplification of DNA can be used to produce amplified DNA products. Amplified DNA products can indicate the presence of target RNA in biological samples. In the case of amplified DNA, any DNA amplification can be adopted. Non-limiting examples of DNA amplification methods include polymerase chain reaction (PCR), variations of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric staggered PCR, decremental PCR), and ligase chain reaction (LCR). In some cases, DNA amplification is linear. In some cases, DNA amplification is exponential. In some cases, DNA amplification is achieved using nested PCR, which can improve the sensitivity of detecting amplified DNA products.

[0265] The denaturation temperature may vary depending on, for example, the specific biological sample analyzed, the specific source of the target nucleic acid (e.g., virions, bacteria) in the biological sample, the reagents used, and / or the desired reaction conditions. In some embodiments, the denaturation temperature may be about 80°C to about 110°C. In some embodiments, the denaturation temperature may be about 90°C to about 100°C. In some embodiments, the denaturation temperature may be about 90°C to about 97°C. In some instances, the denaturation temperature may be about 92°C to about 95°C. In yet other instances, the denaturation temperature may be about 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C.

[0266] The denaturation duration may vary depending on, for example, the specific biological sample being analyzed, the specific source of the target nucleic acid (e.g., virions, bacteria) in the biological sample, the reagents used, and / or the desired reaction conditions. In some embodiments, the denaturation duration may be less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, the denaturation duration may not exceed about 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

[0267] The stretching or extension temperature may vary depending on, for example, the specific biological sample being analyzed, the specific source of the target nucleic acid (e.g., viral particles, bacteria) in the biological sample, the reagents used, and / or the desired reaction conditions. In some embodiments, the stretching temperature may be about 30°C to about 80°C. In some embodiments, the stretching temperature may be about 35°C to about 72°C. In some embodiments, the stretching temperature may be about 45°C to about 68°C. In some embodiments, the stretching temperature may be about 35°C to about 65°C. In some embodiments, the stretching temperature may be about 40°C to about 67°C. In some embodiments, the stretching temperature may be about 50°C to about 68°C. In some embodiments, the stretching temperature may be about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 34°C, 33°C, 32°C, 31°C, 30°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 35°C, 36°C, 37°C, 38°C, 39°C , 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C or 80°C.

[0268] The stretch duration may vary depending on, for example, the specific biological sample analyzed, the specific source of the target nucleic acid (e.g., viral particles, bacteria) in the biological sample, the reagent used, and / or the required reaction conditions. In some embodiments, the stretch duration may be less than or equal to about 360 seconds, less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. In some embodiments, the stretch duration may not exceed about 120 seconds, 90 seconds, 80 seconds, 70 seconds, 65 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

[0269] In some embodiments, multiple cycles of primer extension reactions can be performed. Any suitable number of cycles can be performed. In some embodiments, the number of cycles performed may be less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 cycles. The number of cycles performed may depend on, for example, the number of cycles (e.g., cycle threshold (Ct)) required for obtaining a detectable amplified product (e.g., indicating the presence of a detectable amount of amplified DNA product of a target RNA in a biological sample). In some embodiments, the number of cycles required for obtaining a detectable amplified product (e.g., indicating the presence of a detectable amount of a DNA product of a target RNA in a biological sample) may be less than about or about 100 cycles, 75 cycles, 70 cycles, 65 cycles, 60 cycles, 55 cycles, 50 cycles, 40 cycles, 35 cycles, 30 cycles, 25 cycles, 20 cycles, 15 cycles, 10 cycles, 8 cycles, 7 cycles, 5 cycles or 4 cycles. In addition, in some embodiments, a detectable amount of amplifiable product (e.g., a detectable amount of DNA product indicating the presence of target RNA in a biological sample) can be obtained at a cycle threshold (Ct) of less than 100, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.

[0270] In some embodiments, the amplification step (e.g., primer amplification, template amplification, nucleic acid amplification) includes a PCR step. In some embodiments, each PCR cycle may include a denaturation step, an annealing step, and an extension step. In some embodiments, each PCR cycle may include a denaturation step and an extension step. In some embodiments, PCR includes at least about or about or up to about 1 cycle, at least about or about or up to about 4 cycles, at least about or about or up to about 5 cycles, at least about or about or up to about 10 cycles, at least about or about or up to about 15 cycles, at least about or about or up to about 20 cycles, at least about or about or up to about 25 cycles, at least about or about or up to about 30 cycles, at least about or about or up to about 35 cycles, at least about or about or up to about 40 cycles, at least about or about or up to about 45 cycles, at least about or about or up to about 50 cycles, at least about or about or up to about 55 cycles, at least about or about or up to about 55 cycles, at least about or about or up to about 50 cycles. At most about 60 cycles, at least about or about or at most about 65 cycles, at least about or about or at most about 70 cycles, at least about or about or at most about 75 cycles, at least about or about or at most about 80 cycles, at least about or about or at most about 90 cycles, at least about or about or at most about 95 cycles, at least about or about or at most about 100 cycles, at least about or about or at most about 110 cycles, at least about or about or at most about 120 cycles, at least about or about or at most about 130 cycles, at least about or about or at most about 140 cycles, at least about or about or at most about 150 cycles, at least about or about or at most about 160 cycles. In some embodiments, PCR comprises about 10 cycles to 40 cycles, about 20 cycles to 40 cycles, about 20 cycles to 38 cycles, about 20 cycles to 35 cycles, about 10 cycles to 35 cycles, about 10 cycles to 30 cycles, about 25 cycles to 30 cycles, about 20 cycles to 30 cycles, about 4 cycles to 8 cycles, or about 28 cycles to 32 cycles. In some embodiments, the reaction is heated to 95°C for 3 minutes before the PCR cycle begins. In some embodiments, each PCR cycle comprises holding 95°C for 3 seconds and holding 62°C for 20 seconds. In some embodiments, each PCR cycle comprises holding 95°C for 3 seconds, holding 54°C for 10 seconds, and holding 64°C for 20 seconds. In some embodiments, each PCR cycle comprises holding 95°C for 3 seconds and holding 64°C for 20 seconds. In some embodiments, each PCR cycle comprises holding 95°C for 3 seconds and holding 64°C for 20 seconds. In some embodiments, each PCR cycle comprises holding 95°C for 3 seconds and holding 62°C for 60 seconds. In some embodiments, each PCR cycle comprises holding 95°C for 3 seconds, holding 54°C for 10 seconds, and holding 64°C for 10 seconds. In some embodiments, PCR comprises 30 cycles. In some embodiments, the reaction is heated to 68°C after the PCR cycles are completed.In some embodiments, the reaction is heated to 68° C. for about 1 second to about 5 seconds, about 1 second to about 5 minutes, about 1 minute to about 5 minutes after the PCR cycles are completed. In some embodiments, the PCR methods described herein comprise an extension or stretching step that is at least about 5 seconds long, at least about 6 seconds long, at least about 7 seconds long, at least about 8 seconds long, at least about 9 seconds long, at least about 10 seconds long, at least about 11 seconds long, at least about 12 seconds long, at least about 13 seconds long, at least about 14 seconds long, at least about 15 seconds long, at least about 20 seconds long, at least about 30 seconds long, at least about 40 seconds long, at least about 50 seconds long, at least about 60 seconds long, at least about 90 seconds long, at least about 120 seconds long, at least about 150 seconds long, at least about 180 seconds long, at least about 210 seconds long, at least about 240 seconds long, at least about 270 seconds long, at least about 300 seconds long, at least about 330 seconds long, at least about 360 seconds long, at least about 390 seconds long, or longer.

[0271] The time for amplification to produce a detectable amount of amplified product indicating the presence of an amplified target nucleic acid can vary depending on the biological sample from which the target nucleic acid is obtained, the specific nucleic acid amplification reaction to be performed, and the specific number of cycles of the amplification reaction required. In some embodiments, amplification of a target nucleic acid can produce a detectable amount of amplified product indicating the presence of a target nucleic acid within the following time periods: 120 minutes or less; 90 minutes or less; 60 minutes or less; 50 minutes or less; 45 minutes or less; 40 minutes or less; 35 minutes or less; 30 minutes or less; 25 minutes or less; 20 minutes or less; 15 minutes or less; 10 minutes or less; or 5 minutes or less.

[0272] In some embodiments, the biological sample can be preheated before performing the primer extension reaction. The temperature (e.g., preheating temperature) and duration (e.g., preheating duration) of the preheated biological sample can vary depending on, for example, the specific biological sample analyzed. In some instances, the biological sample can be preheated for no more than about 60 minutes, 50 minutes, 40 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or 5 seconds. In some instances, the biological sample can be preheated at a temperature of about 80°C to about 110°C. In some instances, the biological sample can be preheated at a temperature of about 90°C to about 100°C. In some instances, the biological sample can be preheated at a temperature of about 90°C to about 97°C. In some instances, the biological sample can be preheated at a temperature of about 92°C to about 95°C. In some embodiments, the biological sample can be preheated at a temperature of about 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C or 100°C.

[0273] In certain embodiments, the reagents required for nucleic acid amplification may also include a reporter that generates a detectable signal, and the presence or absence of the detectable signal indicates the presence of the amplified product. The intensity of the detectable signal may be proportional to the amount of the amplified product. In some cases where an amplified product of a nucleic acid different from the target nucleic acid type initially amplified is produced, the intensity of the detectable signal may be proportional to the amount of the target nucleic acid initially amplified. For example, in the case of amplifying a target RNA by parallel reverse transcription and amplifying the DNA obtained from reverse transcription, the reagents required for the two reactions may also include a reporter that generates a detectable signal indicating the presence of the amplified DNA product and / or the amplified target RNA. The intensity of the detectable signal may be proportional to the amount of the amplified DNA product and / or the amplified original target RNA. The use of a reporter also realizes a real-time amplification method, including real-time PCR for DNA amplification.

[0274] The reporter can be connected to the nucleic acid (including the amplified product) by covalent or non-covalent bonding or interaction. Non-limiting examples of non-covalent bonding or interaction include ionic interactions, van der Waals forces, hydrophobic interactions, hydrogen bonding, and combinations thereof. In some embodiments, the reporter can be bound to the initial reactant, and changes in the reporter content can be used to detect the amplified product. In some embodiments, the reporter may be detectable (or undetectable) only when nucleic acid amplification is performed. In some embodiments, optically active dyes (e.g., fluorescent dyes) can be used as reporters. Non-limiting examples of dyes include SYBR green, SYBR blue, DAPI, propidium iodide, Hoeste, SYBR gold, ethidium bromide, acridine, proflavine, acridine orange, acridine yellow, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyl, anthramycin, phenanthridine, and acridine, ethidium bromide, propidium iodide, hexidium iodide, iodide), dihydroethidium, ethidium homodimer-1 and ethidium homodimer-2, ethidium monoazide and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOXOrange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-P RO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBRDX, SYTO-40, SYTO-41, SYTO-42, SYTO-43, SYTO-44, SYTO-45 (blue), SYTO-13, SYTO-16, SYTO-24, SYTO-21, SYTO-23, SYTO-12, SYTO-11, SYTO-20, SYTO-22, SYTO-15, SYTO-14, SY TO-25 (green), SYTO-81, SYTO-80, SYTO-82, SYTO-83, SYTO-84, SYTO-85 (orange), SYTO-64, SYTO-17, SYTO-59, SYTO-61, SYTO-62, SYTO-60, SYTO-63 (red), fluorescein, fluorescein isothiocyanate isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosin, coumarin, methylcoumarin, pyrene, malachite green, stilbene, lucifer yellow, cascade blue, blue), dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes (e.g., complexes containing europium and terbium), carboxytetrachlorofluorescein, 5-carboxy and / or 6-carboxyfluorescein (FAM), 5-(or 6-)iodoacetamidofluorescein, 5-{[2(and 3)-5-(acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein), lissamine rhodamine B sulfonyl chloride, 5 and / or 6 carboxyrhodamine (ROX), 7-amino-methyl-coumarin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophore, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3,6-disulfonate-4-amino-naphthalimide, phycobiliprotein, AlexaFluor 350, AlexaFluor405, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 755, and DyLight 800, or other fluorophores.

[0275] In some embodiments, the reporter can be a sequence-specific oligonucleotide probe that is optically active when hybridized with the amplified product. Due to the sequence-specific binding of the probe to the amplified product, the use of the oligonucleotide probe can increase specificity and detection sensitivity. The probe can be connected to any optically active reporter (e.g., dye) and can also include an optically active quencher that can block the associated dye. Non-limiting examples of probes that may be suitable for use as reporter agents include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes. In some embodiments, the reporter can be a radioactive species. Non-limiting examples of radioactive species include 14C, 1231, 1241, 1251, 1311, 99mTc, 35S, or 3H. In some embodiments, the reporter can be an enzyme that can produce a detectable signal. The detectable signal can be produced by the activity of the enzyme using its substrate or using a specific substrate when the enzyme has multiple substrates. Non-limiting examples of enzymes that can be used as reporters include alkaline phosphatase, horseradish peroxidase, 12-galactosidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, and luciferase.

[0276] In some embodiments, the amplified product (e.g., amplified DNA product, amplified RNA product) can be detected. The detection of the amplified product (including amplified DNA) can be achieved using any suitable detection method. The specific type of detection method used can depend on, for example, the specific amplified product, the type of reaction vessel used for amplification, other reagents in the reaction mixture, whether a reporter is included in the reaction mixture and whether a reporter is used, and the specific type of the reporter used. Non-limiting examples of detection methods include optical detection, spectral detection, electrostatic detection, electrochemical detection, and the like. Optical detection methods include, but are not limited to, fluorescence determination and UV-Vis light absorption. Spectral detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel-based techniques, such as gel electrophoresis, SDS-PAGE gel. Electrochemical detection methods include, but are not limited to, electrochemical detection of the amplified product after high performance liquid chromatography separation of the amplified product.

[0277] In some embodiments, the time required for the element of the method can vary depending on the specific steps of the method. In some embodiments, the amount of time for completing the element of the method can be about 5 minutes to about 120 minutes. In some embodiments, the amount of time for completing the element of the method can be about 5 minutes to about 60 minutes. In some embodiments, the amount of time for completing the element of the method can be about 5 minutes to about 30 minutes. In some embodiments, the amount of time for completing the element of the method can be less than or equal to 120 minutes, less than or equal to 90 minutes, less than or equal to 75 minutes, less than or equal to 60 minutes, less than or equal to 45 minutes, less than or equal to 40 minutes, less than or equal to 35 minutes, less than or equal to 30 minutes, less than or equal to 25 minutes, less than or equal to 20 minutes, less than or equal to 15 minutes, less than or equal to 10 minutes or less than or equal to 5 minutes.

[0278] In some embodiments, the reaction may have a pH suitable for producing a product, suitable for primer extension, protein expression, PCR amplification, or template jumping. In some embodiments, the pH of the reaction may range from about 5 to about 9, about 6 to about 9, about 7 to about 9, about 8 to about 9. In some embodiments, the pH range is about pH 2 to about pH 10, about pH 4 to about pH 10, about pH 2 to about pH 8, about pH 4 to about pH 8, about pH 5 to about pH 8, about pH 5 to about pH 7, about pH 6 to about pH 11, about pH 6 to about pH 12, about pH 5 to pH 13, about pH 5 to about pH 14. In some embodiments, the pH is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14.

[0279] In some embodiments, any of the methods of the present disclosure may include a detergent. In some embodiments, the detergent is a nonionic and / or zwitterionic detergent. In some embodiments, the nonionic detergent is selected from the group consisting of: Tween, Triton, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-SM, Triton N-101 (polyoxyethylene branched nonylphenyl ether), Triton QS-15, Triton QS-44, Triton RW-75 (polyethylene glycol 260 mono (hexadecyl / octadecyl) ether and 1-octadecyl alcohol), Triton X-100 (polyethylene glycol tert-octylphenyl ether), Triton X-102, Triton X-15, Triton X-151, Triton X-200, Triton X-207, Triton X-114, Triton X-165, Triton X-305, Triton X-405 (polyoxyethylene (40) isooctylphenyl ether), reduced Triton X-405 (polyoxyethylene (40) isooctylphenyl ether), Triton X-45 (polyoxyethylene glycol 4-tert-octylphenyl ether), Triton X-705-70, any form of TWEEN (including: TWEEN 20 (polyoxyethylene sorbitan monolaurate), TWEEN 21 (polyoxyethylene sorbitan monolaurate), TWEEN 40 (polyoxyethylene (20) sorbitan monopalmitate), TWEEN 60 (polyoxyethylene glycol sorbitan monostearate), TWEEN 61 (polyoxyethylene glycol sorbitan monostearate), TWEEN 65 (polyoxyethylene sorbitan tristearate), TWEEN 80 (polyoxyethylene sorbitan monooleate), TWEEN 81 (polyoxyethylene sorbitan monooleate), TWEEN 85 (polyoxyethylene (20) sorbitan trioleate)), Brij, Brij Brij 30 (polyoxyethylene 4 lauryl ether), Brij 35 (polyoxyethylene 23 lauryl ether), Brij 52 (polyoxyethylene 2 cetyl ether), Brij 56 (polyoxyethylene 10 cetyl ether), Brij 58 (polyoxyethylene 20 cetyl ether), Brij 72 (polyoxyethylene 2 stearyl ether), Brij 76 (polyoxyethylene 10 stearyl ether), Brij 78 (polyoxyethylene 20 stearyl ether), Brij 92 (polyoxyethylene 2 oleyl ether), Brij 97 (polyoxyethylene 10 oleyl ether), Brij 98 (polyoxyethylene 20 oleyl ether), Brij 700 (polyoxyethylene 100 stearyl ether), octylthioglucoside, maltoside and combinations thereof.

[0280] In certain embodiments, any method for producing any molecule disclosed herein includes at least one salt. In certain embodiments, the salt is at least one member selected from the group consisting of: NaCl, LiCl, AlCl3, CuCl2, MgCl2, InCl3, SnCl4, CrCl2, CrCl3, KCl, NaI, KI, TMACl (tetramethylammonium chloride), TEACl (tetraethylammonium chloride), KSCN, CsSCN, KCH3COO, CH3COONa, C5H8KNO4, C5H8NNaO4, CsCl and any combination thereof. In certain embodiments, any method for producing any molecule disclosed herein includes NaCl. In certain embodiments, the condition that is enough to produce molecule or library includes NaCl. In certain embodiments, reaction may have a salt concentration and / or a NaCl concentration suitable for producing product, suitable for primer extension, protein expression, pcr amplification or template jump. In some embodiments, the NaCl concentration is about 50 mM to about 1000 mM, about 100 mM to about 500 mM, about 200 mM to about 300 mM, about 200 mM to about 600 mM. In some embodiments, the NaCl concentration is at least about, at most about, or about 50 mM, at least about, at most about, or about 100 mM, at least about, at most about, or about 150 mM, at least about, at most about, or about 200 mM, at least about, at most about, or about 250 mM, at least about, at most about, or about 300 mM, at least about, at most about, or about 350 mM, at least about, at most about, or about 400 mM, at least about, at most about, or about 450 mM, at least about, at most about, or about 500 mM. In some embodiments, NaCl can improve the enzyme activity and / or template jumping of an enzyme or polypeptide (e.g., reverse transcriptase) of the present invention.

[0281] In some embodiments, the reaction may have a nucleotide (e.g., dNTP) concentration suitable for producing a product, suitable for primer extension, protein expression, PCR amplification, or template jumping. In some embodiments, the total concentration of dNTPs in the reaction may be about 50 μM to about 1000 μM, about 100 μM to about 500 μM, about 200 μM to about 300 μM, about 200 μM to about 600 μM. In some embodiments, the total concentration of dNTPs is at least about, at most about, or about 50 μM, at least about, at most about, or about 100 μM, at least about, at most about, or about 150 μM, at least about, at most about, or about 200 μM, at least about, at most about, or about 250 μM, at least about, at most about, or about 300 μM, at least about, at most about, or about 350 μM, at least about, at most about, or about 400 μM, at least about, at most about, or about 450 μM, at least about, at most about, or about 500 μM. μM, at least about, at most about, or about 550 μM, at least about, at most about, or at least about, at most about, or about 600 μM, at least about, at most about, or about 650 μM, at least about, at most about, or about 700 μM, at least about, at most about, or about 750 pM, at least about, at most about, or about 800 μM, at least about, at most about, or about 850 μM, at least about, at most about, or about 900 μM, at least about, at most about, or about 950 μM, or at least about, at most about, or about 1000 μM. In some embodiments, the total concentration of each dNTP is at least about, at most about, or about 1 μM; at least about, at most about, or about 2 μM; at least about, at most about, or about 3 μM; at least about, at most about, or about 4 μM; at least about, at most about, or about 5 μM; at least about, at most about, or about 6 μM; at least about, at most about, or about 7 μM; at least about, at most about, or about 8 μM; at least about, at most about, or about 9 μM; at least about, at most about, or about 10 μM; at least about, at most about, or about 15 μM; at least about, at most about, or about 20 μM; at least about, at most about, or about 25 μM; at least about, at most about, or about 30 μM; at least about, at most about, or about 35 μM; at least about, at most about, or about 40 μM. M; at least about, at most about, or about 45 μM; at least about, at most about, or about 50 μM; at least about, at most about, or about 55 μM; at least about, at most about, or about 60 μM; at least about, at most about, or about 65 μM; at least about, at most about, or about 70 μM; at least about, at most about, or about 75 μM; at least about, at most about, or about 80 μM; at least about, at most about, or about 85 μM; at least about, at most about, or about 90 μM; at least about, at most about, or about 95 μM; at least about, at most about, or about 100 μM; at least about, at most about, or about 250 μM; at least about, at most about, or about 500 μM; at least about, at most about, or about 1000 μM; at least about, at most about, or about 10000 μM.In some embodiments, the total concentration of each dNTP is about 2 μM to about 5 μM, about 2 μM to about 10 μM, about 2 μM to about 20 μM, about 2 μM to about 50 μM, about 2 μM to about 100 μM, about 2 μM to about 250 μM, about 5 μM to about 10 μM, about 5 μM to about 50 μM, about 5 μM to about 250 μM, about 5 μM to about 1000 μM.

[0282] In some embodiments, the concentration of each dNTP may be independent of and different from the concentration of one or more dNTPs. In some embodiments, the concentration of each dNTP (e.g., the concentration of each dCTP, dGTP, dTTP, or dATP) may be independent of and different from the concentration of at least one other dNTP. In some embodiments, the concentration of one dNTP (e.g., dCTP, dGTP, dTTP, or dATP) is different from the concentration of at least one other dNTP (e.g., dCTP, dGTP, dTTP, or dATP) by at least about or at most about or about 1, 2, 3, 4, 5, 7, 10, 20, 35, 50, 75, 90, 100, 200, 500, or 1000 times.

[0283] In some embodiments, the reaction mixture comprises a pH adjuster. Relevant pH adjusters include but are not limited to sodium hydroxide, hydrochloric acid, phosphate buffer solution, tris buffer, citric acid buffer solution, etc. For example, the pH of the reaction mixture can be adjusted to a desired range by adding an appropriate amount of a pH adjuster.

[0284] The temperature range suitable for producing the product can vary depending on factors such as the particular polymerase employed, the melting temperature of any optional primers employed, etc. In some embodiments, the polymerase can include, but is not limited to, a reverse transcriptase, a Moloney murine leukemia virus (MMLV) reverse transcriptase, an R2 reverse transcriptase, an RNA-guided DNA polymerase, a DNA-guided DNA polymerase, a non-LTR retrotransposon, an R2 non-LTR retrotransposon, a polypeptide having reverse transcriptase activity, or any variant thereof, or any combination thereof.

[0285] In some embodiments, conditions sufficient to produce a product comprise allowing the reaction mixture to reach a temperature in the range of about 4°C to about 72°C, about 16°C to about 70°C, about 37°C to about 50°C, about 40°C to about 45°C, about 30°C to about 42°C, about 25°C to about 42°C, about 25°C to about 30°C, about 28°C to about 32°C, about 29°C to about 31°C. In some embodiments, the temperature is about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about In some embodiments, the temperature is about or up to about 42°C. In some embodiments, the temperature is about or up to about 50°C. In some embodiments, the temperature is about or up to about 35°C. In some embodiments, the temperature is about or up to about 25°C. In some embodiments, the temperature is about or up to about 30°C. In some embodiments, the reactants are incubated for about 20 minutes to about 3 hours, about 30 minutes to about 1.5 hours, about 30 minutes to about 1 hour, about 30 minutes to about 2 hours, about 1 hour to about 2 hours, about 1 hour to about 1.5 hours, about 30 minutes to about 5 hours, about 1 hour to about 3 hours, about 1 hour to about 4 hours, about 1 hour to about 5 hours. In some embodiments, the reactants are incubated for about 1 hour. In some embodiments, the reactants are incubated for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours. In some embodiments, the reactants are incubated for at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 1 hour, at least about 1.5 hours, at least about 2 hours, at least about 2.5 hours, at least about 3 hours, at least about 3.5 hours, at least about 4 hours, at least about 4.5 hours, or at least about 5 hours. In some embodiments, the reactants are incubated at about 30° C. for about 1 hour or at about 42° C. for about 1 hour. In some embodiments, conditions sufficient to produce a molecule or nucleic acid molecule include maintaining a temperature of about 12° C. to about 42° C. for about 1 minute to about 5 hours.In some embodiments, conditions sufficient to produce a molecule or nucleic acid molecule include maintaining a temperature of about 8°C to about 50°C for about 1 minute to about 24 hours.

[0286] In certain embodiments, primers can be designed to a certain length. In certain embodiments, the length of primers can be about 6 to about 100 nucleotides, about 6 to about 90 nucleotides, about 6 to about 80 nucleotides, about 6 to about 70 nucleotides, about 6 to about 60 nucleotides, about 6 to about 50 nucleotides, about 6 to about 40 nucleotides, about 6 to about 30 nucleotides, about 6 to about 20 nucleotides, or about 6 to about 10 nucleotides. In certain embodiments, the length of primers can be about 25 to about 80, about 25 to about 75, about 25 to about 70, about 25 to about 65, about 25 to about 60, about 25 to about 55, about 25 to about 50, about 25 to about 45, about 25 to about 40, about 25 to about 35, or about 25 to about 30 bases. In some embodiments, the length of a primer can be at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases. In some embodiments, the length of the primer can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 bases. In some embodiments, the primer can be at least about, no more than about, or about 120, 130, 140, 150, 160, 170, 180, 190, 200, 230, 250, 270, 290, 300, 320, 340, 350, 370, 400, 420, 450, 470, 490, or 500.

[0287] In some embodiments, primers can be designed to anneal to a target at a given melting temperature (Tm). In some embodiments, Tm can be about 20°C to about 100°C, about 20°C to about 90°C, about 20°C to about 80°C, about 20°C to about 70°C, about 20°C to about 60°C, about 20°C to about 50°C, about 20°C to about 40°C, or about 20°C to about 30°C. In some embodiments, the Tm can be a minimum of about, a maximum of about, or about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C , 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 83°C, 84°C, 85°C, 96°C, 97°C, 98°C, 99°C or 100°C. Multiple primers can be designed to have Tm within a range, for example, across 15°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C or 1°C. Multiple primers can be designed to have consistent Tm.

[0288] In some embodiments, the enzyme of the present disclosure, or a modified enzyme (e.g., a modified reverse transcriptase), or a protein, or a polypeptide, or a variant or a PCR product, or a cDNA molecule, or a template, or a nucleic acid molecule, or any component can be purified. In some embodiments, fragmented or degraded nucleic acids (e.g., RNA or DNA) can be purified. In some embodiments, a reverse transcriptase or a modified reverse transcriptase can be purified. In some embodiments, an R2 reverse transcriptase or a modified R2 reverse transcriptase can be purified. In some embodiments, a non-LTR retrotransposon protein, or a polypeptide with reverse transcriptase activity, or a modified non-LTR retrotransposon protein, or a modified polypeptide with reverse transcriptase activity can be further purified. In some embodiments, a cDNA molecule can be purified. In some embodiments, a template can be purified. In some embodiments, a receptor nucleic acid molecule can be purified.

[0289] Purification can include precipitation, ultracentrifugation, chromatography based on size, charge, hydrophobicity, affinity, metal binding, HPLC. In some embodiments, purification includes column chromatography. In some embodiments, column chromatography can be size exclusion (SEC), ion exchange (IEX), affinity chromatography, immobilized metal ion affinity chromatography (IMAC), Ni-IMAC chromatography and / or hydrophobic interaction (HIC). In some embodiments, purification includes His tag affinity resin. In some embodiments, purification can include one step. In some embodiments, purification can include two steps. In some embodiments, two-step purification includes nickel and heparin. In some embodiments, two-step purification includes nickel affinity purification and heparin affinity purification. In some embodiments, two purification steps provide higher activity and / or increased template jump compared to one-step purification. In some embodiments, purification includes heparin affinity purification. In some embodiments, purification can include affinity purification, Ni-NTA affinity purification, fast protein liquid chromatography (FPLC) (AKTA FPLC system and Bio-Rad FPLC system), high pressure liquid chromatography (HPLC) (e.g., Beckman HPLC and Waters HPLC). In some embodiments, purification may include, but is not limited to, ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradient, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reverse phase purification, Ceramic HyperD (registered trademark) ion exchange chromatography, and hydrophobic interaction column (HIC). Also included are analytical methods such as SDS-PAGE (e.g., Coomassie, silver staining), immunoblotting, Bradford, and ELISA, which may be utilized during any step of the production or purification process and are typically used to measure the purity of a protein or enzyme composition.

[0290] In some embodiments, the overall activity of the purified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein or polypeptide with reverse transcriptase activity, reverse transcriptase, or variants thereof, or products thereof using two-step purification is at least about 2%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% higher than the overall activity using a single-step purification. In some embodiments, the overall activity of the purified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein or polypeptide having reverse transcriptase activity, reverse transcriptase, or variants thereof, or products thereof is at least about 0.5%, at least about 1%, at least about 2%, or more than the overall activity of the unpurified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein or polypeptide having reverse transcriptase activity, reverse transcriptase, or variants thereof, or products thereof. 1.5%, at least about 2%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%.In some embodiments, the purified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein, or polypeptide having reverse transcriptase activity, reverse transcriptase, modified enzyme, modified reverse transcriptase, modified polypeptide having reverse transcriptase activity, or variants thereof, or products thereof have at least about 0.5%, at least about 1%, at least about 3%, at least about or about 5%, at least about or about 10%, at least about or about 15%, at least about or about 20%, at least about or about 25%, at least about or about 30%, at least about or about 35%, at least about or about 40%, at least about or about 45%, at least about or about 50%, at least about or about 55%, at least about or about 60%, at least about or about 61%, at least about or about 62%, at least about or about 63%, at least about or about 64%, at least about or about 65%, at least about or about 66%, at least about or about 67%, at least about or about 68%, at least about or about 69%, at least about or about 70%, at least about or about 71%, at least about or about 72%, at least about or about 73%, at least about or about 74%, at least about or about 75%, at least about or about 76%, at least about or about 77%, at least about or about 78%, at least about 7%, at least about or about 68%, at least about or about 69%, at least about or about 70%, at least about or about 71%, at least about or about 72%, at least about or about 73%, at least about or about 74%, at least about or about 75%, at least about or about 76%, at least about or about 77%, at least about or about 78%, at least about or about 79%, at least about or about 80%, at least about or about 81%, at least about or about 82%, at least about or about 83%, at least about or about 84%, at least about or about 85%, at least about or about 86%, at least about or about 87%, at least about or about 88%, at least about or about 89%, at least about or about 90%, at least about or about 91%, at least about or about 92%, at least about or about 93%, at least about or about 94%, at least about or about 95%, at least about or about 96%, at least about At least about or about 84%, at least about or about 85%, at least about or about 86%, at least about or about 87%, at least about or about 88%, at least about or about 89%, at least about or about 90%, at least about or about 91%, at least about or about 92%, at least about or about 93%, at least about or about 94%, at least about or about 95%, at least about or about 96%, at least about or about 97%, at least about or about 98%, or at least about or about 99% pure.

[0291] In some embodiments, the purified enzyme, protein, polypeptide, R2 reverse transcriptase, non-LTR retrotransposon protein or polypeptide with reverse transcriptase activity, reverse transcriptase, or variants thereof, or its product, produces template hopping at least about or about one-fold, at least about or about two-fold, at least about or about three-fold, at least about or about four-fold, at least about or about five-fold, at least about or about six-fold, at least about or about seven-fold, at least about or about eight ... At least about or about nine times, at least about or about ten times, at least about or about fifteen times, at least about or about twenty times, at least about or about twenty-five times, at least about or about thirty times, at least about or about forty times, at least about or about fifty times, at least about or about seventy times, at least about or about eighty times, at least about or about ninety times, at least about or about 100 times, at least about or about 150 times, at least about or about 200 times, at least about or about 250 times, at least about or about 300 times, at least about or about 350 times, at least about or about 400 times, at least about or about 500 times, at least about or about 700 times, at least about or about 1000 times, at least about or about 10000 times.

[0292] Cell-free DNA and circulating tumor DNA

[0293] In some embodiments, obtaining a certain amount of circulating tumor (ctDNA) can include PCR. In some embodiments, obtaining a certain amount of ctDNA can include digital PCR. In some embodiments, obtaining a certain amount of ctDNA can include quantitative PCR. In some embodiments, obtaining a certain amount of ctDNA can include obtaining sequencing information on ctDNA. Sequencing information can include information related to one or more genomic region bases. In some embodiments, obtaining a certain amount of ctDNA can include hybridizing ctDNA with an array.

[0294] Determining the amount of free DNA can include determining the absolute amount of free DNA. The amount of free DNA can be determined by counting sequencing reads associated with the free DNA. The amount of free DNA can be determined by quantitative PCR.

[0295] Determining the amount of free DNA (cfDNA) can be performed by molecular barcoding of cfDNA. Molecular barcoding of cfDNA can include connecting an adapter to one or more ends of the cfDNA. The adapter can include a plurality of oligonucleotides. The adapter can include one or more deoxyribonucleotides. The adapter can include ribonucleotides. The adapter can be single-stranded. The adapter can be double-stranded. The adapter can include double-stranded and single-stranded portions. For example, the adapter can be a Y-shaped adapter. The adapter can be a linear adapter. The adapter can be a circular adapter. The adapter can include a molecular barcode, a sample index, a primer sequence, a linker sequence, or a combination thereof. The molecular barcode can be adjacent to the sample index. The molecular barcode can be adjacent to the primer sequence. The sample index can be adjacent to the primer sequence. The linker sequence can connect the molecular barcode to the sample index. The linker sequence can connect the molecular barcode to the primer sequence. The linker sequence can connect the sample index to the primer sequence.

[0296] The adapter may include a molecular barcode. The molecular barcode may include a random sequence. The molecular barcode may include a predetermined sequence. Two or more than two adapters may include two or more than two different molecular barcodes. The molecular barcode may be optimized to minimize dimerization. The molecular barcode may be optimized to enable even identification of amplification or sequencing errors. For example, the amplification of the first molecular barcode may introduce a single base error. The first molecular barcode may include a difference greater than a single base from other molecular barcodes. Therefore, the first molecular barcode with a single base error may still be identified as the first molecular barcode. The molecular barcode may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. The molecular barcode may include at least 3 nucleotides. The molecular barcode may include at least 4 nucleotides. The molecular barcode may include less than 20, 19, 18, 17, 16 or 15 nucleotides. The molecular barcode may include less than 10 nucleotides. The molecular barcode may include less than 8 nucleotides. The molecular barcode may include less than 6 nucleotides. The molecular barcode may include 2 to 15 nucleotides. The molecular barcode may include 2 to 12 nucleotides. The molecular barcode may include 3 to 10 nucleotides. The molecular barcode may include 3 to 8 nucleotides. The molecular barcode may include 4 to 8 nucleotides. The molecular barcode may include 4 to 6 nucleotides.

[0297] The adapter may include a sample index. The sample index may include a random sequence. The sample index may include a predetermined sequence. Two or more groups of adapters may include two or more than two different sample indexes. An adapter within a group of adapters may include a consistent sample index. The sample index may be optimized to minimize dimerization. The sample index may be optimized to enable even identification of amplification or sequencing errors. For example, amplification of a first sample index may introduce a single base error. The first sample index may include a difference greater than a single base from other sample indexes. Therefore, a first sample index with a single base error may still be identified as a first molecular barcode. The sample index may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. The sample index may include at least 3 nucleotides. The sample index may include at least 4 nucleotides. The sample index may include less than 20, 19, 18, 17, 16 or 15 nucleotides. The sample index may include less than 10 nucleotides. The sample index may include less than 8 nucleotides. The sample index may include less than 6 nucleotides. The sample index may include 2 to 15 nucleotides. The sample index may include 2 to 12 nucleotides. The sample index may include 3 to 10 nucleotides. The sample index may include 3 to 8 nucleotides. The sample index may include 4 to 8 nucleotides. The sample index may include 4 to 6 nucleotides.

[0298] The adaptor may include a primer sequence. The primer sequence may be a PCR primer sequence. The primer sequence may be a sequencing primer.

[0299] The adapter can be connected to one end of the cf DNA. The adapter can be connected to both ends of the cf DNA. The adapter can be connected to one or more ends of the single-stranded cf DNA. The adapter can be connected to one or more ends of the double-stranded cfDNA.

[0300] The adapter may be attached to the cf DNA by ligation. The ligation may be a blunt-end ligation. The ligation may be a sticky-end ligation. The adapter may be attached to the cf DNA by primer extension. The adapter may be attached to the cf DNA by reverse transcription. The adapter may be attached to the cf DNA by hybridization. The adapter may include a sequence that is at least partially complementary to the cf DNA. Alternatively, in some cases, the adapter does not include a sequence that is complementary to the cf DNA.

[0301] The cf-DNA may be derived from a tumor of the subject. The cf-DNA may be derived from any sample of the subject. The cf-DNA may be derived from any organ of the subject. The cf-DNA may be derived from any body fluid (e.g., blood, saliva, urine, and mucus) of the subject. The method may further include detecting cancer in the subject based on the detection of the cf-DNA.

[0302] In some embodiments, the presence or absence of a sequence may be related to a DNA or RNA expression profile or cancer. For example, the presence or absence of a sequence may be related to a transcriptional expression profile, a microRNA expression profile, a cancer expression profile, or a genomic mutation expression profile of a sample (e.g., a single cell). In some embodiments, cfDNA can be used for cancer expression profiling, and / or monitoring disease progression, and / or monitoring the occurrence of previous mutations and new mutations. The method may further include diagnosing cancer in a subject based on the detection of cf-DNA. Diagnosing cancer may have a sensitivity of at least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99%. Diagnosing cancer may have a specificity of at least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. The method may further include prognosing cancer in the subject based on the detection of cf-DNA. Prognosing cancer may have a sensitivity of at least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. Prognosing cancer may have a specificity of at least about 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. The method may further include determining a treatment regimen for the subject based on the detection of cf-DNA. The method may further include administering an anticancer therapy to the subject based on the detection of cf-DNA. The method may further include detecting mutations (e.g., mutations in a specific region) based on sequencing information. Diagnosis of cancer may be based on the detection of mutations. Detection of at least 3 mutations may indicate cancer. Detection of one or more mutations in three or more regions may indicate cancer.

[0303] Enzyme mutation

[0304] In some embodiments, the modified enzymes or derivatives and variants can be prepared during peptide synthesis or by post-production modification. In some embodiments, the modified enzymes or derivatives and variants can be prepared by site-directed mutagenesis (e.g. Site-directed mutagenesis kit protocol), random mutagenesis or enzyme cleavage and / or nucleic acid ligation. In some embodiments, derivatives and variants or modified enzymes are generated by random mutagenesis. In some embodiments, rational design and / or mutations are based on sequence alignment analysis. In some embodiments, rational design / mutation is based on sequence alignment analysis of determined and known enzymes and proteins. In some embodiments, enzymes and / or elements are subjected to sequence alignment analysis for homology to R2, including but not limited to non-LTR retrotransposons, telomerase, type II introns, LTR retrotransposons, reverse transcriptases, retroviral reverse transcriptases (e.g., HIV, MMLV) and viral RNA-dependent RNA polymerases.

[0305] In some embodiments, the variants or modified enzymes disclosed herein can be produced by methods including, but not limited to, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution, as well as various other recombination methods. Methods for preparing modified enzymes, polynucleotides, and proteins (e.g., variants) include: DNA shuffling methods; methods based on non-homologous recombination of genes, such as ITCHY (see Ostermeier et al., 7:2139-44

[1999] ), SCRACHY (see Lutz et al. 98:11248-53

[2001] ), SHIPREC (see Sieber et al. 19:456-60

[2001] ), and NRR (see Bittkeret et al., 19:456-60

[2001] ). al, 20:1024-9

[2001] ; Bittkeretak, 101:7011-6

[2004] ); and methods that rely on the use of oligonucleotides to insert random and targeted mutations, deletions and / or insertions (see Ness et al., 20:1251-5

[2002] ; Coco et al., 20:1246-50

[2002] ; Zha et al., 4:34-9

[2003] ; Glaser et al., 149:3903-13

[1992] ). In some embodiments, the polynucleotides, polypeptides, proteins or enzymes of the present disclosure may be altered by random mutations prior to recombination by means of error-prone PCR, random nucleotide insertion or other methods. The polynucleotides, polypeptides, proteins or enzymes of the present disclosure may be produced by DNA shuffling, gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling involves assembling two or more DNA segments by homologous or site-directed recombination to produce polynucleotide sequence changes. DNA shuffling can be used to modulate the activity of the polynucleotides, polypeptides, proteins or enzymes of the present disclosure, and these methods can be used to generate polypeptides with altered activity.See generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; 5,837,458; and 6,444,468; and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998). The polynucleotides, polypeptides, proteins or enzymes of the present disclosure may contain one or more components, motifs, segments, parts, domains, fragments,...

Claims

1. A purified polypeptide comprising one or more domains from an R2 reverse transcriptase, wherein the purified polypeptide generates one or more copies of a complementary deoxyribonucleic acid molecule from a template nucleic acid molecule with an error rate of up to about 5%.

2. The polypeptide of claim 1, wherein the polypeptide reverse transcribes the template nucleic acid molecule with a processivity of at least about 80% per base.

3. The polypeptide of claim 1, wherein the polypeptide reverse transcribes the template nucleic acid molecule at a temperature of about 12°C to about 42°C.

4. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:

67.

5. A non-naturally occurring enzyme comprising one or more domains from an R2 reverse transcriptase, wherein the non-naturally occurring enzyme produces a complementary deoxyribonucleic acid product in a period of less than three hours with a performance index greater than 1.0 for at least one enzyme property, wherein the at least one enzyme property is selected from the group consisting of: improved stability, specific activity, protein expression, purification, processivity, strand displacement, template jumping, increased DNA / RNA affinity, and fidelity compared to a purified enzyme having SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67.

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