RU2811465C2 - Method of amplification and identification of nucleic acids - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of obtaining labeled amplification fragments of a nucleic acid matrix is described. A nucleic acid template is provided. At least two oligonucleotide primers are annealed to a nucleic acid template, where at least one underlying primer is an elongation stopper. Elongation of at least two oligonucleotide primers is carried out in a template-specific manner. The elongation reaction stops when the elongation product reaches 5'-end of the nucleic acid template or oligonucleotide primer as a nucleic acid elongation stopper, hybridized on the nucleic acid template below the elongation product. A plurality of at least two adapter nucleic acids are provided, wherein the said plurality of adapters contain different identification sequences on their 5'-end, and these identification sequences do not hybridize with the oligonucleotide primer, which is an elongation stopper, or with the template. The adapter nucleic acid is ligated at its 5'-end with 3'-end of the elongation product, and for ligation, the elongation products are hybridized with the matrix, thereby obtaining a labeled amplification fragment. Also the following is described: a set containing at least two oligonucleotide primers capable of hybridizing with the template nucleic acid and triggering the elongation reaction on its 3'-end, where at least one primer is an elongation stopper, a plurality of at least two adapter nucleic acids, wherein the said plurality of adapters contain different identification sequences on their 5'-end, wherein such identification sequences do not hybridize with oligonucleotide primers, preferably where the adapter nucleic acid either binds and hybridizes, or does not bind and does not hybridize with the oligonucleotide primer, reverse transcriptase and oligonucleotide ligase.

EFFECT: invention expands the arsenal of agents for delivering nucleic acids.

15 cl, 5 dwg, 5 ex

Description

Field of technology to which the invention relates

The present invention relates to the field of nucleic acid analysis and amplification.

State of the art

US 20100273219 A1 describes a multiprimer amplification method for barcoding target nucleic acids.

WO 2012/134884 A1 describes barcoding of template nucleic acids in a multiplex amplification reaction.

WO 2013038010 A2 describes a method for producing an amplified nucleic acid portion from a nucleic acid template using oligonucleotide primers and stoppers to prevent strand displacement and polymerase readout, which is used to obtain nucleic acid portions for sequencing. This method eliminates errors in the form of preference for one sequence in nucleic acid amplification.

WO 2014071361 A1 describes a method for producing double-barcoded nucleic acids using barcoded adapter nucleic acids.

US 20140274729 A1 describes a method for preparing cDNA libraries using DNA polymerases with strand displacement activity.

EP 3119886 B1 describes a quantitative method for obtaining nucleic acid products from an RNA template.

US 2018163201 A1 discloses a reverse transcription method in which a C-tail is added to the 3' end of the cDNA strand.

WO 2016138500 A1 describes a method for barcoding nucleic acids for sequencing. Stochastic, that is, random barcodes are used as molecular tags.

Molecular tags or unique molecular identifiers (UMIs), also called molecular barcodes, have been developed to identify PCR duplicates to reduce PCR bias expressed as preference for certain sequences and to identify rare mutations. Attaching unique molecular identifiers to RNA molecules prior to any PCR amplification when generating a sequencing library provides a distinctive identifier for each input molecule. This makes it possible to eliminate the effects of sequence preference errors in subsequent PCR amplification, which is especially important where many PCR cycles are required, such as when generating sequencing libraries from small starting quantities of template, as in single cell studies. After PCR, molecules sharing the same sequence as well as the same UMI are assumed to be identical copies derived from the same parent molecule (Sena et al., Scientific Reports (2018) 8: 13121).

The essence of the invention

An object of the invention is to provide an improved method for generating template nucleic acid sequence fragments that facilitates the isolation and assembly of these sequence fragments into a combined sequence corresponding to the template nucleic acid sequence. Desirable improvements would also reduce sequence preference errors in fragment acquisition and increase coverage of sequence fragments along the entire length of the array, increasing the correct assembly of the resulting combined sequence.

Accordingly, the invention provides a method for producing labeled amplification fragments of a nucleic acid template, comprising the steps of providing such a nucleic acid template, annealing at least one oligonucleotide primer to the given nucleic acid template, elongating at least one oligonucleotide primer in a template-specific manner, thereby obtaining elongation product, wherein this elongation reaction stops when the elongation product reaches the 5' end of the nucleic acid template or a nucleic acid elongation stopper hybridized on the nucleic acid template in the 3' direction from the elongation product, providing an adapter nucleic acid containing the identification sequence on at its 5' end, wherein the identification sequence does not hybridize with the elongation stopper upon contact with it, and preferably does not hybridize with the template, ligating the adapter nucleic acid at its 5' end with the 3' end of the elongation product, thereby obtaining a labeled amplification fragment.

The invention also provides a method for producing labeled amplification fragments of a template nucleic acid, including the steps of providing such a template nucleic acid, annealing at least one oligonucleotide primer with a given template nucleic acid, elongating at least one oligonucleotide primer in a template-specific manner, obtaining this is an elongation product, providing an adapter nucleic acid containing an identification sequence, such an identification sequence not hybridizing with the template, ligating the adapter nucleic acid at its 5' end with the 3' end of the elongation product, thereby obtaining a labeled amplification fragment.

The invention also provides kits suitable for carrying out the method. The kit of the invention may contain at least one oligonucleotide primer capable of hybridizing to a template nucleic acid and triggering an elongation reaction at its 3' end, one or more elongation stoppers capable of hybridizing to a template nucleic acid, preferably capable of triggering an elongation reaction at at its 3' end, one or more adapter nucleic acids containing an identification sequence at its 5' end, wherein such identification sequence does not hybridize to an elongation stopper, preferably wherein the adapter nucleic acid binds, hybridizes or does not bind to an elongation stopper, the reverse transcriptase and oligonucleotide ligase. The different components of the kit can be presented in different containers such as bottles.

The following detailed description sets forth all aspects, including methods and kits, as well as embodiments of the present invention. Those. the method descriptions may accordingly describe the contents of the sets. Any components described in the methods may be included in kits. The components of the kit can be used in the methods of the invention.

Disclosure of the invention

The present invention provides a method for producing labeled amplification fragments of a template nucleic acid, in which an identification sequence is introduced as a tag before amplification of these fragments. The nucleic acid template may be present in multiple copies. According to the invention, fragmentation is a process that typically occurs during amplification, that is, based on a matrix of a given length, one or more (usually several) fragments are created when parts of this matrix are amplified. The sequences of the resulting fragments can overlap when fragments are simultaneously formed from different copies of the template, and primers for the synthesis of these complementary nucleic acid fragments are placed in different places on different copies of the template. Although the concepts of the invention work for one fragment per template, preferably many fragments are generated from one template molecule, typically by using multiple primers that bind to the template at different locations.

The invention improves existing methods by linking an identification sequence to the resulting fragment. Identification sequences can be introduced using a primer or after elongation, i.e. synthesis of a complementary nucleic acid fragment. The identification sequence is then introduced by ligating the elongation product to an adapter nucleic acid. Unexpectedly, the ligation reaction proceeds with single-stranded identification sequences, i.e. portions of the identification sequences containing the unhybridized (or “free”) 5' end can be ligated to the 3' end of the elongation product. The ligation reaction usually involves a phosphate residue, which is preferably located at the 5' end of the identification sequence. It is not expected that the adapter nucleic acid, depending on the template or hybridization stopper sequence, will be located in the vicinity of the 3' end of the elongation product (as shown in the examples). Although such proximity can be achieved by providing the adapter nucleic acid with a complementary portion of the sequence (downstream, i.e., in the 3' direction of the identification sequence) for hybridization with a template-bound oligonucleotide (which is also referred to herein as an elongation stopper or simply a stopper and may also serve as an additional primer if more than one fragment is formed per template), but directional proximity is not required and can result from a non-directional simple diffusion process. In particular, it has been shown that an adapter nucleic acid can ligate to an elongation product that has reached the 5' end of the nucleic acid template, where there are no other elongation stoppers. This ligation reaction can reach this end of the elongation product directly or after the polymerase has added one or more non-template nucleotides based on its terminal transferase activity, which some polymerases possess. This ligation to the elongation product that corresponds to the 5' end of the template has some unexpected and useful advantages: it increases the number of fragments at the 5' end of the template and thereby significantly increases sequence coverage, which prior art methods lack. In previous methods, the distribution of the initial fragment sites is constant, which leads to high fragment coverage in the middle part of the template with much lower coverage, close to zero, at its 3' and 5' ends (which depends on the number of copies of the template, the average size of the fragments and sequencing read lengths). This effect at the 5' end is attenuated by the method of the invention. In addition, the invention also provides embodiments to increase coverage at the 3' end of the template.

Amplified fragments (formed one fragment molecule per elongation reaction) are usually subject to further amplification, that is, copying. This means that the ligated identification sequence is amplified, that is, copied. Typically, identification sequences are so diverse that a random selection process allows the unambiguous identification of individual fragments that carry the same sequence but originate from different copies of the same template. In all embodiments of the invention, the identification sequence helps determine whether the copies of the fragments after sequencing come from different copies of the template because they have different identification sequences, or whether they originate from the same template molecule and are simply copies resulting from such further amplification.

The following method provides for obtaining labeled amplification fragments of a template nucleic acid, including the steps of providing such a template nucleic acid, annealing at least one oligonucleotide primer with a given nucleic acid template, elongating at least one oligonucleotide primer in a template-specific manner, thereby obtaining elongation product, providing an adapter nucleic acid containing an identification sequence, wherein the identification sequence does not hybridize to the template, ligating the adapter nucleic acid preferably at its 5' end to the 3' end of the elongation product, thereby obtaining a labeled amplification fragment. This method is essentially the same as above, and all of the preferred embodiments described herein also apply, except stoppers are not used. Several primers can be used, possibly without a stopper function. Adapter nucleic acids can still ligate to elongation products through a process of diffusion. For ligation, elongation products can still hybridize with the matrix or as a single strand. However, stoppers are preferably used.

The method according to the invention begins with the step of providing nucleic acid templates. Matrix molecules for use in the method of the invention should be available to those skilled in the art. Typically the template is provided in the form of a sample of nucleic acid molecules. Such template nucleic acids can be isolated from cells such as eukaryotic or prokaryotic cells. In particularly preferred embodiments, the template is RNA. Total RNA or a fraction of RNA such as mRNA or RNA from cells depleted of rRNA may be provided. The amount of RNA that is easy to handle is, for example, 0.1 pg to 500 ng, 1 pg to 200 ng, 10 pg to 100 ng, or 0.1 pg to 100 ng rRNA-depleted RNA, or from 0.1 ng to 1000 ng of total RNA. In some embodiments, the amount of total RNA may be, for example, 10 pg, and the amount of RNA without rRNA may be less than 1 pg. Primers, stoppers and adapters are preferably DNA.

The method also includes annealing at least one oligonucleotide primer to a template nucleic acid. An oligonucleotide primer is an oligonucleotide molecule, preferably DNA, that hybridizes to a template and is capable of priming an elongation reaction, which is standard practice in the art. The oligonucleotide primer (or simply “primer”) preferably hybridizes to the template in at least one region along its length, eg, from 4 nucleotides to 30 nucleotides (nt). Annealing occurs through this hybridization. The primer may contain a portion that does not hybridize to the template. Such additional portions may be used for hybridization with other oligonucleotides and/or for additional amplification as mentioned above, where amplification fragments are further amplified to produce copies thereof. However, such additional parts or regions may contain a sequence to which other primers for this amplification/copy reaction bind. Such portions are also referred to as a primer linker sequence. The primer linker sequence preferably has a length of from 4 to 30 nt.

Returning to the basic method of the invention, at least one oligonucleotide primer is elongated in a template-specific manner, thereby generating an elongation product (complementary sequence). Such reactions are standard in the art and typically use polymerases. If the template is RNA, then an RNA-dependent polymerase such as reverse transcriptase is used. If the template is DNA, then a DNA-dependent polymerase is used. The elongation reaction stops when it reaches a nucleic acid elongation stopper hybridized on the template nucleic acid downstream of the elongation product, or when the elongation product reaches the 5' end of the template nucleic acid. It is clear that when the elongation reaction reaches the 5' end of the template and thereby goes beyond the template, it will stop. Some polymerases at this point can add one or more non-template nucleotides to the elongation product, which is acceptable and may even be useful in the selection of 5'-spanning products when analyzing the sequence of the resulting labeled amplified fragments. However, such addition of non-template nucleotides is not necessary. The elongation reaction will also stop when it reaches a nucleic acid elongation stopper hybridized on the nucleic acid template downstream of the elongation product. Such stopping of the reaction is described in detail in WO 2013/038010 A2 (incorporated here by reference). In this WO document, an elongation stopper is referred to as an “oligonucleotide stopper” or “additional oligonucleotide primer”. In the present invention, the following terms are used: nucleic acid elongation stopper or simply “elongation stopper” or simply “stopper”. Such a stopper according to the invention can also be a primer and then it corresponds to the “additional oligonucleotide primer” in WO 2013/038010 A2. Essentially, such a stopper stops the elongation reaction occurring in front of it (so the stopper is located below the elongation product), creating an obstacle on the matrix. The stopper anneals or hybridizes to the template, and the elongation reaction cannot displace it and therefore stops. Further promotion, i.e. displacement of the stopper would be an adverse reaction. Measures to prevent movement of the stopper are described in detail in WO 2013/038010 A2 and can be applied in accordance with the invention. Briefly, preferred methods and means for preventing stopper displacement (caused by strand displacement activity) are to use elongation stoppers containing one or more modified nucleotides that increase the melting temperature of the hybridized sequence when it has hybridized to the template (the portion of the stopper that anneals/hybridizes to the template ). The melting point increase applies to unmodified natural nucleic acid such as DNA or RNA. Modifications such as LNA (locked nucleic acid), ZNA (zip nucleic acid), 2'-fluoronucleosides/2'-fluoronucleotides or PNA (peptide nucleic acid). Other measures include the use of a polymerase that does not have strand displacement activity or the use of intercalators. Preferably, 1, 2, 3, 4, 5 or 6 nucleotides are modified. Preferably, the modified nucleotides are located on the 5' side of the portion of the stopper sequence that hybridizes to the template. There may be other parts of the stopper in the 5' direction that do not hybridize, such as amplification sequences that act in the same way as described for the oligonucleotide primer described above for amplification/copying in a further amplification reaction (“linker primer sequence”), but in fact, such an additional part is preferable for binding/hybridization with the adapter nucleic acid - see below. The adapter may bind/hybridize to a “primer linker sequence” or another portion of the oligonucleotide stopper. In preferred embodiments, the elongation stopper, and also preferably the oligonucleotide primer, contains one or more modified nucleotides that increase the melting temperature of the annealing sequence (linker) when annealed to the template.

Preferably, after the elongation reaction, those primers and stoppers that are not bound to the template are removed in a purification step. Those. elongation products hybridized with the matrix are purified and stored for further processing. Other embodiments of the invention are performed in one volume without purification. Such purification can be carried out by methods known in the art, eg, immobilizing the template or elongation products on a solid phase (eg, beads) and washing to remove unbound primers and stoppers. An example of such a method is solid phase reversible immobilization (SPRI; DeAngelis et al., Nucleic Acids Research, 1995, 23(22): 4742-4743).

The method of the invention includes the step of producing an adapter nucleic acid containing an identification sequence at its 5' end. The adapter nucleic acid may also include other sequence tags such as amplification sequences (amplification sequences). It is the 5' end that is intended for ligation with the 3' end of the elongation product to label the latter with an identification sequence. The identification sequence must not hybridize to either the elongation stopper or the template. Therefore, it is usually single-stranded and does not hybridize. The term “identification sequence” is used to refer to the 5'-terminal part of the adapter nucleic acid that does not hybridize or anneal, even if only portions of the identification sequence are later used for identification. Other portions of the adapter nucleic acid may hybridize with or anneal to the elongation stopper. The adapter nucleic acid may also contain a complementary primer sequence that serves as a target for a further amplification reaction of labeled amplification fragments as described above (the so-called adapter linker sequence). Hybridization of an identification sequence to an elongation stopper or to a template can be prevented by selecting a sequence for the identification sequence that is not complementary to the elongation stopper. It is also possible to select a sequence for the identification sequence that is not complementary to the template. This is easy to do if the sequence of the matrix is known. If it is not known, but comes from a biological source, then the identification sequence can be selected from sequences that are not or rarely found in biological nucleic acids. Such sequences are known among “spike-in” nucleic acids such as ERCC (External RNA Controls Consortium) sequences or SIRV (spike-in RNA variants) sequences (e.g., see ERCC, BMC Genomics 2005, 6:150; Jiang et al. , Genome Res. 2011, 21(9): 1543-1551; WO 2016/005524 A1, all of which are incorporated herein by reference). If the identification sequence hybridizes to the template in a side reaction, this usually prevents ligation in the next step and thus does not produce labeled fragments and is therefore not reported as a result. Such adverse reactions are acceptable, but not preferred. The easiest and most preferred method of preventing the identification sequence (and preferably the entire adapter nucleic acid) from annealing to the template is simply to introduce the adapter nucleic acid after the elongation reaction. The template after the elongation reaction is in a double-stranded form with elongation products (as well as primer and stoppers). In this form, the adapter nucleic acid can no longer bind to the template, since the template is already covered by hybridization partners. In this preferred method, the identification sequence may even have a sequence that is complementary to the template and is capable of hybridizing to the template, but this is prevented by the sequence of steps of the method. Thus, in this embodiment there is no need to consider the matrix sequence.

The most preferred option for preventing annealing of the identification sequence on the stopper is to have the stopper portions and the adapter portions having complementary sequences to each other. Because when the adapter approaches the stopper, complementary sequences will hybridize first, and the identification sequence will remain single-stranded.

The method of the invention also involves ligating the adapter nucleic acid at its 5' end to the 3' end of the elongation product, thereby generating a labeled amplification fragment. Ligation is usually carried out using the enzyme ligase. The type of ligase depends on the nature of the ligated oligonucleotides and can be selected by one skilled in the art. Examples of ligases include DNA ligases or RNA ligases. The ligase may also be an RNA ligase, especially an RNA ligase that has DNA ligation activity of the T4 RNA ligase-2 type. Other ligases are T4 DNA ligase, T4 RNA ligase-1, DNA ligase I, DNA ligase III, DNA ligase IV, E. coli DNA ligase, DNA ampligase, truncated Rnl2, truncated Rnl2 K227Q, Thermus scotoductus ligase, Methanobacterium thermoautotrophicum RNA ligase, thermostable App ligase (NEB), Chlorella virus DNA ligase or SplintR ligase. The ligase may be a single-stranded ligase or a double-stranded ligase. Combinations of ligases for different reactions in the same reaction volume are also possible and must proceed in parallel, for example when different elongation products and/or adapter nucleic acid molecules are present which must be ligated simultaneously. Preferred combinations are DNA ligase and RNA ligase or single-stranded ligase and double-stranded ligase. The ligase reaction typically involves a phosphate residue, which is preferably located at the 5' end of the adapter nucleic acid identification sequence. Other 5' enzymes can also be used for ligation, eg ligation of adenylated ends. They can be ligated using truncated ligases or App ligases.

The resulting labeled amplification fragments after ligation will have the following structure from the 5' to the 3' end: primer sequence - elongation product sequence - adapter sequence with an identification sequence adjacent to the elongation product sequence. The primer sequence may include a “primer linker sequence” and/or the adapter sequence may include an “adapter linker sequence”. The products obtained by the method according to the invention, i.e. the resulting labeled amplification fragments are preferably further amplified. With such further amplification, copies of the resulting labeled amplification fragments are formed by methods known in the art such as PCR (polymerase chain reaction) or linear amplification. Such further amplification typically involves additional primers that bind to the labeled amplification fragments, preferably on linker sequences, especially linker sequences located at the ends of the fragments, i.e. within part of the primer sequence and part of the adapter sequence, especially preferably at the 5' end of the primer sequence and at the 3' end of the adapter sequence. As stated above with respect to these primers and adapters, they may include regions of known sequence for binding such primers upon further amplification (“primer linker sequence” and “adapter linker sequence”). These sections (or “parts”) may be so long and specific that they will not bind to the matrix; they may be universal primer binding sites, i.e. non-selective between different adapters/primers, unlike the identification sequence, which is preferably unique.

The identification sequence provides a unique tag for the amplification fragment and is therefore also referred to herein as a unique molecular identifier (UMI). Identification sequences can identify repeats during further amplification (eg, PCR) and reduce the effects of sequence-dependent amplification error, which is expressed in the preferential amplification of one sequence. In preferred embodiments, the identification sequences are oligonucleotides, with a substantially random distribution of nucleotides at each position, that are ligated to elongation products (fragments) before further amplification. If the identification sequences are evenly distributed and their number greatly exceeds the number of identical elongation products, then it is unlikely that the same identification sequence will ligate to two identical elongation products (different copies). In this case, the number of different identification sequences after further amplification will be the same as their number before further amplification. The identification sequences of the invention can also be used as described for UMI in Sena et al. (Scientific Reports (2018) 8:13121). The entire sequence or portions of the entire sequence of a tagged fragment may be considered a “read” in next generation sequencing methods and further sequence analysis. Data analysis involves assembling one or more reads to produce a combined sequence template. Subsequently, data analysis can also evolve into quantitative analysis of template molecules and fragments, which can provide insight into whether certain template copies are over- or under-represented, e.g. indicating different rates of expression of RNA splice variants. In preferred embodiments, the present invention further includes the step of assembling sequences of such amplification fragments that are unique, wherein tags are used to identify the unique amplification fragments. Various identification sequences in the amplified tagged amplification fragments identify unique amplification fragments. Identification sequences provide the ability to duplicate and repeat identification and removal during assembly or at any other stage of data analysis.

In preferred embodiments, the identification sequence is 3 nucleotides (nt) or more in length, preferably 3 nt to 20 nt, particularly preferably 4 nt to 15 nt or 5 nt to 10 nt, such as 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt or more. This length is short enough for ease of handling and efficient ligation reactions, but still provides a sufficiently large number of different identification sequences due to the rearrangement of nucleotides within them, providing the required identification of single amplified fragments, preferably providing unique tags for them.

In preferred embodiments, when the elongation product reaches the 5' end of the nucleic acid template, the nucleotide polymerase is able to add non-template nucleotides to the elongation product, preferably through the terminal transferase activity of the polymerase, and/or preferably at least 1 to 15 non-template nucleotides are added. in at least 70% of elongation products. As stated above, such addition of non-template nucleotides is a property of some polymerases (see Chen et al., Biotechniques 2001, 30(3): 574-582). This activity is most pronounced in reverse transcriptases such as M-MLV (murine leukemia virus) reverse transcriptase or AMV (alfalfa mosaic virus) reverse transcriptase. These non-template nucleotides usually represent any type of nucleotide (A, T(U), G, C) and may appear randomly. This means that the 5' end elongation products of different templates may have the same sequence corresponding to the 5' end, but may then be continued by different, apparently random additional nucleotides that are the products of such non-template addition. These various additions can be used to determine the exact position of the 5' end of the template sequence at the transition between the repeated template sequence and random non-template additions. The non-template nucleotides of the tagged fragment are followed by an identification sequence that can be used as described above. In case the identification sequence is (also) random, then non-template random nucleotides can be considered as part of the identification sequence. The position of the identification sequence relative to the constant portion of the adapter sequence uniquely defines the identification sequence.

In particularly preferred embodiments, a plurality of adapter nucleic acids are provided that are used in the ligation step. Adapters from this set may have different identification sequences. This allows the adapters and the resulting fragments to which they are ligated to be unambiguously identified. Preferably, at least 10, more preferably at least 50 or even 100 or more or 200 or more adapter nucleic acids with different identification sequences are provided and used in the ligation step. In particularly preferred embodiments, as many adapters with different identification sequences are used as there are expected different resulting fragments with the same sequence, and preferably even more adapters with different identification sequences. The expected copy number can be based on the type of samples, e.g. RNA from whole cells, mRNA from whole cells (transcriptome), amount of RNA and complexity of the samples (how many different transcript variants are identified, which could mean the whole transcriptome or just selected genes or transcripts, as in the case of gene panels), etc.

In particularly preferred embodiments, the identification sequence is a random sequence. “Random sequences” should be understood as a mixture of different sequences with high variance due to the random synthesis of at least part of the identification sequence. Random sequences potentially cover the entire range of combinations for a given sequence across 4 natural nucleotides (A, T (U), G, C). A random sequence may span 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides chosen at random from A, G, C or T(U ). In terms of hybridization ability, T and U nucleotide sequences are used interchangeably here. The entire range of possible combinations for a random part of the sequence is equal to ^mn , where m is the number of nucleotide types used (preferably all four of A, G, C, T(U)) and n is the number of random nucleotides. Therefore, a random hexamer in which every possible sequence is represented consists of 4 ⁶ = 4096 different sequences. The identification sequence must not be associated with the matrix. In all cases, but especially for random identification sequences, preferably the adapter nucleic acid is added after the elongation reaction. When the elongation product reaches the stopper (or end of the template) and substantially all of the template is double-stranded with the elongation products, the adapter nucleic acid is prevented from binding to the template.

In further embodiments of the invention, primers and stoppers are selected to bind to one or more specific target sequences of interest in the template nucleic acid (with the stoppers being downstream of the elongation products) to produce an elongation sequence of a specific portion of the template. This site targeting is preferentially used for transcripts (RNA) or genes (gDNA) as templates. Identification sequences are particularly useful when used in gene panels. For example, to analyze sequence variants in different types of matrices such as splice variants or other variable template sequences.

In particularly preferred embodiments of the invention for all its embodiments and aspects, elongation stoppers have primer activity and are also elongated during the elongation step. This means that more than one primer is used and most of them have a stopper function (ie they prevent displacement - see above). The use of several primers means that the template produces many fragments, i.e. coverage improves. Although each primer binds to a single template, they will provide complete coverage if different primers bind to different regions of the same template. The method of the invention using multiple primers (which are preferably also stoppers) will increase coverage since the new elongation product will start at the position on the template where the previous elongation product just left off. This produces many fragments covering the entire matrix. In addition, this means that stoppers/primers (in this embodiment these are synonyms) are used that bind to different parts of the template molecule. In general, binding to a template molecule is determined by the annealing sequences of the primers and stoppers. These sequences hybridize to the template and can be varied to bind to different regions of the template. Preferably, at least 9, at least 10, more preferably at least 49, at least 50, eg 100 or more or 200 or more elongation stoppers are used, which have different annealing sequences for landing on the matrix. Thus, they will potentially hybridize with different regions of the template nucleic acid. Preferably, the annealing sequence is a random sequence. Random sequences are described above with respect to the identification sequence and the same applies to the annealing sequences of primers, stoppers and stoppers with primer function. Preferably, the random annealing sequence may span 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides selected randomly from A, G, C or T(U).

Preferably, the adapter nucleic acid binds, hybridizes, or does not bind to the elongation stopper. This binding reaction, e.g. by chemical reaction, complex formation or hybridization, facilitates the positioning of the adapter nucleic acid near the 3' end of the upstream elongation product to which the identification sequence is bound, which itself does not hybridize to the stopper or template and is not expected to required for the ligation reaction to occur. Preferably, when the adapter nucleic acid binds to or hybridizes to an elongation stopper, the identification sequence is selected independently of the annealing sequence of the elongation stopper to fit the elongation stopper onto the template. Both the annealing sequence and the identification sequence may be a random sequence, preferably independently of each other. This is usually ensured by the fact that the stopper and adapter nucleic acid portions are universal sequences, i.e. any adapter can bind to any stopper (which is preferred for all embodiments of the invention), and also in that the adapter nucleic acid does not bind to the stopper, eg, the adapter is provided only after the elongation reaction. In another embodiment or in other parts of the reaction, they do not bind such that when the elongation reaction reaches the 5' end of the template, to which the stopper would not normally hybridize, because the stopper requires at least a minimal annealing sequence on the template, which shifts the lowest stop position to several nucleotides upstream from the 5' end. The adapter can also be ligated to the elongation product without binding or hybridizing to the elongation stopper. However, in all embodiments, it is preferred that when an adapter nucleic acid is ligated to an elongation product, the elongation stopper and/or elongation product, especially preferably its 3' end, still hybridizes to the template. It is also preferred that the adapter nucleic acid hybridizes to the elongation stopper, particularly preferably after the elongation reaction and/or, particularly preferably, for ligation.

In preferred embodiments of the method and kit of the invention, the oligonucleotide primer, and preferably, but not necessarily, the elongation stopper, contains a universal amplification sequence (“linker primer sequence”, see above), and/or the adapter nucleic acid contains a universal amplification adapter sequence (“ adapter linker sequence”, see above). Such an amplification sequence or “linker” can be used to link primers for further amplification, as discussed above. A universal sequence means that it is the same for all primers, stoppers or adapters, respectively. This allows the same type of primer to bind to these oligonucleotides. In particularly preferred embodiments, the universal amplification sequence (linker sequence) is also the same for primers, stoppers and adapters, i.e. the primer for further amplification can equally bind to oligonucleotide primers, elongation stoppers and adapter nucleic acids. This simplifies use as only one type of primer is needed for further amplification. In other embodiments, the primers, stoppers and adapters have different universal amplification sequences (linker sequences), i.e. a primer for further amplification can bind only to oligonucleotide primers, another primer for further amplification can bind only to elongation stoppers, and yet another primer for further amplification can bind only to adapter nucleic acids. Within these groups, primers are preferably universal. This still simplifies the use, but provides better control since the primers for both ends of the labeled fragment will be different and can be selected specifically.

In preferred embodiments, a specific oligonucleotide primer is used to select and hybridize to a selected template sequence, preferably at the 3' end of the template. In the case of mRNA or any other type of RNA containing an oligo(A) tail, such a 3' end can hybridize with a complementary oligonucleotide primer, for example, containing an oligo(dT) annealing sequence complementary to that oligo(A) tail. Preferably, the at least one oligonucleotide primer contains an annealing sequence for hybridization to a selected template sequence, which may be at or near the 3' end of the template. This selected sequence is any known oligo(A) tail template sequence, but any other sequence may be used if known. Preferably, the oligonucleotide primer for the selected sequence contains an oligo(dT) sequence for hybridization with an oligo(A) sequence in the template. Preferably, such an oligo(dT) sequence contains one or more 3' anchor nucleotides different from the oligo(dT) sequence. This ensures correct localization and binding to the 5' end of the oligo(A) template sequence. The anchor nucleotide will bind to the nearest nucleotide other than A (eg T, G, C) on the template near the oligo(A) site. If the nearest non-A nucleotide is unknown, then a mixture of oligonucleotide primer with different anchor primers can be used, e.g. using three oligonucleotide primers, each without a T nucleotide (e.g. A, G, C) (complementary to the nearest non-A (e.g. , T, G, C) on the matrix). In preferred embodiments, two anchor nucleotides are used. The anchor nucleotide closest to a given non-T nucleotide can be selected from any type of nucleotide (A, T(U), G, C) since it is not adjacent to an oligo(T). Such a special oligonucleotide primer may not be a stopper and may not contain a sequence to hybridize to an adapter, since these are not needed if the special oligonucleotide primer hybridizes at or near the 3' end of the template, which means that the overlying elongation product will not reach this position. Of course, for simplicity or consistency in the production of primers/stoppers, such a sequence and/or stopper function may be present.

Preferably, the ligation reaction is carried out in the presence of a crowding agent. The crowding agent increases the likelihood of the adapter and elongation product interacting with each other by reducing the effective volume of the reaction, see Zimmerman et al., Proc. Natl. Acad. Sci. USA 1983, 80(19):5852-6. Other crowding agents are given, for example, in US 5,554,730, US 8,017,339 and WO 2013/038010 A2. Preferably, the crowding agent is a macromolecule, a polymer or a polymer-containing compound such as a polyalkyl glycol, preferably PEG, octoxynol or Triton X or a polysorbate, preferably Tween. In preferred embodiments, the crowding agent is used in concentrations of 5% to 35% (v/v), particularly preferably 10% to 25% (v/v). Preferably the crowding agent has a molecular weight of from 200 to 35,000 g/mol, preferably from 1000 to 10,000 g/mol. Particularly preferred is a polyalkylglycol of the PEG type, especially with the indicated molecular weight. The crowding agent is preferably provided in the kit of the invention, preferably in a ligation buffer.

Other components of the kit include buffers, salts, enzyme cofactors and metals such as Mn ²⁺ and Mg ²⁺ for polymerases and ligases, solvents, containers.

The present invention provides kits for performing the method of the invention. Such a set may include any of the compounds and agents already described. Preferably, the set includes: (i) at least one oligonucleotide primer capable of hybridizing with a template nucleic acid and triggering an elongation reaction at its 3' end, (ii) one or more elongation stoppers capable of hybridizing with a template nucleic acid, preferably capable of triggering an elongation reaction at its 3' end, (iii) one or more adapter nucleic acids containing an identification sequence at its 5' end, wherein such identification sequence does not hybridize to the elongation stopper, preferably wherein the adapter nucleic acid binds, hybridizes, or does not bind to the elongation stopper, (iv) reverse transcriptase and (v) oligonucleotide ligase, and (iv) and (v) may be optional since they may be available in many laboratories, independent of the present invention. The important parts are the adapter/stopper designs, in particular the identification sequences in the adapters. Preferably, the set contains a plurality of adapters with different identification sequences, as described above. All of these kit components have been described above, and any preferred embodiments thereof are also applicable to kits. Preferably the kit contains at least 10, more preferably at least 50 adapter nucleic acids with different identification sequences. The reasons for this preferred embodiment have been given above. Preferably, the oligonucleotide primer contains a template annealing sequence that includes an oligo(dT) sequence for annealing to an oligo(A) sequence in the template, wherein preferably such oligo(dT) sequence includes one or more 3' anchor nucleotides other than oligo(dT) sequences. The kit may also include a solid phase for purification such as beads, preferably magnetic beads (see method details above, which also discusses the suitability of kit components and embodiments).

All preferred embodiments described above can be combined. This method uses a random primer (with a linker sequence), which is also a stopper (and is also called a “strand displacement stopping primer”). After the elongation reaction, purification of the elongation products (hybridized with the template) is preferably carried out to remove unrelated primers and stoppers. The adapters, with their linkers and identification sequences, are then ligated to the elongation products. The identification sequence is a random sequence, preferably between 4 and 12 nt in length. One preferred option is to use mixtures of identification sequences of varying lengths, since ligases tend to cause uneven ligation, favoring some 5'-located nucleotides in the last and penultimate positions. Since such unevenness can affect the quality of the reads during sequencing, the distribution of nucleotides in such mixtures during sequencing in the region of the ligation junctions is equalized. However, the variability of the identification sequences provides much less ligation unevenness than any other established sequence, while also serving as a UMI (unique molecular identifier). A UMI-type identification sequence allows one to determine whether sequencing reads that have an identical sequence or are identified at an identical position in a reference annotation that accounts for minor sequencing errors will come from different template molecules or from the same template molecule and are simply the result of further amplification (PCR). duplications). The adapter hybridizes with the primer if one is present.

UMI-type identification sequences can also distinguish actual SNPs (single nucleotide polymorphisms) between individuals from errors (mutations) introduced during reverse transcription or early PCR cycles that are then amplified. All of these randomly occurring and amplified errors should have the same identifiers, whereas the “real SNPs” in the samples have various other identifiers. Or, it may be possible to more reliably identify those RNA editing events that introduce modified bases, leading to incorrect incorporation and thus RT errors.

UMI-type identification sequences can also be used to reliably determine and quantify allele frequencies in populations, molecular markers, and disease-causing mutations in inherited diseases. Preferably, DNA templates are used for this embodiment.

Another preferred combination is the method of the invention wherein at least one, and preferably at least 9 elongation stoppers have primer activity and are elongated during the elongation step, and at least two, and preferably at least 10 adapter nucleic acids are used. acids containing different identification sequences, thereby producing at least two, and preferably at least 10 different tagged fragments, which are optionally amplified, further comprising assembling those amplification fragment sequences that are unique, wherein the tags are used to identify the unique amplification fragments . Various labels can be used to identify unique amplification fragments within the amplified tagged fragments.

Another preferred method uses stoppers with primer functions. Preferably, a plurality of such primers is used. In such a method, without distinguishing between stoppers and primers, one embodiment of the invention can be defined as follows: A method for producing labeled amplification fragments from a template nucleic acid, comprising the steps of providing such a template nucleic acid, annealing a plurality of oligonucleotide primers with a given template nucleic acid, elongating the oligonucleotides primers in a template-specific manner, thereby producing a plurality of elongation products, the elongation reactions stopping when the elongation products reach the 5' end of the template nucleic acid or an oligonucleotide primer hybridized to the template nucleic acid downstream of such elongation product, providing a plurality of adapter nucleic acids containing identification sequence at their 5' ends, and these identification sequences do not hybridize with the oligonucleotide primer or template, ligating adapter nucleic acids from this set at their 5' ends with the 3' end of the elongation products, thereby obtaining a plurality of labeled amplification fragments . This is a preferred embodiment and can be combined with any of the specifically described aspects in the claims and those described above. In this embodiment, everything described above for stoppers also applies to primers, since these primers are stoppers with the function of primers. The term “multiple” is used for oligonucleotide primers, elongation products (which are the result of elongation of primers), adapter nucleic acids, and tagged amplification fragments (which are the result of elongation and ligation to adapters). As indicated, the number of some of these sets is the result of the method. The number of oligonucleotide primers and adapter nucleic acids can be selected as described above. Such amounts can be selected independently, but preferably they are approximately the same for pairwise combination with a given elongation product. Preferably, the set is, for example, 10 or more, 50 or more, 100 or more, 200 or more, etc. Many different oligonucleotide primers and adapter nucleic acids can be used: so that the oligonucleotide primers bind to many different locations on the template, so that the adapter nucleic acids have different identification sequences, preferably unique identification sequences for the tagged amplification fragments. Although in this embodiment the primers and stoppers are the same, a special primer may be added that does not need (but may have) stopper function, such as a 5' end specific primer, or an oligo-targeting type primer. A) as described above.

The present invention will now be described in the following figures and examples, but without limiting the present invention to these embodiments.

Brief description of the figures

Fig. 1. Scheme for obtaining a library of short cDNAs labeled with a UMI linker using a primer with SDS properties and a partially complementary UMI-containing linker oligonucleotide in the total mass of RNA.

a) The general strand-stopping primer Pn hybridizes to the RNA transcript, and the primer Pn+1 hybridizes to a more upstream (5') position of the RNA template than the Pn primer. When the Pn elongation reverse transcriptase reaches the Pn+1 primer, the polymerase reaction will stop using the strand displacement stopping technology described in WO 2013/038010 A2. A UMI-containing linker oligonucleotide spanning L2, which is complementary to L1, hybridizes with primers Pn and Pn+1. b) Upon ligation, the elongation product is now ligated to the UMI preceding the L2 linker strand. In the same way, a cDNA library is created that contains two linker sequences (L1, L2) at the ends and contains unique molecular identifiers. c) Finally, PCR is performed to amplify these libraries.

Fig. 2. Obtaining libraries containing UMI.

In fig. a) Libraries obtained by SDS + ligation are presented. Ligation of the UMI-containing partially complementary adapter L2 (see Fig. 1 for reference) can be accomplished using either ss ligase or ds ligase (lanes 2, 3). In the absence of ligase, no libraries are formed (lane 1). After ligation, cDNA fragments containing the L1 and L2 linkers were amplified by PCR and analyzed. Pictures of gels obtained by analyzing the HS DNA Assay on a bioanalyzer (Agilent Technologies, Inc.) are presented. b) Scheme for obtaining libraries containing UMI using the SDS + ligation method with non-hybridizing primer and adapter oligonucleotides. In this case, the L2' adapter oligonucleotide does not contain sequences complementary to the Pn elongation primer. c) Gel snapshot and electropherogram of replicate libraries generated with a non-hybridizing elongation primer and UMI-containing adapter oligonucleotides (SEQ ID No. 10). Images were obtained using the HS DNA Assay on a bioanalyzer (Agilent Technologies, Inc.).

Fig. 3. Improved coverage of the 5' end of transcripts, achieved by ligating L2 linkers with cDNA at the 5' end of the RNA template.

a) Scheme of the RT reaction at the 5' end of the transcripts. Without SDS at the downstream primers Pn+1, the terminal deoxynucleotidyl transferase (TdT) activity of RT adds non-template nucleotides to the 3' end of the cDNA, forming an overhang. b) Non-template nucleotides can serve as a hybridization site for the L1-containing primer Pn+1. In combination with partially hybridized L2, ligation of the UMI to the L2 linker in the double strand can occur. c) On the other hand, in the absence of priming, UMI can ligate to the L2 linker as a single strand. d) Libraries obtained as shown schematically in FIG. 3 ac, sequenced on an Illumina NextSeq 500 (single read, 75 bp). Reads related to the 5' end of ERCC-0130 (which is present in the SIRV 3 kit, Lexogen, cat. no. 051.0N) are shown. Reads were analyzed without trimming additional or mismatched bases. Nucleotides in gray correspond to the ERCC-0130 annotation, and nucleotides in black are derived from non-template addition due to the TdT activity of RT. Below are 30 representative sequence reads obtained for the 5' end of ERCC-0130. Sequence reads are presented as SEQ ID NO: 12 to 42, from top to bottom. e) Improved 5' end coverage by SDS/ligation compared to standard technique. Libraries were prepared using standard methods (NEBNext ^® Ultra™ II directional RNA Library Prep Kit for Illumina ^® , New England Biolabs, cat. no. E7760S) or SDS/ligation method and sequenced on Illumina NextSeq 500 (paired-end reads, 150 bp .). Reads related to ERCC-0130 were overlaid and compared with the expected coverage represented as a box; left: standard RNA library preparation procedure, right: coverage obtained using the new SDS/ligation technology.

Fig. 4. Schematic of the reaction used to improve 3' end coverage using the SDS/ligation method and a combination of common (Pn) and oligo(dT) primers (PdT).

a) The general primer Pn hybridizes with the RNA template in the total RNA mass. In addition, the oligo(dT) primers (PdT) present hybridize to the poly(A) tail at the 3′ end of polyadenylated transcripts. RT will continue PdT elongation until it reaches the downstream primer Pn and stops strand displacement. b) Upon ligation, the UMI-containing L2 linker will ligate to cDNA fragments spanning the 3' end, forming L1- and L2-linked, UMI-containing cDNA libraries spanning the 3' ends of the transcripts. c) Gene body coverage plot showing the increase in 3′ end coverage of transcripts throughout the transcriptome. Libraries were obtained using the SDS + ligation method using a mixture of random primers and an oligo(dT) primer for the synthesis of the first strand, as described in example 3. The libraries were sequenced on a NextSeq 500 installation and graphs of gene body coverage were plotted across the entire transcriptome in comparison with that described previously using the SDS + ligation technique. d) Example coverage for an endogenous housekeeping gene (HSP90) for the standard library preparation method (top panel) and for the SDS+ ligation technique with oligo-dT titration (bottom panel), which results in improved 3' end coverage.

Fig. 5. Global improvement in coverage of the 5' and 3' ends of transcripts. Transcription start sites, i.e. the true 5' ends of transcripts, and transcription termination sites, i.e. true 3' ends of transcripts are resolved by SDS + ligation, but are not resolved using two typical traditional library preparation methods. Libraries obtained by the SDS + ligation technique, as shown schematically in FIG. 3a-c, sequenced on Illumina NextSeq 500 (paired ends, 150 bp). Traditional libraries were prepared according to the manufacturer's instructions using either the TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat kit, Illumina, cat. No. 20020596 or 20020597 (=regular 1), or NEBNext ^® Ultra™ II directional RNA Library Prep Kit for Illumina ^® , New England Biolabs, cat. No. E7760S (= regular 2). a) Reads corresponding to the true 5' and 3' ends of the detected ERCCs (which are present in SIRV set 3, Lexogen, cat. no. 051.0N) are shown. Reads were assigned to ERCC RNA inserts of known sequence. Normalized coverage values of the accumulated mapped reads for all detected ERCCs were plotted against the absolute nucleotide positions relative to the transcription start sites (TSS) and transcription termination sites (TES), indicated by dotted lines. b) Broad coverage of 5' ends reveals typical TSSs. Top panel: coverage profile for gapdh with condensed intron imaging obtained by SDS+ligation or traditional library preparations as described above. Reads related to gapdh were analyzed without trimming additional or mismatched bases. Sequence reads are provided as SEQ ID Nos. from 43 to 67, from top to bottom. Nucleotides highlighted in black correspond to gapdh annotation, while nucleotides highlighted in gray are mismatched or obtained by template-free addition due to the TdT activity of RT. Start site clusters obtained by superimposing reads at the 5′ end of transcripts can be used to re-annotate TSSs. Annotated and manually identified TSSs are indicated by arrows on the annotated consensus sequence in bold.

Examples

Example 1: Ligation of Unique Molecular Identifiers (UMIs) to First Strand cDNA Fragments

Libraries were prepared from universal human reference RNA (Agilent Technologies, cat. no. 740000) containing SIRV Set 3 spike-in control mixture (Lexogen, cat. no. 051.0N) according to the manufacturer's instructions.

After cDNA synthesis, downstream primers (Pn+1 (L2)), containing a unique molecular identifier of 2 to 24 nucleotides in length, preferably 6 to 12 nucleotides, can be ligated to the newly transcribed cDNA strand as a hybrid with the RNA template. Reverse transcription was performed using oligonucleotides, templates and conditions described in WO 2013/038010 A2. Various ligases and their combinations can be used to ligate oligonucleotides such as:

SEQ ID No: 1: (Phos)(5'-NNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3'(3InvdT)),

SEQ ID No: 2: (Phos)(5'-NNNNNNNNNNGAATCGGAAGAGCACACGTCTGAA-3'(3InvdT)),

SEQ ID No: 3: (Phos)(5'-NNNNNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAA GAGTG-3'(3InvdT)),

SEQ ID No: 4: (Phos)(5'-NNNNNNNNNNGAATCGGAAGAGCGTCGTGTAGG-3'(3InvdT)),

SEQ ID No: 5: (Phos)(5'-NNNNNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTG-3'(3InvdT)),

SEQ ID No: 6: (Phos)(5'-NNNNNNNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTG-3'(3InvdT)),

SEQ ID No: 7: (Phos)(5'-NNNNNNNNNNNNNAGATCGGAAGAGCGTCGTGTAGG-3'(3InvdT)),

SEQ ID No: 8: (Phos)(5'-+NNNNNNNNNNNAGATCGGAAGAGCGTCGTGTAGG-3'(3InvdT)),

SEQ ID No: 9: (Phos)(5'-+NNNNNNNNNNNNNAGATCGGAAGAGCGTCGTGTAGG-3'(3InvdT)).

After reverse transcription (RT), samples were purified by solid-phase reversible immobilization (SPRI) using magnetic beads (AMPure Beads; Agentcourt) according to the manufacturer's instructions. cDNA:RNA hybrids were eluted in 20 μl of water or 10 mM Tris, pH 8.0, and then 17 μl of supernatants were transferred to a new PCR plate. Ligation reactions were then performed in 60 μl with 20% PEG-8000, 50 mM Tris-HCl (pH 7.5 at 25°C), 10 mM MgCl ₂ , 5 mM DTT, 0.4 mM ATP, 0.01% Triton -X100, 50 μg/ml BSA and 20 units of ligase, which can be single-strand-specific ligase and/or double-strand-specific ligase. Unligated small fragments and remaining oligonucleotides were removed by SPRI. All remaining primary cDNA libraries were amplified in a PCR reaction using high-fidelity polymerase according to the following program: 30 sec at 98°C, and then 10-25 PCR cycles of 10 sec at 98°C, 20 sec at 65°C and 30 sec at 72°C. Then the final elongation was carried out at 72°C for 60 seconds. In fig. 1b shows the general principle underlying the ligation of elongated cDNA to a UMI-containing linker oligonucleotide (L2), the sequence of which is complementary to the strand displacement stop primer (L1).

In fig. Figure 2 provides an example of how various ligases can perform a ligation reaction on a UMI-containing oligonucleotide and thereby produce cDNA fragments containing both PCR linkers and capable of being amplified by PCR (Figure 2a, lane 2-3). In contrast, a control experiment without any ligase shows that the libraries cannot be amplified, highlighting the specificity of the reaction (Fig. 2a, lane 1).

Example 2. Preparation of libraries using non-hybridizing oligonucleotides - elongation initiator and adapter

Libraries were prepared from universal human reference RNA (Agilent Technologies, cat. no. 740000) containing SIRV Set 3 spike-in control mixture (Lexogen, cat. no. 051.0N) according to the manufacturer's instructions.

Reverse transcription (RT) was performed as described in Example 1. After RT, samples were purified by solid-phase reversible immobilization (SPRI) using magnetic beads (AMPure Beads; Agentcourt) according to the manufacturer's instructions and cDNA:RNA hybrids were eluted in 20 μl of 10 mM Tris. pH 8.0, and then 17 μl of supernatants were transferred to a new PCR plate. Ligation was carried out under the conditions described in Example 1, but an adapter oligonucleotide was used that did not contain a sequence complementary to the elongation initiator used for priming the reverse transcription reaction. Therefore, the adapter oligonucleotide cannot hybridize and therefore does not come into the vicinity of the newly formed 3' ends of the elongation products upon recruitment (Fig. 2b). Oligonucleotides of the type SEQ ID No. 10: (Phos)(5'-NNNNNNNNNNNNTGGAATTCTCGGGTGCCAAGG-3'(SpcC3)) do not contain sequences complementary to elongation initiators. Fragments containing both linker sequences were amplified after purification as described in Example 1. FIG. Figure 2c shows gel snapshots and electropherograms for two SDS+ ligation replicate libraries prepared with non-hybridizing elongation initiator and adapter oligonucleotides.

Example 3: Improved 5'-end coverage resulting from terminal transferase activity and ss-ligation of the UMI linker to first-strand cDNA fragments

Libraries were prepared from universal human reference RNA (Agilent Technologies, cat. no. 740000) containing SIRV Set 3 spike-in control mixture (Lexogen, cat. no. 051.0N) according to the manufacturer's instructions.

Synthesis of the first strand of cDNA stops at the 5' ends of the RNA template molecules. The terminal transferase activity of reverse transcriptases catalyzes template-free addition of nucleotides at the 3' end of the cDNA strand (Fig. 3a).

Ligation of UMI linker oligonucleotides (eg, SEQ IDs 1-9) after reverse transcription can occur in double-stranded form (Fig. 3b) and on single-stranded overhangs (Fig. 3c). After SPRI purification and PCR amplification, the libraries were sequenced on a NextSeq 500 in single-read or paired-end mode. Reads related to the 5′ end of ERCC-0130 were analyzed without prior trimming of mismatched nucleotides. Reads spanning the 5' end of ERCC-0130 are shown as an example in FIG. 3d. Addition of terminal nucleotides and UMI ligation along elongated single-stranded strands results in improved coverage of the 5' ends. In fig. 3e shows a comparison of coverage profiles between a conventional RNA-seq library preparation and the present invention. Coverage is represented as an overlay of all aligned reads (gray profile) and compared to expected uniform coverage, represented as a box. While sequencing data generated by standard techniques cover the 5' and 3' ends less efficiently, as evidenced by the slopes towards both ends (Fig. 3e, left), the new technique produces more reads related to the very 5' end of the transcripts (Fig. 3e, right).

Example 4: Improving 3' End Coverage when Titrating Oligo(dT) Primers for First Strand Synthesis

Coverage of the 3' ends of transcripts can be modified, preferably increased, by using oligo(dT)-containing first-strand primers (L1-containing Pn) added to a random priming SDS oligonucleotide mixture that already contains a portion of T-rich and T-only primer sequences (type SEQ ID No. 11: 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT+TTT TTT TTT TTT TTT TTT+V-3') according to the normal distribution of random nucleotides to increase coverage at the 3' ends. Depending on the selected ratio between random primers and L1 poly(dT) primers, the sequencing depth at the 3' ends can be adjusted (Fig. 4). The ratio between random SDS primers and specific oligo(dT) primers, as well as primer length and LNA content, can vary and will determine the extent to which 3' end coverage is increased.

Libraries were prepared by SDS+ ligation using either random stalled primers alone or mixtures with varying numbers of first-strand oligo(dT) primers (SEQ ID No: 11). The resulting libraries were sequenced on NextSeq 500, data analyzed, and transcriptome-wide gene body coverage plotted from mapped reads using the Python script geneBody_coverage available from RSeqC (Figure 4c). Coverage of 3' ends can be significantly increased by the addition of oligo(dT) primers during reverse transcription.

In addition, gene coverage was visualized for example for endogenous genes using a special script to estimate the coverage of individual genes. In fig. Figure 4d shows the coverage of the housekeeping gene HSP90 obtained using the standard method for preparing RNA libraries (top panel), with greatly reduced coverage of the 5' and 3' ends. In contrast, the SDS+ ligation technique with oligo-dT titration results in improved coverage of the 5′ and 3′ ends (lower panel).

Example 5: Improved coverage of 5' and 3' ends makes it easier to determine the true transcription start and stop sites

Libraries were prepared by SDS + ligation from ribosomal RNA-depleted human universal reference RNA (Agilent Technologies, cat. no. 740000) containing the SIRV Set 3 spike-in control mixture (Lexogen, cat. no. 051.0N), as described in Examples 3 and 4. Ribosomal RNA removal was performed using RiboCop (Lexogen, cat. no. 037.96) according to the manufacturer's instructions. For comparison, two traditional methods were used on the same universal ribosomal RNA-depleted human reference RNA: TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat kit, Illumina, cat. No. 20020596 or 20020597 (=regular 1) or ^NEBNext® Ultra™ II directional RNA Library Prep Kit for ^Illumina® , New England Biolabs, cat. No. E7760S (=regular 2), according to manufacturer's instructions. The resulting libraries were sequenced on a NextSeq 500 and the data analyzed. Gene body coverage plots were generated for all detected ERCCs from SIRV Set 3. FIG. Figure 5a shows the normalized coverage across the accumulated mapped ERCC reads for absolute nucleotide positions relative to known transcription start sites (TSS) and transcription termination sites (TES), which are both indicated by dotted lines. Coverage at the 5' and 3' ends is significantly increased for samples derived from SDS+ligation libraries, compared to both traditional library preparations exhibiting decreased 3' end coverage and imprecise 5' end resolution.

In addition, gene coverage was visualized for the example of the endogenous housekeeping gene gapdh using a special script to estimate coverage for individual genes. In fig. Figure 5b shows the coverage profile for gapdh with condensed intron imaging. Reads related to gapdh (SEQ ID Nos. 43 to 67) were analyzed without trimming additional or mismatched bases. Nucleotides matching the consensus sequence (top row) are marked in black, and nucleotides deviating from the annotated consensus sequence or resulting from template-free addition are marked in grey. Based on the read matches observed for samples obtained from SDS+ligation library preparations, the true transcription start sites can be determined and transcripts of interest can be re-annotated. In the example shown in FIG. 5b, TSS was manually set to position -15 (relative to the annotated position +1). Likewise, the true transcription start and stop sites can be re-established for other transcripts of interest, allowing for comprehensive analysis of full transcripts, including single nucleotide-level resolution of true TSSs for high-throughput NGS experiments. This can be achieved by simply using the SDS+ligation method to generate libraries, as opposed to specialized and more complex approaches such as 5'-capture sequencing (CAGE-Seq) or low-throughput methods such as 5'-RACE (rapid amplification of ends). cDNA).

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/note="5' phosphorylated; a or g or c or t"

<220>

<221> misc_difference

<222> 2,3,4,5,6,7,8,9,10,11,12

<223> /note="a or g or c or t"

<220>

<221> modified_base

<222> 45

<223> /mod_base="OTHER"

/note="3' Inverted dT (reverse linkage)"

<400> 6

nnnnnnnnnn nnagatcgga agagcgtcgt gtagggaaag agtgn 45

<210> 7

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> oligo

<220>

<221> modified_base

<222> 1

<223> /mod_base="OTHER"

/note="5' phosphorylated; a or g or c or t"

<220>

<221> misc_difference

<222> 2,3,4,5,6,7,8,9,10,11,12

<223> /note="a or g or c or t"

<220>

<221> modified_base

<222> 36

<223> /mod_base="OTHER"

/note="3' Inverted dT (reverse linkage)"

<400> 7

nnnnnnnnnn nnagatcgga agagcgtcgt gtaggn 36

<210> 8

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> oligo

<220>

<221> modified_base

<222> 1

<223> /mod_base="OTHER"

/note="5' phosphorylated; a or g or c or t"

<220>

<221> misc_difference

<222> 2,3,4,5,6,7,8,9,10

<223> /note="a or g or c or t"

<220>

<221> modified_base

<222> 34

<223> /mod_base="OTHER"

/note="3' Inverted dT (reverse linkage)"

<400> 8

nnnnnnnnnn agatcggaag agcgtcgtgt aggn 34

<210> 9

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> oligo

<220>

<221> modified_base

<222> 1

<223> /mod_base="OTHER"

/note="5' phosphorylated; a or g or c or t"

<220>

<221> misc_difference

<222> 2,3,4,5,6,7,8,9,10,11,12

<223> /note="a or g or c or t"

<220>

<221> modified_base

<222> 36

<223> /mod_base="OTHER"

/note="3' Inverted dT (reverse linkage)"

<400> 9

nnnnnnnnnn nnagatcgga agagcgtcgt gtaggn 36

<210> 10

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> oligo

<220>

<221> modified_base

<222> 1

<223> /mod_base="OTHER"

/note="5' phosphorylated; a or g or c or t"

<220>

<221> misc_difference

<222> 2,3,4,5,6,7,8,9,10,11,12

<223> /note="a or g or c or t"

<220>

<221> modified_base

<222> 33

<223> /mod_base="OTHER"

/note="g, 3' Spacer C3"

<400> 10

nnnnnnnnnn nntggaattc tcgggtgcca agn 33

<210> 11

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> oligo

<400> 11

gtgactggag ttcagacgtg tgctcttccg atcttttttt tttttttttt ttv 53

<210> 12

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 12

cgatttctaa agggaattcg agctcgcatt ttgaaaattc tatggaagag ctagcatctc 60

tgacgaaaac agcag 75

<210> 13

<211> 68

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 13

cctttgggga attcgagctc gcattttgaa aattctatgg aagagctagc atctctgacg 60

aaaaccag 68

<210> 14

<211> 68

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 14

caaaacggga attcgagctc gcattttgaa aattctatgg aagagctagc atctctgacg 60

68

<210> 15

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 15

agtggtggga attcgagctc gcattttgaa aattctatgg aagagctagc atctctgacg 60

aaatgc 66

<210> 16

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 16

caaaatggga attcgagctc gcattttgaa aattctatgg aagagctagc atctctgacg 60

aaaacagcgt 70

<210> 17

<211> 65

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 17

tcggacggga attcgagctc gcattttgaa aattctatgg aagagctagc atctcttacg 60

aaaac 65

<210> 18

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 18

ggggacggga attcgagctc gcattttgaa aattctatgg aagagctagc atctctgaca 60

aaaaca 66

<210> 19

<211> 73

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 19

cccgagggga attcgagctc gcattttgaa aattctatgg aagagctagc atctctgacg 60

aaaacggcag aca 73

<210> 20

<211> 71

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 20

aatacaggga attcgagctc gcattttgaa aattctatgg aagagctagc atctctgacg 60

aaaacagaga g 71

<210> 21

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 21

caaaatggga attcgagctc gcattttgaa aattctatgg aagagctagc atctctgacg 60

aaaacagcgt 70

<210> 22

<211> 74

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 22

atttggggaa ttcgagctcg cattttgaaa attctatgga agagctagca tctctgacga 60

aaacagcagg cgga 74

<210> 23

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 23

aatggggaat tcgagctcgc attttgaaaa ttctatggaa gagctagcat ctctgacgaa 60

aacagcaatc ggaaa 75

<210> 24

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 24

aaggggaatt cgagctcgca ttttgaaaat tctctggaag agctagcatc tctgacgaaa 60

acagcagaac agaaa 75

<210> 25

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 25

ggggaattcg agctcgcatt ttgaaaatac tatggaagag ctagcatctc tgacgaaaac 60

agcagacgaa aaagt 75

<210> 26

<211> 61

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 26

gggaattcga gctcgcattt tgaaaattct atggaagagc tagcatctct gactactaca 60

g 61

<210> 27

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 27

aagatctcgc attttgaaaa ttctatggaa gagctagcat ctctgacgaa aacagcagaa 60

<210> 28

<211> 74

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 28

cgagctcgca ttttgaaaat tctatggaag agctagcatc tctgacgaaa acagcagacg 60

gaaaaggaga gacc 74

<210> 29

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 29

cgagctcgca ttttgaaaat tctatggaag agctagcatc tctgacgaaa acagcagacg 60

gaaaagtact gacca 75

<210> 30

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 30

cgagctcgca ttttgaaaat tctatggaag agctagcacc tctgacgaaa acagcagacg 60

gaaaaggact gaaaa 75

<210> 31

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 31

cgagctcgca ttttgaaaat tctatggaag agctagcatc tctgacgaaa acagcagacg 60

gaaaagtact gagcc 75

<210> 32

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 32

cgagctcgca ttttgaaaat tctatggaag agctagcacc tctgacgaaa acagcagacg 60

gaaaaggact gaaaa 75

<210> 33

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 33

cgagctcgca ttttgaaaat tctatggaag agctagcatc tctgacgaaa acagcagacg 60

gaaaagtact gactc 75

<210> 34

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 34

cgagctcgca ttttgaaaat tctatggaag agctagcatc tctgacgaaa acagcagacg 60

gaaaagtact gacca 75

<210> 35

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 35

cgagctcgca ttttgaaaat tctatggaag agctagcatc tctgacgaaa acagcagacg 60

gaaaagtaca aaacc 75

<210> 36

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 36

gagctcgcat tttgaaaatt ctatggaaga gctagcatct ctgacgaaaa cagcagacgg 60

aaaagtagct gacca 75

<210> 37

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 37

agctcgcatt ttgaaaattc tatggaagag ctagcatctc tgacgaaaac agcagacgga 60

aaagtactga ccaga 75

<210> 38

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 38

gctcgcattt tgaaaattct atggaagagc tagcatctct gacgaaaaca gcagacggaa 60

aagtacagac ccaac 75

<210> 39

<211> 74

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 39

cgcattttga aaattctatg gaagagctag catctctgac gaaaacagca gacggaaaag 60

tactgaccag ctag 74

<210> 40

<211> 73

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 40

cgcattttga aaattctatg gaagagctag catctctgac gaaaacagca gacggaaaag 60

tactgaccat gca 73

<210> 41

<211> 74

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 41

cgcattttga aaattctatg gaagagctag catctctgac gaaaacagca gacggaaaag 60

tactgaccag ccac 74

<210> 42

<211> 73

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 42

cgcattttga aaattctatg gaagagctag catctctgac gaaaacagca gacggaaaag 60

tactgaccag cat 73

<210> 43

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> consensus sequence

<400> 43

ataaattgag cccgcagcct cccgcttcgc tctctgctcc tcctgttcga cagtcagccg 60

catcttcttt tgcgtcgcca gccgagccac atcgctcaga caccatgggg aaggtgaagg 120

tcggagtca 129

<210> 44

<211> 104

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 44

acgtgtgctc gtcactacct ccccgggtgc tctctgctcc tcctgttcga cagtcagccg 60

catcttcttt tgcgtcgcca gccgagccac atcgctcaga cacc 104

<210> 45

<211> 129

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 45

gaaaattgag cccgcagcct cccgcttcgc tctctgctcc tcctgttcga cagtcagccg 60

catcttcttt tgcgtcgcca gccgagccac atcgctcaga caccatgggg aaggtgaagg 120

tcggagtca 129

<210> 46

<211> 122

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 46

aaaatgcatt agaggaactg taaaaatctg ctcctccgtt cgacagtcag ccgcatcttc 60

ttttgcgtcg ccagccgagc cacatcgctc agacaccatg gggaaggtga aggtcggagt 120

ca 122

<210> 47

<211> 122

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 47

ctagaggaga ttggccaacg agattcactg gactcctgtt cgacagtcag ccgcatcttc 60

ttttgcgtcg ccagccgagc cacatcgctg agacaccatg gggaaggtga aggtcggagt 120

ca 122

<210> 48

<211> 118

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 48

ttttctgaac gctctggccg ctctgctcct cctgttcgac agtcagccgc ctcttcgttt 60

gcgtcgccag ccgagccaca tagctcagac accaagggga aggtgaaggt cggagtca 118

<210> 49

<211> 114

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 49

accgagcacc agctttctcc gacgccggga agtcgcagtc agccgcatct tcttttgcgt 60

cgccagccga gccacatcgc tcagacacca tggggaaggt gaaggtcgga gtca 114

<210> 50

<211> 112

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 50

aacgtgtgct ggctctctgc tcctcctgtt cgacagtcag ccgcatcttc ttttgcgtcg 60

ccagccgagc cacatcgctc agacaccatg gggaaggtga aggtcggagt ca 112

<210> 51

<211> 110

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 51

ttgctttggg ctctctgctc ctcctgttcg acagtcagcc gcatcttctt ttgcgtcgcc 60

agccgagcca catcgctcag acaccatggg gaaggtgaag gtcggagtca 110

<210> 52

<211> 106

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 52

gatgggctct ctgctcctcc tgttcgacag tcagccgcat cttcttttgc gtcgccagcc 60

gagccacatc gctcagacac catggggaag gtgaaggtcg gagtca 106

<210> 53

<211> 106

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 53

atagggctct ctgctcctcc tgttcgacag tcagccgcat cttcttttgc gtcgccagcc 60

gagccacatc gctcagacac catggggaag gcgaaggtcg gagtca 106

<210> 54

<211> 106

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 54

atctggctct ctgctcctcc tgttcgacag tcagccgcat cttcttttgc gtcgccagcc 60

gagccacatc gctgagacac catggggaag gtgaaggtcg gagtca 106

<210> 55

<211> 106

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 55

acgtggctct ctgctcctcc tgttcgacag tcagccgcat cttcttttgc gtcgccagcc 60

gagccacatc gctcagacac catggggaag gtgaaggtcg gagtca 106

<210> 56

<211> 106

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 56

acgtggctct ctgctcctcc tgttcgacag tcagccgcat cttcttttgc gtcgccagcc 60

gagccacatc gctcagacac catggggaag gtgaaggtcg gagtca 106

<210> 57

<211> 97

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 57

ttgcggctct ctgctcctcc tgttcgacag tcagccgcat cttcttttgc gtcgccagcc 60

gagccacatc gctcagacac catggggaag cggaaca 97

<210> 58

<211> 82

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 58

gggggctctc tgctcctcct gttcgacagt cagccgcatc ttcttttgcg tcgccagccg 60

agccacatcg ctcagacccc ac 82

<210> 59

<211> 105

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 59

aatggctctc tgctcctcct gttcgacagt cagccgcatc ttcttttgcg tcgccagccg 60

agccacatcg ctcagacacc atggggaagg tgaaggtcgg agtca 105

<210> 60

<211> 105

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 60

atcggctctc tgctcctcct gttcgacagt cagccgcatc ttcttttgcg tcgccagccg 60

agccacatcg ctcagacacc atggggaagg tgaaggtcgg agtca 105

<210> 61

<211> 105

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 61

attggctctc tgctcctcct gttcgacagt cagccgcatc ttcttttgcg tcgccagccg 60

agccacatcg ctcagacacc atggggaagg tgaaggtcgg agtca 105

<210> 62

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 62

gttggctctc tgctcctcct gttcgacagt cagccgcatc ttcttttgca atcgcca 57

<210> 63

<211> 104

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 63

atggctctct gctcctcctg ttcgacagtc agccgcatct tcttttgcgt cgccagccga 60

gccacatcgc tcagacacca tggggaaggt gaaggtcgga gtca 104

<210> 64

<211> 104

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 64

ttggctctct gctcctcctg ttcgacagtc agccgcatct tcttttgcgt cgccagccga 60

gccacatcgc tcagacacca tggggaaggt gaaggtcgga gtca 104

<210> 65

<211> 82

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 65

ggggctctct gctcctcctg ttcgacagtc agccgcatct tcttttgcgt cgccagccga 60

gccacatcgc tcagaacagc ca 82

<210> 66

<211> 104

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 66

gtggctctct gctcctcctg ttcgacagtc agccgcatct tcctttgcgt cgccagccga 60

gccacatcgc tcagacacca tggggaaggt gaaggtcgga gtca 104

<210> 67

<211> 103

<212> DNA

<213> Artificial Sequence

<220>

<223> sequencing read

<400> 67

gggctctctg ctcctcctgt tcgacagtca gccgcatctt cttttgcgtc gccagccgag 60

ccacatcgct cagacaccat ggggaaggtg aaggtcggag tca 103

<---

Claims (23)

1. A method for obtaining labeled amplification fragments of a nucleic acid matrix, including the stages: providing such a nucleic acid template, annealing at least two oligonucleotide primers to a given nucleic acid template, where at least one downstream primer is an elongation stopper, elongating at least two oligonucleotide primers in a template-specific manner, thereby producing an elongation product, wherein such elongation reaction is stopped when the elongation product reaches (a) the 5′ end of the nucleic acid template or (b) the oligonucleotide primer as a nucleic acid elongation stopper hybridized on the nucleic acid template below the elongation product, providing a plurality of at least two adapter nucleic acids, wherein said plurality of adapters contain different identification sequences at their 5′ end, and these identification sequences do not hybridize with an oligonucleotide primer that is an elongation stopper or with a template, ligation of the adapter nucleic acid at its 5'-end with the 3'-end of the elongation product, and for ligation, the elongation products are hybridized with the template, thereby obtaining a labeled amplification fragment. 2. The method according to claim 1, wherein when the elongation product reaches the 5' end of the nucleic acid template, the nucleotide polymerase is able to add non-template nucleotides to the elongation product, preferably due to the terminal transferase activity of the polymerase, and/or preferably is added from 1 to 15 non-template nucleotides in at least 70% of elongation products. 3. The method according to claim 1 or 2, wherein at least 10, more preferably at least 50 adapter nucleic acids with different identification sequences are provided and used in the ligation step. 4. Method according to any one of paragraphs. 1-3, wherein the identification sequence is a random sequence. 5. Method according to any one of paragraphs. 1-4, using at least 9, more preferably at least 49 oliconucleotide primers as elongation stoppers, which have different annealing sequences for hybridization to the template and therefore can hybridize to different regions of the nucleic acid template. 6. The method according to claim 5, wherein the annealing sequence is a random sequence. 7. Method according to any one of paragraphs. 1-6, wherein the adapter nucleic acid(s) bind(s), hybridize(s), or do not bind(s), or do not hybridize) with the oligonucleotide primer(s) which(s) is an elongation stopper(s), and preferably the adapter nucleic acid(s) binds or hybridizes to the oligonucleotide primer(s) that is the stopper (s) of elongation, then the identification sequence is independent of the annealing sequence from the oligonucleotide primer(s) that is(are) the elongation stopper(s) for hybridization of the primer(s) that is(are) the stopper(s) with a matrix. 8. Method according to any one of paragraphs. 1-7, wherein the template is RNA and reverse transcriptase is preferably used for elongation. 9. Method according to any one of paragraphs. 1-8, wherein each oligonucleotide primer contains a universal amplification sequence and/or the adapter nucleic acid contains a universal amplification sequence of the adapter. 10. Method according to any one of paragraphs. 1-9, wherein the oligonucleotide primer contains a template annealing sequence that includes an oligo(T) sequence for annealing to an oligo(A) sequence in the template, preferably such oligo(T) sequence containing one or more 3'- anchors nucleotides other than the oligo(T) sequence. 11. Method according to any one of paragraphs. 1-10, wherein the ligation reaction is carried out in the presence of a crowding agent, preferably a polymer or a polymer-containing compound such as a polyalkylglycol, preferably PEG, octoxynol or Triton X, or a polysorbate, preferably Tween, and/or the oligonucleotide primer contains one or more modified nucleotides that increase melting temperature of the annealed sequence during annealing with the matrix. 12. Method according to any one of paragraphs. 1-11, wherein at least two, and preferably at least 10 adapter nucleic acids containing different identification sequences are used, thereby producing at least two, and preferably at least 10 different labeled amplified fragments, optionally including amplification of the labeled amplified fragments, and also involves the assembly of those amplification fragment sequences that are unique, using tags to identify the unique amplification fragments. 13. Kit for performing the method according to any one of paragraphs. 1-12 containing: at least two oligonucleotide primers capable of hybridizing with a template nucleic acid and triggering an elongation reaction at their 3′ end, where at least one primer is an elongation stopper, a plurality of at least two adapter nucleic acids, wherein the plurality of adapters contain different identification sequences at their 5' end, such identification sequences not hybridizing to oligonucleotide primers, preferably wherein the adapter nucleic acid binds, hybridizes, or does not bind or not hybridizes with an oligonucleotide primer, reverse transcriptase and oligonucleotide ligase. 14. The kit according to claim 13, containing at least 10, more preferably at least 50 adapter nucleic acids with different identification sequences. 15. The kit according to claim 13 or 14, wherein the at least one oligonucleotide primer contains a template annealing sequence that includes an oligo(T) sequence for annealing to an oligo(A) sequence in the template, preferably such an oligo(T) sequence in the template. T) contains one or more 3′ anchor nucleotides different from the oligo(T) sequence.