Gene specific spatial rolling circle amplification and ngs sequencing

Abstract

To provide methods for simultaneously obtaining both the spatial location and sequence information of a target sequence with a high resolution.SOLUTION: The disclosure provides a method to obtain the spatial location and sequence information of a target sequence of at least one mRNA strand on a tissue sample, the method comprising the steps of: a. providing a locator probe; b. hybridizing the locator probe having an RNA anchor region to the mRNA strand; c. obtaining a reverse-transcribed c-DNA strand; d. obtaining an extended reverse-transcribed c-DNA strand; e. multiplying the circular locator by RCA and creating a first rolony; f. obtaining the spatial and sequence information of the first rolony; g. obtaining a single stranded oligomer; h. creating a circular single stranded oligomers; and i. multiplying the circular single stranded oligomer by RCA into second rolonies; sequencing the second rolonies and linking the spatial information of the first rolonies with the sequence information of the second rolonies.SELECTED DRAWING: None

Description

[Technical field]

[0001] background It is difficult to detect all expressed genes at the subcellular level in a tissue and at the same time obtain sequence and / or spatial information for these genes.

[0002] The present invention describes a method to recognize and amplify specific regions on messenger RNA and copy the sequence information of the UMI using locator circles (or open circles that are later ligated) containing Unique Molecular Identifier (UMI) probes, followed by reverse transcription of the region of interest containing the mutation or sequence variant. The resulting cDNA with UMI information is searched, extracted, and sequenced by NGS of the Rolling Circle Amplified (RCA) products generated on tissue sections.

[0003] WO2020076976 discloses a method for obtaining sequence information of nucleic acids, including spatial positions, by FISSEQ. WO2019199579 teaches a Snail probe system for a similar purpose.

[0004] Summary of the Invention It was therefore an object of the present invention to provide a method for simultaneously obtaining both the spatial position and sequence information of a target sequence with higher resolution than known techniques.

[0005] The following methods are described herein: (1) Spatial localization of expressed mRNAs in tissues using hybrid circular / linear DNA probes with UMIs. (2) The nucleotide information of the adjacent region downstream of the region where the circular / linear DNA probe with UMI is annealed is copied by reverse transcriptase, and at the same time, the circular part of the hybrid probe carrying the UMI sequence is copied by Phusion DNA polymerase, and the resulting copied RNA strand is ligated to the reverse transcribed cDNA. (3) The UMI-containing cDNA strand is then extracted from the tissue. (4) The extracted UMI-containing cDNA strand is then PCR amplified with PCR primers that have known adapter ends. (5) The PCR product is circularized and amplified by RCA. (6) The RCA products can be sequenced by NGS.

[0006] The object of the present invention is to provide a method for obtaining spatial location and sequence information of a target sequence of at least one m-RNA strand on a tissue sample, comprising: a. providing a locator probe, the locator probe comprising: a) a linear locator hybridized to a circular locator via a locator anchor region capable of binding the circular locator; b) an RNA anchor region capable of binding to at least one m-RNA strand; and c) a primer sequence, the circular locator comprising a UMI region, first and second primer regions, and a region complementary to the locator anchor region of the linear locator; b. hybridizing a locator probe having an RNA anchor region to the m-RNA strand; using the cm-RNA strand as a template to complement the RNA anchor region of the locator probe, thereby obtaining a reverse transcribed c-DNA strand; d. complementing the circular locator starting from the first primer region using the circular locator as a template and ligating the resulting oligomer to the locator anchor region of the linear locator, thereby obtaining an extended reverse transcribed c-DNA strand that includes the UMI region and the target sequence of the m-RNA strand; e. amplifying the circular locator by RCA starting from the second primer region on the tissue sample to generate at least one first rolony (where L is only one); f. spatially analyzing and sequencing at least one first Lorony, thereby obtaining spatial and sequence information of at least one first Lorony (in other words, obtaining spatial coordinates and circular locator UMI by sequencing at least one first Lorony); g. removing the extended reverse-transcribed c-DNA strand from the tissue and dehybridizing the extended reverse-transcribed c-DNA strand from the m-RNA strand to obtain single-stranded oligomers; h. providing a first and a second adaptor primer at the 3' and 5' ends of the single-stranded oligomer, obtaining a primed single-stranded oligomer, amplifying the primed single-stranded oligomer by PCR, and circularizing the primed single-stranded oligomer by ligation of the first and second adaptor primers to one another, thereby creating a circular single-stranded oligomer; i. amplifying the circular single-stranded oligomer by RCA to a second Lorony, sequencing the second Lorony, and linking the spatial information of the first Lorony and the sequence information of the second Lorony via a UMI sequence; The method includes:

[0007] The present invention is directed to a method for performing spatial localization of a gene of interest in a given tissue using a hybrid probe that includes either a circular (Figure 1A) or open circular (Figure 1B) molecule with a random, non-predefined unique molecular identifier (UMI) and two priming regions. The circle / open circle can be pre-bound to a linear DNA primer (Figure 1A and Figure 1B) or the circular locator can be hybridized to a linear DNA primer after the linear primer binds to a sequence upstream of the desired region on the mRNA directly on the tissue (Figure 1C). The circular portion of the hybrid probe can then be amplified by rolling circle amplification (RCA) using oligonucleotide primers directly on the tissue, resulting in an RCA product at the exact location where the mRNA was expressed on the given tissue. The random, non-predefined UMI can then be decoded by sequencing the RCA product.

[0008] Next, the desired sequence information of a specific region of interest, that is a specific nucleotide change or variant on the same mRNA spatially localized on the tissue, can be simultaneously captured by reverse transcription of the mRNA with the same hybrid probe as a primer.

[0009] Concurrent with reverse transcription, the UMI information can be captured on the same reverse transcribed cDNA strand by Phusion DNA polymerase primed by a DNA primer specifically designed to copy the circular strand. Once Phusion DNA polymerase has copied the DNA circle, it can be ligated to the same cDNA strand that captured the sequence information of the region of interest. If an open circular locator probe is used, it is ligated at this point to form a closed circle.

[0010] The UMI-ligated cDNA is then physically retrieved and extracted from the tissue.

[0011] The recovered cDNA is then PCR amplified using PCR primers that anneal to the 5' and 3' ends of the cDNA and carry DNA adapters.

[0012] The PCR amplified product can then be circularized using a splint bridge oligonucleotide primer that brings the two ends together.

[0013] The circles are then amplified by rolling circle amplification (RCA). The RCA products are then sequenced.

[0014] NGS sequencing allows the spatial identifier UMI and linked mRNA sequences of interest to be assigned to a tissue location.

[0015] NGS sequencing can analyze targeted regions, mutations, or nucleotide variants on genomic DNA or mRNA. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1A shows three embodiments of a hybrid circular / linear locator probe; the closed circular portion (FIG. 1A) contains a random UMI sequence, two priming regions 1 and 2, and an anchor sequence that anneals to the probe in the 5' half of the linear locator probe; FIG. 1B shows an open circle version of this probe, where the locator circle can be closed later in the process by T4 DNA ligase; and FIG. 1C shows a version where the linear probe hybridizes to the target first, followed by reverse transcription, and once the linear probe is stably bound to the target, the circular locator is hybridized to the linear probe. For all three designs, the 3' half of the linear probe has a sequence that anneals to a specific region upstream of the region of interest in the target mRNA gene that needs to be sequenced, where the region of interest contains a sequence suspected to have a nucleotide change due to a variant difference. [Figure 2a]Diagram showing how a hybrid locator probe binds to a region upstream of a target region of a specific mRNA and reverse transcribes the target region. A hybrid (closed) open circular locator / linear hybrid probe is used to reverse transcribe the target region of the mRNA into cDNA. The locator circle containing the UMI is then copied and ligated into the cDNA, closing the locator circle. [Figure 2b] Figure 1 shows how a hybrid locator probe binds to a region upstream of a region of interest in a specific mRNA and reverse transcribes the region of interest, the closed locator circle is amplified by RCA, and the UMI portion is sequenced in situ. The ligated cDNA strand containing the copied UMI is then extracted for sequencing of the region of interest. [Diagram 3] FIG. 1 shows how a locator circle containing a UMI region and a region-of-interest specific sequence can be amplified by PCR prior to sequencing. [Figure 4] FIG. 1 shows how cDNA strands extracted from tissues can be PCR amplified and later circularized by adding adapter regions (P1 and P2) to the PCR primers. [Diagram 5] FIG. 1 shows how PCR amplicons with P1 / P2 adapter ends can be circularized and RCA amplified for analysis by NGS sequencing. [Figure 6] FIG. 1 illustrates the primers required to perform paired-end sequencing to match and pair the UMI region with the "region of interest."

[0017] Detailed Description Spatial localization of mRNA molecules by RCA followed by ex-situ sequencing of the region of interest from the same RNA molecule involves RCA, which consists of a nine-step process, performed directly on tissue: (1) Hybridization of linear or linear / circular locator probes to mRNA on tissue; (2) Reverse transcription of the region of interest in downstream mRNA by the linear part of hybrid locator probe hybridized to the target mRNA molecule (2A) and DNA replication of the circular part of hybrid probe by Phusion DNA polymerase with circle-specific oligonucleotide primer (2B); (3) T4 DNA ligation to connect the copied circular UMI and cDNA. If an open circular locator is used, it is also ligated in this step; (4) RCA with circle-specific RCA primers to generate rolony (DNA concatemer nanoballs) and in situ sequencing of random UMI on the generated rolony; (5) Extraction of cDNA-UMI first strand from tissue; (6) Second strand cDNA synthesis. (7) PCR amplification of cDNA using P1 / P2 adaptor primers, (8) Circularization of the PCR amplicon and RCA amplification of a circle containing the UMI and region of interest, (9) Identification of the location and sequence of the region of interest by NGS sequencing and matching to the UMI.

[0018] In a first embodiment of the invention, the circular locator is provided in step a) as a closed circle. In another embodiment, the circular locator is provided in step a) as an open circle, where in step d) the 3' and 5' ends of the open circle are ligated to each other to create a circular locator before propagating the circular locator.

[0019] In embodiments having an open circle, the 3' and 5' ends of the open circle can be ligated together using T4 DNA ligase, where the second primer region of the circular locator functions as a bridge splint.

[0020] Step d) (complementation of the circular locator) can be performed by Phusion DNA polymerase using the first primer region of the circular locator as an initiator primer. Additionally, the ligation step of step d) can be performed by DNA ligase.

[0021] The locator probe can be provided in the following variants: - A method in which a linear locator is first hybridized to a circular locator. - a method in which a linear locator is first hybridized to at least one m-RNA strand and then a circular locator is hybridized to the linear locator. - a method in which a linear locator is first hybridized to at least one m-RNA strand, the RNA anchor region of the locator probe is made complementary to the reverse transcribed c-DNA strand, and then a circular locator is hybridized to the linear locator.

[0022] Preferably, the single stranded oligomers are physically sheared into smaller fragments prior to the addition of first and second adapter primers to the 3' and 5' ends.

[0023] These embodiments are shown in the figures. Figure 1 shows three possible designs of locator probe setup. In Figure 1A, a hybrid locator probe is generated by hybridizing a circular probe containing the UMI with a linear probe pre-attached to the circle via a region-of-interest specific sequence (SP). In Figure 1B, an open circular probe is used. The pre-assembled probes in both Figures 1A and 1B are stable and are purified before use. In Figure 1C, a linear probe is used first, and a locator circle is added after the linear probe hybridizes to the mRNA target. The size of the circle is 20-400 bp long, and the region-of-interest SP portion is about 10-40 bp, which can form a stable double-stranded structure with the linear probe. The 3' end of the linear probe consists of a sequence about 30 bp long and can hybridize to a specific region of the desired target of the mRNA on the tissue.

[0024] Figure 2 shows the process of how locator probes are used. In step 1, with the tissue fixed on a slide, a hybrid locator probe is added, binding to a region upstream from the region of interest of a particular mRNA (dotted line). In one embodiment of the present invention, in step 1, a linear probe is added first, binding to a region upstream from the region of interest of a particular mRNA (dotted line), and a circle is added later.

[0025] Then, in step 2A, reverse transcription of the region of interest is initiated by a locator probe used as a primer.However, in one embodiment, once reverse transcription is completed, a circular locator is added to hybridize to the anchor region of the linear probe.Then, in step 2B, DNA synthesis of the locator circle is initiated by a circle-specific primer that binds to the primer 1 region.

[0026] The locator circle is then copied by a non-strand-displacing enzyme, such as Phusion DNA polymerase, and then joined to the reverse-transcribed cDNA strand by T4 DNA ligation (step 3). If an open circular locator probe version is used, the circle is closed by T4 DNA ligation in this step as well.

[0027] Next, isothermal rolling circle amplification (RCA) of the locator circles is performed on the tissue using Phi29 DNA polymerase and RCA oligonucleotides bound to primer region 2 (step 4). As a result, the location of the RCA products on the tissue is determined by sequencing the randomly generated UMIs on the tissue and obtaining the coordinates.

[0028] Next, in step 5, cDNA is extracted from the tissue after the locator circle RCA is visualized.

[0029] Figure 3 illustrates the workflow after extraction of first strand cDNA from tissue section slides. Once the cDNA is extracted and purified from the tissue, second strand cDNA (step 6) followed by a PCR reaction (step 7) is performed using a pair of primers P1 and P2. Primer P1 is located upstream of the circular locator region and primer P2 is located downstream of the "region of interest".

[0030] Figure 4 describes the workflow for generating RCA products that are sequenced by NGS. In step 8, the PCR product library is used to generate circle and RCA products, which are used for sequencing to identify UMIs and correspond to the DNA sequence of the region of interest. In step 9, RCA of both sense and antisense strands can be generated independently using sense and antisense bridge guides.

[0031] Figure 5 shows an example of UMI sequencing of L. rolonyi on a tissue section.

[0032] 6 illustrates the primers required to perform paired-end sequencing to match and pair the UMI region with the "region of interest." The first segment of paired-end sequencing is performed on the region of interest using paired-end segment 1 sequencing primers SQ-S primer 1 and SQ-AS primer 1. The second segment of paired-end sequencing is performed on the UMI region using paired-end segment 2 sequencing primers SQ-S primer 2 and SQ-AS primer 2.

[0033] NGS sequencing can determine if a mutation / nucleotide variant is present in the targeted region of interest, and by sequencing the UMI, the location of each locus on the substrate can later be matched with its original location in the tissue, i.e., the location of a gene on the tissue can be determined and then sequenced to confirm if that gene has a mutation.

Claims

1. A method for obtaining the spatial location and sequence information of a target sequence of at least one mRNA strand on a tissue sample, a. A step of providing a locator probe, wherein the locator probe comprises a) a linear locator hybridized to a circular locator via a locator anchor region capable of binding to the circular locator, b) an RNA anchor region capable of binding to at least one mRNA strand, and c) a primer sequence, wherein the circular locator comprises a UMI region, first and second primer regions, and a region of the linear locator complementary to the locator anchor region. b. A step of hybridizing the locator probe having the RNA anchor region to the mRNA strand, c. The step of using the mRNA strand as a template to complement the RNA anchor region of the locator probe, thereby obtaining a reverse-transcribed cDNA strand; d. Using the circular locator as a template, complement the circular locator starting from the first primer region, ligate the resulting oligomer with the locator anchor region of the linear locator, thereby obtaining an extended reverse-transcribed cDNA strand containing the UMI region and the target sequence of the mRNA strand; e. A step of growing the annular locator with RCA starting from the second primer region on the tissue sample to produce at least one first loronee (only one L), f. A step of spatially analyzing and sequencing the at least one first lorony, thereby obtaining spatial information and sequence information of the at least one first lorony, g. The steps of removing the extended reverse-transcribed c-DNA strand from the tissue, dehybriding the extended reverse-transcribed c-DNA strand from the mRNA strand, and obtaining a single-stranded oligomer, h. Providing first and second adapter primers to the 3' and 5' ends of the single-stranded oligomer to obtain a primed single-stranded oligomer, amplifying the primed single-stranded oligomer by PCR, cyclizing the primed single-stranded oligomer by mutual ligation of the first and second adapter primers, thereby producing a cyclic single-stranded oligomer; i. The steps of growing the cyclic single-stranded oligomer by RCA to form a second lorony, sequencing the second lorony, and linking the spatial information of the first lorony and the sequence information of the second lorony via the UMI sequence. Methods that include...

2. The method according to claim 1, characterized in that the annular locator is provided as a closed circle in step a).

3. The method according to claim 1, characterized in that the annular locator is provided as an open circle in step a), and in step d), the 3' and 5' ends of the open circle are ligated together to form an annular locator before the annular locator is propagated.

4. The method according to claim 3, characterized in that the 3' end and the 5' end of the open circle are ligated to each other with T4 DNA ligase, and the second primer region of the circular locator functions as a bridge splint.

5. The method according to claim 1, characterized in that the complementation of the circular locator in step d) is carried out by Phusion DNA polymerase using the first primer region of the circular locator as an initiator primer.

6. The method according to claim 1, characterized in that the ligation step in step d) is carried out by DNA ligase.

7. The method according to claim 1, characterized in that the locator probe is provided by first hybridizing the linear locator to the annular locator.

8. The method according to claim 1, characterized in that the locator probe is provided by first hybridizing the linear locator to at least one mRNA strand, and then hybridizing the circular locator to the linear locator.

9. The method according to claim 8, characterized in that the locator probe is provided by first hybridizing the linear locator to at least one mRNA strand, complementing the RNA anchor region of the locator probe with a reverse-transcribed cDNA strand, and then hybridizing the circular locator to the linear locator.

10. The method according to claim 1, characterized in that the single-stranded oligomer is physically sheared into smaller fragments before adding the first and second adapter primers to the 3' and 5' ends.

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