Ex-situ sequencing of rca product created in-situ

Abstract

To provide a method for acquiring sequence information of a target sequence from a tissue sample at high resolution.SOLUTION: A method for acquiring sequence information of a target sequence from a tissue including at least one RNA or c-DNA strand includes: a. a step of providing at least one first oligonucleotide which includes 50 to 1,000 nucleic acids having 5' and 3' terminals; b. a step of hybridizing 5' and 3' terminals of the first oligonucleotide into a complementary portion of at least one RNA or c-DNA strand; c. a step of binding the hybridized 3' and 5' terminals of the first oligonucleotide with each other to obtain a first single stranded circular template; d. a step of increasing the first single stranded circular template to a plurality of concatemers by a polymerase capable of performing rolling circle amplification to obtain a primary rolony; and e. a step of taking out the primary rolony from a sample.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the recovery, extraction, and sequencing of rolling circle amplification (RCA) products generated on tissue sections from circular or padlock probes that hybridize to the target region of in-situ expressed mRNA and are ligated with or without an intended reverse transcription target region that may contain a nucleotide change / variant or any other sequence.

[0002] Background Padlock oligonucleotides have proven to be highly successful in polymerizing short portions of nucleic acids to which they hybridize. Most padlock approaches begin with reverse transcription of the target into cDNA.

[0003] The padlock method is disclosed, for example, in "Highly multiplexed subcellular RNA sequencing in situ" by Lee et al., Science. 2014 March 21; 343(6177): 1360-1363. doi:10.1126 / science.1250212 or "Efficient In Situ Detection of mRNAs using the Chlorella virus DNA ligase for Padlock Probe Ligation" by Nils Schneider and Matthias Meier; February 5, 2020 - Cold Spring Harbor Laboratory Press.

[0004] An inclusive assay for target multiplex amplification of human DNA sequences is disclosed in Sujatha Krishnakumar et al.; PNAS sent for review February 19, 2008.

[0005] Furthermore, International Publication No. 2017143155 discloses the multiple changes in cells and their analysis using a pooled nucleic acid library, and International Publication No. 2018045181 discloses a method for generating a library of nucleic acid sequences for detection by fluorescence in situ sequencing.

[0006] While publicly available padlock methods allow for DNA or RNA sequencing, they are in situ only and cannot sequence entire target regions. In recent years, in situ genome sequencing (IGS) has been described as a method for simultaneously sequencing and imaging the genome within a sample. This method involves localizing unique molecular identifiers (UMIs) by short-read in situ sequencing, followed by ex situ sequencing of amplicons associated with genomic sequences containing UMIs by amplicon dissociation, PCR, and paired-end sequencing. This workflow was published by AC Payne et al., Science 10.1126 / science.aay3446 (2020).

[0007] Microscopic imaging, which enables the analysis of numerous mRNAs at the single-cell level, provides valuable information on transcriptional volume and localization, which are crucial elements for understanding tissue heterogeneity, molecular development, and disease treatment. Furthermore, the ability to identify potential mutations from mRNAs with known spatial information is also extremely valuable.

[0008] A method for obtaining spatial information and sequencing of RNA or c-DNA is disclosed in European Patent Application Publication No. 3936623. In this method, an oligonucleotide hybridizes to RNA or c-DNA to form a circular template, where the oligonucleotide (and subsequently the circular template) includes at least one region having a known sequence that is recognized by a detection probe having a suitable complementary sequence. Detection of the detection probe allows for obtaining specific information about the RNA or c-DNA on a tissue. In one variation of this method, the circular template is fragmented and re-circulated to obtain a second circular template for further amplification. However, since this method aims to obtain spatial information, the first and second circularization / amplification steps are performed on a tissue.

[0009] Subject of the invention The present invention relates to a method for recovering rolling circle amplification (RCA) products generated on a tissue having a desired target nucleotide (genomic DNA or mRNA) and a barcode or unique molecular identifier that can optionally function as a spatial identifier.

[0010] This is achieved by using a circular or padlock molecule to detect the desired target nucleotide (genomic DNA or mRNA) and hybridize to it. The desired sequence information is captured by the circular or padlock molecule used to detect the desired target nucleotide (genomic DNA or mRNA) on the tissue and hybridize to it. These circular or padlock molecules have the desired target nucleotide (genomic DNA or mRNA), or a barcode or unique molecular identifier that functions as a spatial identifier. These circular or padlock molecules are directly RCA amplified on the tissue. Subsequently, the RCA product is physically recovered and extracted from the tissue, fragmented, the region of interest is amplified by PCR, and then a second circularization and RCA amplification is performed. The RCA product is then sequenced using an NGS sequencing platform.

[0011] NGS sequencing of a target region of genomic DNA or mRNA allows for the analysis of mutations or nucleotide variants. Furthermore, a spatial identifier is assigned to the location of the target sequence in the tissue.

[0012] overview Therefore, an object of the present invention was to provide a method for obtaining sequence information of a target sequence from a tissue sample with higher resolution than known techniques.

[0013] The subject of the present invention is a method for obtaining sequence information of a target sequence from a tissue comprising at least one RNA or c-DNA strand, a. A step of providing at least one first oligonucleotide comprising 50 to 1000 nucleic acids having 5' and 3' ends, b. A step of hybridizing the 5' and 3' ends of the first oligonucleotide to a complementary portion of at least one RNA or c-DNA strand, c. A step of obtaining a first single-stranded cyclic template by joining the 3' and 5' ends of the hybridized first oligonucleotide to each other, d. A step to obtain primary Rollonie by increasing the first single-stranded cyclic template into multiple concatemers using a polymerase capable of rolling circle amplification, e. The step of removing the primary loroney from the sample, f. The steps of obtaining a third oligonucleotide by fragmenting the primary loroney into multiple second oligonucleotides and hybridizing the 3 and 5' ends of the second oligonucleotides with the first and second PCR primers, g. A step of increasing the third oligonucleotide using a polymerase capable of polymerase chain reaction (PCR), h. Obtaining a second single-stranded cyclic template by ligating the first PCR primer to the second PCR primer of the increased third oligonucleotide, i. A second single-stranded cyclic template is increased into multiple concatemers using a polymerase capable of rolling circle amplification to obtain a secondary Rollonie; j. A step to obtain sequence information of the target sequence by determining the sequence of secondary Rollonie. This method includes [something].

[0014] The method of the present invention is particularly useful for quality control of sequencing methods. Therefore, a further subject of the present invention is a method for quantifying gene expression profiles using sequence information of obtained target sequences, or a method for confirming the effectiveness of oligonucleotide hybridization and target sequence selection using sequence information of obtained target sequences.

[0015] Herein, the inventors describe a method for (1) recovering an RCA product from a tissue section and (2) performing targeted PCR amplification of the RCA product containing a region of interest having a nucleotide change / variant and / or a barcode / unique molecular identifier.

[0016] The methods of the present invention are partially carried out directly on tissue and partially carried out after removing molecules containing the target sequence from the tissue. The “on tissue” step is: 1. Recovery and extraction of DNA from RCA products from tissue sections. 2. Targeted PCR amplification of RCA products 3. Cyclization and RCA amplification of PCR products 4. NGS sequencing Includes. [Brief explanation of the drawing]

[0017] [Figure 1]This figure shows the design of the ring or padlock probe used in this method. The ring / padlock may contain a barcode or UMI identifier. The ring / padlock is hybridized to a tissue section expressing the target mRNA or genomic DNA. After hybridization, the padlock is ligated with SplintR ligase to generate a ring. Next, rolling circle amplification (RCA) can be performed using this ring to generate a detectable RCA product on the tissue. [Figure 2] This figure shows a strategy for PCR amplification of the target region of the RCA generated from the ring / padlock. PCR primers containing P1 and P2 receptors can be amplified and sequenced by hybridizing to the flanking region of the target region. [Figure 3a] This figure shows successful ex-situ sequencing of four gene transcripts from padlocks extracted from tissue. Each padlock, designed to detect the target transcript, was clearly identified by sequencing of the target region. [Figure 3b] This figure shows successful ex-situ sequencing of four gene transcripts from padlocks extracted from tissue. Each padlock, designed to detect the target transcript, was clearly identified by sequencing of the target region.

[0018] Detailed explanation Ex-situ sequencing of RCAs performed directly on tissue consists of a six-step process: (1) RCA generation on tissue. (2) Recovery of RCA loronyi and extraction of DNA from loronyi obtained from tissue sections. (3) Targeted PCR amplification of the region of interest. The region of interest may be either a barcode / UMI or a nucleotide change / variant of the target sequence. (4) Cyclization of the PCR product. (5) RCA or cyclic product. (6) NGS sequencing.

[0019] In FIG. 1, two types of padlocks that hybridize to mRNA (dotted line) are shown together with a specific region of the target of interest. The region of interest can include either the nucleotide position of the region that hybridizes to mRNA having a base mutation or variant, or the barcode of the UMI in the padlock backbone region. The circular body generated following ligation can function as a substrate for directly generating RCA colonies on tissue (step 1 in FIG. 1).

[0020] In the first embodiment, the 5' and 3' ends of the first oligonucleotide are hybridized adjacent to complementary portions of at least one RNA or cDNA strand, and a first single-stranded circular template is obtained by direct ligation of the 5' and 3' ends of the first oligonucleotide to each other.

[0021] In the second embodiment, the 5' and 3' ends of the first oligonucleotide are hybridized to complementary portions of at least one RNA or cDNA strand with a gap of 2 to 100 nucleotides between the 5' and 3' ends of the first oligonucleotide, and the first single-stranded circular template is obtained by filling this gap with nucleotides complementary to the RNA or cDNA strand.

[0022] Preferably, the first oligonucleotide contains a fragmentation sequence that enables fragmentation of the primary colony by a restriction enzyme or chemically.

[0023] Extraction of DNA from RCA A method for recovering the RCA product from a tissue section is shown (steps 1 and 2 in FIG. 2). First, tissue digestion is performed by heating the sample on a tissue section containing the RCA product in the presence of a lysis buffer and proteinase K.

[0024] The sample can be removed from the heat source and incubated using solid-phase reversible immobilization (SPRI) beads, and by using a magnetic body, the beads containing nucleic acids can be easily washed.

[0025] Preferably, the eluate is added to SPRI beads, washed three times, the SPRI beads are removed using a magnetic material, and the supernatant is transferred to a tube. This tube contains the extracted RCA DNA eluate.

[0026] The extracted nucleic acids can be quantified using Nanodrop or Qubit.

[0027] Targeted PCR amplification is performed on the recovered RCA product containing the target padlock junction region with the above nucleotide changes / variants. After extracting and quantifying the RCA DNA, a PCR reaction is performed using the RCA DNA as a template with a primer set (first PCR primer and second PCR primer) specific to the region adjacent to the target region (Step 3 in Figure 2).

[0028] For example, the sequence of the first PCR primer is: [ka] The sequence of the second PCR primer is: [ka] This may be the case. The bolded type of sequence hybridizes to the region adjacent to the target region shown in step 3 of Figure 2. The P1 and P2 receptor moieties are unique sequences that can be used for cyclization in a later step.

[0029] Perform 25 cycles of PCR.

[0030] The PCR products were purified using a QiaQuick PCR product purification column, and the DNA was quantified using a Qubit assay.

[0031] The obtained PCR products containing P1 and P2 receptors are cyclized using a sprint oligonucleotide that brings both ends together (Step 4 in Figure 2). Preferably, the first PCR primer is ligated to the second PCR primer by providing the sprint DNA.

[0032] The ring-shaped body is amplified via RCA (step 5 in Figure 2), and a Rollonie is formed.

[0033] NGS sequencing can be performed using sequencing primers that bind to the P1 or P2 receptor region (Step 5 in Figure 2).

[0034] NGS sequencing allows for the determination of whether mutations / nucleotide variants exist in the target region, and sequencing of the padlock ID region allows for the subsequent correlation of the Rollony gene's position on the substrate with its original position in the tissue. In other words, sequencing allows for the investigation of gene locations and the presence or absence of mutations in tissues. Furthermore, by quantifying the number of sequencing reads obtained, gene expression profiles can also be presented.

[0035] Furthermore, the spatial location of the first loronie in the tissue is determined by imaging the luminescence of at least one fluorescently labeled oligonucleotide bound to the first loronie.

[0036] For this purpose, the first lorony can be obtained by decorating (binding) the first lorony with at least one fluorescently labeled oligonucleotide.

[0037] Furthermore, the first oligonucleotide includes an identification region comprising a UMI sequence and / or a barcode sequence to which at least one fluorescently labeled oligonucleotide is bound.

[0038] Examples Four mouse genes were tested, and for these genes, five different padlock oligonucleotide probes were designed to be complementary to a portion of the corresponding mRNA transcript. A total of 20 probes (five probes for each of the four genes) were hybridized to their respective targets in mouse tissue sections. The probes were supplied in excess. The padlock probes were then enzymatically ligated, and RCA was performed directly on the tissue (in situ). Gene-specifically generated loroney was detected by hybridizing fluorescently labeled oligonucleotides to the padlock recognition region containing the UMI and / or barcode sequence. As described in Step 1 above, the RCA loroney was then extracted from the tissue and randomly fragmented. As described in Step 2, the PCR reaction was performed using primers (P1 and P2) in which one portion contained a region complementary to the region adjacent to the region of interest and the other portion contained a general-purpose sequence. The resulting linear products containing P1 and P2 receptors were cyclized using sprint oligonucleotides that joined both ends. This cyclized product was RCA amplified to form secondary loroney.

[0039] The target region of the RCA product was ultimately sequenced using a Rollonie-compatible NGS sequencer. Each generated sequence can be aligned and mapped to the four target gene transcripts.

[0040] As shown in Figure 3 below, sequencing reads for all four genes were uniquely detected with different read counts. These results demonstrate that this method can be used as a tool to quantify the number of specific RCA products (Roronie) from extracted tissue. Furthermore, unlike other methods, the effectiveness of hybridization of various padlock probes targeting the same transcript can be evaluated by quantifying the individual sequencing read counts for each probe. For example, for gene 1, some probes hybridize more efficiently than others, and the probe design can be improved by examining the sequencing read count. This can also be used to quantify gene expression profiles, as shown in the graph in Figure 3, and to confirm hybridization approaches using fluorescently labeled oligonucleotides for primary Roronie generated on tissue.

Claims

1. A method for obtaining sequence information of a target sequence from a tissue containing at least one RNA or c-DNA strand, a. Providing at least one first oligonucleotide comprising 50 to 1000 nucleic acids having 5' and 3' ends, b. A step of hybridizing the 5' and 3' ends of the first oligonucleotide to a complementary portion of at least one RNA or c-DNA strand, c. The step of obtaining a first single-stranded cyclic template by bonding the 3' and 5' ends of the hybridized first oligonucleotide to each other, d. The first single-stranded cyclic template is increased into multiple concatemers using a polymerase capable of rolling circle amplification to obtain primary Rollonie; e. The step of removing the primary loroney from the sample, f. The steps of obtaining a third oligonucleotide by fragmenting the primary loroney into a plurality of second oligonucleotides and hybridizing the 3 and 5' ends of the second oligonucleotides with the first PCR primer and the second PCR primer, g. A step of increasing the third oligonucleotide using a polymerase capable of polymerase chain reaction (PCR), h. The step of obtaining a second single-stranded cyclic template by ligating the first PCR primer to the second PCR primer of the increased third oligonucleotide, i. The second single-stranded cyclic template is increased into multiple concatemers using a polymerase capable of rolling circle amplification to obtain secondary Rollonie; j. The step of obtaining the sequence information of the target sequence by determining the sequence of the secondary Rollonie. Methods that include...

2. The method according to claim 1, wherein the 5' and 3' ends of the first oligonucleotide are hybridized adjacent to complementary portions of the at least one RNA or c-DNA strand, and the first single-stranded circular template is obtained by direct ligation of the 5' and 3' ends of the first oligonucleotide.

3. The method according to claim 1, comprising hybridizing the 5' and 3' ends of the first oligonucleotide to a complementary portion of the at least one RNA or c-DNA strand with a gap of 2 to 100 nucleotides between the 5' and 3' ends of the first oligonucleotide, and filling the gap with nucleotides complementary to the RNA or c-DNA strand to obtain the first single-stranded circular template.

4. The method according to claim 1, wherein the first PCR primer is ligated to the second PCR primer by providing sprint DNA.

5. The method according to claim 1, wherein the rolling circle amplification (RCA) is activated by light and / or heat.

6. The method according to claim 1, wherein the first oligonucleotide comprises a fragmentation sequence that enables the primary lorony to be fragmented by restriction enzymes or chemically.

7. The method according to claim 1, wherein the primary lorony is decorated with at least one fluorescently labeled oligonucleotide.

8. The method according to claim 7, wherein the spatial location of the primary loroney on the tissue is determined by imaging the emission of at least one fluorescently labeled oligonucleotide bound to the primary loroney.

9. The method according to claim 7 or 8, wherein the first oligonucleotide includes an identification region comprising a UMI sequence and / or a barcode sequence to which the at least one fluorescently labeled oligonucleotide is bound.

10. The method according to claim 1, wherein the gene expression profile is quantified using the sequence information of the target sequence.

11. The method according to claim 1, wherein the effectiveness of oligonucleotide hybridization and selection of the target sequence is confirmed using the sequence information of the target sequence.

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