Probe design of high-throughput single cell space transcriptome based on multi-round imaging coding

Abstract

The invention relates to a probe design of a high-throughput single cell space transcriptome based on multi-round imaging coding. The invention provides a design method of a readout probe for a high-throughput single-cell space transcriptome, which is simultaneously suitable for more than two species tissues, and comprises the following steps: generating a candidate DNA sequence library with a sequence length of n, filtering out a sequence capable of being compared to a genome of a target species, filtering out a candidate DNA probe containing a short sequence appearing on the genome for more than one time, and obtaining a read-out probe for the high-throughput single-cell space transcriptome. The method comprises the steps of filtering candidate DNA probes capable of being compared to transcriptomes of target species through local BLAST, repeating the steps S20-S40 for all target species, performing probe internal mutual binding screening through the local BLAST, performing probe secondary structure inspection and probe internal mutual binding inspection through two-sequence comparison, performing online BLAST inspection on the probes, and determining the target species according to the BLAST. The final DNA probe sequence is obtained.

Description

Technical Field

[0001] The present invention relates to spatial transcriptomics, and in particular to a method for designing readout probes for high-throughput single-cell spatial transcriptomics encoded by multiple rounds of imaging, and probe products according to the method. Background Art

[0002] Single-cell spatial transcriptomics is an emerging field that provides a new approach to understanding the spatial distribution of gene expression within tissues. In traditional single-cell transcriptomics, spatial information about gene expression in tissues is lost during the extraction process. Therefore, while traditional single-cell transcriptomics can provide a lot of information about the genes being expressed in a sample, it cannot provide information about the location of gene expression in the tissue. Single-cell spatial transcriptomics addresses this limitation by retaining spatial information during RNA extraction (or in situ capture). This allows researchers to determine not only which genes are being expressed, but also where they are expressed in the tissue. This additional spatial information can provide valuable information for understanding the development and function of organs and tissues, as well as the progression of diseases such as cancer and response to treatment.

[0003] Spatial transcriptome technologies are mainly divided into two categories: one is based on probe hybridization and imaging, such as smFISH, MERFISH, etc.; the other is based on sequencing, including 10x VISIUM, DBiT-seq, Stereo-seq, etc. Among them, the methods based on probe hybridization and imaging currently lack publicly published readout probe design methods and products that can be applied to multiple species at the same time. Summary of the invention

[0004] The present invention aims to solve the problem of the lack of publicly published design methods for secondary readout probes (readout probes labeled with fluorescent groups that specifically bind to primary non-fluorescent probes) and secondary probe sequences that are applicable to multiple species at the same time. Secondary readout probes are the key to achieving high throughput, but currently there are only secondary probes that are applicable to a single species, such as mouse or human samples, and there are no secondary probes that are applicable to tissues of multiple species (such as humans and mice) at the same time. Designing secondary probes that are applicable to more than two species at the same time is of great significance in saving experimental costs.

[0005] In some aspects, provided herein are inventions described in one or more of the following.

[0006] 1. A method for designing readout probes for high-throughput single-cell spatial transcriptomes that is applicable to tissues of two or more species, wherein the method comprises:

[0007] Step S10, generating a candidate DNA sequence library with a sequence length of n,

[0008] Step S20, filtering out sequences that can be matched to the genome of the target species,

[0009] Step S30, filtering out candidate DNA probes containing short sequences that appear more than once in the genome,

[0010] Step S40, filtering out candidate DNA probes that can be matched to the transcriptome of the target species through local BLAST,

[0011] Step S50, repeating steps S20-S40 for all target species,

[0012] Step S60, performing internal binding screening of probes by local BLAST,

[0013] Step S70, by comparing the two sequences, checking the secondary structure of the probes and checking the internal binding of the probes.

[0014] Step S80, performing an online BLAST check on the probe to obtain a final DNA probe sequence, and optionally,

[0015] Step S90, connecting the DNA sequence to the fluorescent group to obtain a readout probe.

[0016] 2. The method described in item 1, wherein step S10 comprises: generating a candidate DNA sequence library with a sequence length of n, wherein the length n can be set to any appropriate length, preferably not more than 40 nt and not less than 15 nt, optimally 18 nt-21 nt, and the GC content of each sequence is between 0.4 and 0.6, and the sequence does not contain low-complexity sequence G-quadruplexes.

[0017] 3. The method described in item 1 or 2, wherein step S20 comprises: using the alignment software bbmap to align each sequence in the candidate sequence library to the genome of the target species, and filtering out sequences that can be aligned to the genome of the target species.

[0018] 4. The method described in any one of items 1-3, wherein step S30 comprises: counting the number of occurrences of the short sequence in the genome of the target species, searching the candidate sequence library obtained in step S20 for the short sequence, and filtering out candidate DNA probe sequences containing the short sequence that appears more than once, and optionally, the length of the short sequence can be appropriately set to, for example, 12, 13, 14, 15 or 16 nt, preferably to 13 nt.

[0019] 5. The method described in any one of items 1-4, wherein step S40 comprises: aligning the DNA sequence obtained in step S30 to the reference transcriptome of the target species by local BLAST, and filtering out sequences that can be continuously aligned over a threshold length, wherein the threshold length can be appropriately set to, for example, 11, 12, 13, 14, 15 or 16 nt, preferably 12 nt.

[0020] 6. The method described in any one of items 1-5, wherein step S60 comprises: using local BLAST to compare whether the internal sequences of the probes can bind to each other, and filtering out sequences with more than a continuous threshold base binding, wherein the threshold length can be appropriately set to, for example, 9, 10, 11, 12, 13, 14, 15 or 16 nt, preferably to 10 nt.

[0021] 7. The method described in any one of items 1-6, wherein step S70 comprises: checking whether the sequence itself is consistent with its reverse complementary sequence through a local dynamic programming alignment algorithm of the two sequences, and filtering out sequences with base matches exceeding a threshold, wherein the threshold length can be appropriately set to, for example, 10, 11, 12, 13, 14, 15 or 16 nt, preferably 15 nt.

[0022] 8. The method described in any one of items 1-7, wherein step S80 comprises: performing an online BLAST check on the probes, filtering out probe sequences that have more than a threshold number of consecutive alignments to the transcriptome and the genome, wherein the threshold length can be appropriately set to, for example, 12, 13, 14, 15 or 16 nt, preferably, filtering out probe sequences that have more than 12 consecutive alignments to the transcriptome and 15 consecutive alignments to the genome.

[0023] 9. A computer system comprising:

[0024] processor;

[0025] A memory having instructions stored thereon, which, when executed by the processor, cause the processor to perform a method according to any one of items 1-8.

[0026] 10. A computer-readable storage medium storing instructions which, when executed by a processor, cause the processor to perform a method according to any one of items 1-8.

[0027] In some embodiments, the species to which the present invention is applicable are not limited, for example, mammals, including primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., mice and rats). In some embodiments, the species to which the present invention is applicable include more than 2 species, for example, 2, 3, 4, 5, 6 or more species. In some embodiments, the species to which the present invention is applicable may include humans, mice, rats, hamsters, birds, rabbits, cats, pigs, dogs, horses, cattle, sheep, monkeys, etc.

[0028] In some embodiments, any suitable algorithm and software can be used to carry out sequence alignment in the method of the present invention.For example, software well known in the art such as BLAST, BLAST-2, ALIGN can be used to carry out.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for realizing contrast.In some embodiments, local BLAST and online BLAST can be carried out using the parameters of suitable settings, for example, using default parameters.

[0029] In some embodiments, the probe sequences involved can be linked to any suitable fluorescent groups to prepare readout probes, such fluorescent groups are well known in the art.

[0030] The probe designed by the present invention is applicable to tissues of multiple species such as mice and humans, and has a broader application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the process of designing secondary probes for high-throughput single-cell spatial transcriptome based on multiple rounds of imaging encoding in the present invention.

[0032] Figure 2 It is a structural diagram of the secondary probe designed for the high-throughput single-cell spatial transcriptome secondary probe based on multiple rounds of imaging encoding in the present invention.

[0033] Figure 3 This is a hybridization signal image (left) and a background image (right) of the present invention applied to mouse neural tissue.

[0034] Figure 4 The hybridization signal image (left) and background image (right) of the present invention applied to human neural tissue. DETAILED DESCRIPTION

[0035] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the relevant invention are shown in the accompanying drawings.

[0036] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0037] In some embodiments, the present invention provides a secondary probe design method applicable to multiple species tissues (taking humans and mice as examples), the method comprising:

[0038] Step S10, generating a candidate DNA sequence library with a sequence length of n (n=18 nt);

[0039] Step S20, using the alignment software bbmap to filter out sequences that can be aligned to the genome of the target species;

[0040] Step S30, filtering out candidate DNA probes containing short sequences that appear multiple times on the genome;

[0041] Step S40, filtering out candidate DNA probes that can be aligned to the transcriptome of the target species through local BLAST;

[0042] Step S50, repeating steps S20-S40 for all target species;

[0043] Step S60, performing internal binding screening of probes by local BLAST;

[0044] Step S70, performing a probe secondary structure check and a probe internal binding check by aligning the two sequences;

[0045] Step S80: Online BLAST check of the probe.

[0046] Step S90, the DNA sequence and the fluorescent group part of the probe are Figure 2 The structures shown were assembled to obtain the secondary probe.

[0047] In some embodiments, the present invention provides a method for designing secondary probes for high-throughput single-cell spatial transcriptome based on multiple rounds of imaging encoding, the method comprising:

[0048] Step S10, generating a candidate DNA sequence library with a sequence length of n (n=18 nt), and each sequence should satisfy the GC content between 0.4 and 0.6, and the sequence should not contain low-complexity sequences such as G-quadruplexes;

[0049] Step S20, using the alignment software bbmap to align each sequence in the candidate sequence library to the genome of the target species, and filtering out sequences that can be aligned to the genome of the target species;

[0050] Step S30, counting the number of occurrences of the short sequence of 13 nt in length in the genome of the target species, searching the candidate sequence library obtained in step S20 for these short sequences of 13 nt in length, and filtering out candidate DNA probe sequences containing the short sequence of 13 nt in length that appears more than once, as these sequences have a potential risk of binding to the genome of the target species;

[0051] Step S40, aligning the DNA sequence obtained in step S30 to the reference transcriptome of the target species by local BLAST, setting the evalue value to 1000, word_size to 7, gapopen to 5, gapextend to 2, penalty to -3, reward to 1, and filtering out sequences that can be continuously aligned to more than 12 bases;

[0052] Step S50, repeating steps S20-S40 for all target species;

[0053] Step S60, screening for internal binding of probes, using local BLAST to compare whether the internal sequences of the probes can bind to each other, and filtering out sequences with more than 10 consecutive bases that are completely bound;

[0054] Step S70, probe secondary structure check and probe internal binding check. In order to exclude the possibility that the probe itself may form a secondary structure and the internal binding between secondary probes, a two-sequence local dynamic programming alignment algorithm is used to check whether the sequence itself is consistent with its own reverse complementary sequence, and whether any sequence is consistent with the reverse complementary sequence of other sequences. The alignment parameters are match = 1, mismatch = -1, gapopen = -5, gapextend = -1, and sequences with more than 15 base matches are filtered out;

[0055] Step S80, online BLAST check of the probe, using a larger database online BLAST to obtain the sequence in step S60 to ensure that the sequence absolutely does not match the genome and transcriptome of all target species, and filtering out probe sequences with more than 12 nt continuous alignment to the transcriptome and 15 nt continuous alignment to the genome to obtain the final DNA probe sequence.

[0056] Step S90, the DNA sequence and the fluorescent group part of the probe are Figure 2 The structures shown were assembled to obtain the secondary probe.

[0057] The high-throughput single-cell spatial transcriptome secondary probe design method based on multi-round imaging encoding according to the first embodiment of the present invention includes steps S10 to S90, each of which is described in detail as follows:

[0058] Step S10, generating a candidate DNA sequence library with a sequence length of n (n=18 nt), and each sequence should satisfy the GC content between 0.4 and 0.6, and the sequence should not contain low-complexity sequences such as G-quadruplexes;

[0059] Step S20, using the alignment software bbmap to align each sequence in the candidate sequence library to the mouse genome, and filtering out sequences that can be aligned to the mouse genome;

[0060] Step S30, counting the number of occurrences of the short sequence of 13 nt in length in the mouse genome, searching the candidate sequence library obtained in step S20 with the short sequence of 13 nt in length, and filtering out the candidate DNA probe sequences containing the short sequence of 13 nt in length that appears more than once, as these sequences have a potential risk of binding to the mouse genome;

[0061] Step S40, aligning the DNA sequence obtained in step S30 to the reference transcriptome of mice by local BLAST, setting the evalue value to 1000, word_size to 7, gapopen to 5, gapextend to 2, penalty to -3, reward to 1, and filtering out sequences that can be continuously aligned to more than 12 bases;

[0062] Step S50, applying steps S20-S40 to the human genome and transcriptome to obtain candidate secondary probe DNA sequences suitable for human tissues;

[0063] Step S60, screening for internal binding of probes, using local BLAST to compare whether the internal sequences of the probes can bind to each other, and filtering out sequences with more than 10 consecutive bases that are completely bound;

[0064] Step S70, probe secondary structure check and probe internal binding check. In order to exclude the possibility that the probe itself may form a secondary structure and the internal binding between secondary probes, a two-sequence local dynamic programming alignment algorithm is used to check whether the sequence itself is consistent with its own reverse complementary sequence, and whether any sequence is consistent with the reverse complementary sequence of other sequences. The alignment parameters are match = 1, mismatch = -1, gapopen = -5, gapextend = -1, and sequences with more than 15 base matches are filtered out;

[0065] Step S80, online BLAST check of the probe, using a larger database online BLAST to obtain the sequence in step S60 to ensure that the sequence absolutely does not match the mouse and human genomes and transcriptomes, and filtering out probe sequences with more than 12 nt continuous alignment to the transcriptome and 15 nt continuous alignment to the genome to obtain the final DNA probe sequence.

[0066] Step S90, the DNA sequence and the fluorescent group part of the probe are Figure 2 The structure shown in the figure was assembled to obtain the secondary probe. The product is shown in Table 1.

[0067] The second embodiment of the present invention describes a practical application of a high-throughput single-cell spatial transcriptome secondary probe design based on multi-round imaging encoding in mouse and human tissues, which is described in detail as follows:

[0068] Sample preparation: The target sequence of the target gene is combined with the reverse complementary sequence of the secondary sequence involved in this patent to obtain a primary probe sequence, and the primary probe is hybridized with the mRNA of the target gene in a 37°C hybridization oven with a slice of human or mouse tissue to prepare the sample required for the secondary probe image acquisition experiment;

[0069] Collecting hybridization images of secondary probes: Before collecting all probe images, first collect background signals of all channels in the field of view of interest. After the background signal is collected, the secondary probes are grouped and hybridized, and a group of probes belonging to different fluorescent channels are imaged and hybridized at a time. This group of probes will hybridize with the complementary part of the primary probe of the target gene. After hybridization, non-specific hybridization adsorption is eluted with 20% formamide solution (Sigma, F9027), and the cell nucleus is histologically stained with 0.01mg / mL DAPI (Sigma, D9542) solution. Then, the tissue is immersed in a solution containing 10% D-glucose (Sigma, G5767), 1:100 diluted catalase (Sigma, C3155), 0.5mg / mL glucose oxidase (Sigma G2133), and 1:1000 diluted RNase inhibitor (Ambion, AM2694), and the imaging of this round is collected immediately. After the acquisition of the probe signal of this group is completed, the probe group is washed away with an eluent containing 60% formamide concentration.

[0070] After the hybridization-imaging-elution process of one set of probes is completed, the cycle of the next set of probes is immediately entered, and the cycle is repeated until all probe signals are collected.

[0071] By comparing the background signal with the probe hybridization signal, it can be seen that the probe has the ability to specifically report the target sequence for both human and mouse tissues, with low background signal intensity and good signal-to-noise ratio. The probe designed in this patent is suitable for a variety of species, such as humans and mice, and is suitable for in situ detection of target signals in tissues of a variety of species.

[0072] It should be noted that the embodiments of the present application are only illustrated by the division of the above-mentioned functional modules. In practical applications, the above-mentioned functions can be assigned to different functional modules as needed, that is, the modules or steps in the embodiments of the present invention can be decomposed or combined. For example, the modules in the above-mentioned embodiments can be combined into one module, or further divided into multiple sub-modules to complete all or part of the functions described above. The names of the modules and steps involved in the embodiments of the present invention are only for distinguishing the modules or steps, and are not regarded as improper limitations of the present invention.

[0073] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

[0074] Table 1: Exemplary secondary probes of some embodiments of the present invention (the probe sequences are shown in the table below, wherein each probe sequence is respectively connected to seven fluorescent groups, namely 405fluorophore, 488fluorophore, 532fluorophore, 561fluorophore, 594fluorophore, 647fluorophore, and 750fluorophore, to form seven secondary probes).

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

Claims

1. A method for designing readout probes for high-throughput single-cell spatial transcriptomes that is applicable to tissues of two or more species, wherein the method comprises: Step S10, generating a candidate DNA sequence library with a sequence length of n, Step S20, filtering out sequences that can be matched to the genome of the target species, Step S30, filtering out candidate DNA probes containing short sequences that appear more than once in the genome, Step S40, filtering out candidate DNA probes that can be matched to the transcriptome of the target species through local BLAST, Step S50, repeating steps S20-S40 for all target species, Step S60, performing internal binding screening of probes by local BLAST, Step S70, by comparing the two sequences, checking the secondary structure of the probes and checking the internal binding of the probes. Step S80, performing an online BLAST check on the probe to obtain the final DNA probe sequence.

2. The method of claim 1, wherein step S10 comprises: The GC content of each sequence in the generated sequence is between 0.4 and 0.6, and the sequence does not contain low-complexity sequence G-quadruplex.

3. The method of claim 1 or 2, wherein step S20 comprises: Use the alignment software bbmap to align each sequence in the candidate sequence library to the genome of the target species, and filter out sequences that can be aligned to the genome of the target species.

4. The method according to any one of claims 1 to 3, wherein step S30 comprises: The number of times the short sequence appears in the genome of the target species is counted, and the candidate sequence library obtained in step S20 is searched for the short sequence, and candidate DNA probe sequences containing the short sequence that appears more than once are filtered out.

5. The method according to any one of claims 1 to 4, wherein step S40 comprises: The DNA sequence obtained in step S30 is aligned to the reference transcriptome of the target species by local BLAST, and sequences that can be continuously aligned exceeding a threshold length are filtered out.

6. The method according to any one of claims 1 to 5, wherein step S60 comprises: Through local BLAST, the internal sequences of the alignment probes are compared to see if they can bind to each other, and the sequences with base binding exceeding the continuous threshold are filtered out.

7. The method according to any one of claims 1 to 6, wherein step S70 comprises: The local dynamic programming alignment algorithm of the two sequences is used to check whether the sequence itself is consistent with its reverse complementary sequence, and sequences with base matches exceeding the threshold are filtered out.

8. The method according to any one of claims 1 to 7, wherein step S80 comprises: The probes were subjected to online BLAST check, and all probe sequences that were continuously aligned to the transcriptome and genome exceeding the threshold were filtered out.

9. A computer system comprising: processor; A memory having instructions stored thereon, which, when executed by the processor, cause the processor to perform the method according to any one of claims 1-8.

10. A computer-readable storage medium storing instructions which, when executed by a processor, cause the processor to perform the method according to any one of claims 1-8.

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