JPWO2019157445A5

JPWO2019157445A5 -

Info

Publication number: JPWO2019157445A5
Application number: JP2020542878A
Authority: JP
Publication date: 2022-02-21
Anticipated expiration: 2039-02-11

Claims

A method for spatially detecting the abundance of at least one target sample in at least one cell from a tissue sample, the following:
(1) In the step of contacting at least one target sample in at least one cell in a tissue sample with at least three probes , thereby tiling the probe on the at least one target sample . There,
Where each of the probes is:
A target binding domain that binds to at least one target specimen; and
Single-stranded identifier oligonucleotide
Including
Here, the single-stranded identifier oligonucleotide is
A unique nucleic acid sequence that identifies a target sample bound to the target binding domain,
Nucleic acid sequence containing a unique molecular identifier,
Includes a first amplification primer binding site and a second amplification primer binding site.
Here, each of the probes comprises a photocleavable linker between the single-stranded identifier oligonucleotide and the target binding domain ;
(2) A step of exciting the position of the tissue sample with light of sufficient wavelength to cleave the photocleavable linker , thereby releasing the single-stranded identifier oligonucleotide ;
(3) A step of collecting the released single-stranded identifier oligonucleotide from the position of the tissue sample excited in the step (2) ;
(4) In the step of amplifying the collected single- stranded identifier oligonucleotide, amplifying the single-stranded identifier oligonucleotide collected here includes carrying out PCR, and here. Performing the PCR comprises a first amplification primer and a second amplification primer.
Here, the first amplification primer is:
First flow cell binding site;
A first nucleic acid sequence that identifies the particular location of the tissue sample from which the identifier oligonucleotide has been released;
Nucleic acid sequence complementary to the second amplification primer binding site
Including;
Here, the second amplification primer is:
Second flow cell binding site;
A second nucleic acid sequence that identifies the particular position of the sample from which the identifier oligonucleotide has been released; and
Nucleic acid sequence complementary to the first amplification primer binding site
And (5) the single-stranded identifier oligonucleotide released by sequencing the amplification product produced in step (4), thereby identifying the at least one of said in a tissue sample. A method comprising the step of spatially detecting the abundance of the at least one target sample in a cell.

The method of claim 1, wherein step (1) comprises contacting the at least one target sample in at least one cell in a tissue sample with at least five probes.

The method of claim 1 or 2 , wherein at least one of the flow cell binding sites comprises a flow cell adapter sequence suitable for sequencing.

The P5 flow cell adapter sequence comprises the P5 flow cell adapter sequence, wherein the P5 flow cell adapter sequence comprises the sequence set forth in SEQ ID NO: 3, according to any one of claims 1 to 3 . Method.

The method according to any one of claims 1 to 4 , wherein at least one of the flow cell binding sites comprises a P7 flow cell adapter sequence, wherein the P7 flow cell adapter sequence comprises the sequence set forth in SEQ ID NO: 4.

The method of any one of claims 1-5, wherein at least one of the flow cell binding sites comprises 15-40 nucleotides.

The method of claim 6, wherein at least one of the flow cell binding sites comprises 29 nucleotides.

The method of claim 6, wherein at least one of the flow cell binding sites comprises 24 nucleotides.

The method according to any one of claims 1 to 8, wherein the unique nucleic acid sequence that identifies the target sample bound to the target binding domain comprises 5 nucleotides to 25 nucleotides.

The method according to any one of claims 1 to 9, wherein the nucleic acid sequence that identifies the target sample bound to the target binding domain comprises 12 nucleotides.

The method according to any one of claims 1 to 10, wherein at least one of the amplification primer binding sites comprises 10 to 40 nucleotides.

11. The method of claim 11, wherein at least one of the amplification primer binding sites comprises 34 nucleotides.

11. The method of claim 11, wherein at least one of the amplification primer binding sites comprises 33 nucleotides.

The method according to any one of claims 1 to 13 , wherein at least one of the amplification primer binding sites comprises an i7 sequence, wherein the i7 sequence comprises the sequence set forth in SEQ ID NO: 1.

The method according to any one of claims 1 to 14 , wherein at least one of the amplification primer binding sites comprises an i5 sequence, wherein the i5 sequence comprises the sequence set forth in SEQ ID NO: 2.

The expression according to any one of claims 1 to 15, wherein at least one of the nucleic acid sequences identifying the particular position of the tissue sample from which the identifier oligonucleotide has been released comprises 1 to 20 nucleotides. the method of.

16. The method of claim 16, wherein at least one of the nucleic acid sequences identifying the particular location of the tissue sample from which the identifier oligonucleotide has been released comprises 8 nucleotides.

The method according to any one of claims 1 to 17, wherein the nucleic acid sequence containing a unique molecular identifier comprises 5 to 20 nucleotides.

18. The method of claim 18, wherein the nucleic acid sequence comprising a unique molecular identifier comprises 14 nucleotides.

The method according to any one of claims 1 to 19, wherein the target binding domain comprises 10 nucleotides to 70 nucleotides.

The method of claim 20, wherein the target binding domain comprises 35 to 50 nucleotides.