Detecting polynucleotides with barcoded oligonucleotide probes

Patent metadata

Field	Value
Publication number	US-2025027143-A1
Application number	US-202418889834-A
Country	US
Kind code	A1
Filing date	Sep 19, 2024
Priority date	Dec 31, 2019
Publication date	Jan 23, 2025
Grant date	—

Abstract

Provided herein are methods of detecting polynucleotides with a set of barcoded oligonucleotide probes.

First claim

What is claimed:

1 . A method of sequencing, said method comprising:
contacting a sample with a set of barcoded probes, wherein the set of barcoded probes comprises a first oligonucleotide probe comprising a first target hybridization sequence and a first barcode sequence; a second oligonucleotide probe comprising a second target hybridization sequence and a second barcode sequence; and a third oligonucleotide probe comprising a third target hybridization sequence and a third barcode sequence; wherein each target hybridization sequence specifically binds to a different sequence of a target polynucleotide; binding the set of barcoded probes to the sample, wherein the sample comprises one or more target polynucleotides; sequencing each barcode sequence of the set of barcoded probes; and identifying the polynucleotide in the sample based on the sequenced barcode sequences of the set of barcoded probes.

2 . The method of claim 1 , wherein sequencing generates a sequencing read for each barcode sequence.

3 . The method of claim 1 , wherein prior to sequencing the barcode sequences, the method comprises amplifying the barcode sequences and forming amplification products, and sequencing the amplification products.

4 . The method of claim 2 , wherein sequencing produces a plurality of sequencing reads, the method further comprises computationally grouping the plurality of sequencing reads based on the co-occurrence of the barcode sequences in the set of barcoded probes.

5 . The method of claim 2 , wherein sequencing comprises sequencing-by synthesis (SBS), pyrosequencing, sequencing by ligation (SBL), or sequencing by hybridization (SBH).

6 . The method of claim 1 , wherein each barcode sequence comprises about 5 to about 20 nucleotides.

7 . The method of claim 1 , wherein each barcode sequence comprises 10 to 16 nucleotides.

8 . The method of claim 1 , wherein each barcode sequence differs from every other barcode sequence by at least two nucleotide positions.

9 . The method of claim 1 , wherein the target polynucleotide comprises a cancer-associated gene, T cell receptor (TCR) gene, or a B cell receptor (BCR) gene.

10 . The method of claim 9 , wherein the cancer-associated gene is an ALK gene, BCL2 gene, HER2 gene, MYC gene, KRAS gene, MMP-10 gene, VEGF-D gene, PlGF gene, or P-Cadherin gene.

11 . The method of claim 9 , wherein the TCR gene is a TCR alpha (TCRA) chain, a TCR beta (TCRB) chain, or a TCR delta (TCRD) chain.

12 . The method of claim 9 , wherein the TCR gene is a TCRA gene or a TCRB gene.

13 . The method of claim 1 , wherein the target polynucleotide is messenger RNA (mRNA).

14 . The method of claim 13 , wherein the sample comprises a plurality of mRNA molecules.

15 . The method of claim 1 , wherein the sample comprises cells or tissues.

16 . The method of claim 15 , wherein the cells are cancer cells.

17 . The method of claim 1 , further comprising:
contacting the sample with a second set of barcoded probes, wherein the second set of barcoded probes comprises a fourth oligonucleotide probe comprising a fourth target hybridization sequence and a fourth barcode sequence; a fifth oligonucleotide probe comprising a fifth target hybridization sequence and a fifth barcode sequence; and a sixth oligonucleotide probe comprising a sixth target hybridization sequence and a sixth barcode sequence; wherein each target hybridization sequence specifically binds to a different sequence of a second target polynucleotide; binding the second set of barcoded probes to the sample; sequencing each barcode sequence of the second set of barcoded probes; and identifying the polynucleotide in the sample based on the sequenced barcode sequences of the second set of barcoded probes.

18 . The method of claim 17 , wherein the second target polynucleotide comprises an ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, or VHL gene.

19 . The method of claim 1 , wherein each oligonucleotide probe of the set comprises a sequence common to all of the oligonucleotide probes in the set.

20 . The method of claim 1 , wherein the set of barcoded probes comprises 6 to 12 oligonucleotide probes comprising a barcode sequence.

Assignees

• Singular Genomics Systems Inc

Inventors

• Glezer Eli N
• Fabani Martin Maria
• Shultzaberger Ryan
• Sridhar Bharat
• Stengel Gudrun
• Wei Christopher Jen-Yue

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2525/161

  incorporating target specific and non-target specific sites · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title