In situ detection of barcodes

Abstract

The current disclosure provides a method suitable for detecting barcode sequences in a biological sample in situ. Advantageously, the method can be effectively used to detect barcode sequences in formalin-fixed paraffin-embedded (FFPE) tissue samples.

Description

This disclosure provides a method suitable for in-situ detection of barcode sequences in biological samples. Advantageously, the method can be effectively used to detect barcode sequences in formalin-fixed paraffin-embedded (FFPE) tissue samples. Abstract

Claims

CLAIMSWhat is claimed is:Claim 1. A method for detecting barcode sequences in a biological sample in situ, said method comprising the steps of: c) processing a biological sample comprising a plurality of cells, wherein each cell of the plurality of cells comprises an oligonucleotide having a single barcode sequence of 20 to 60 nucleotides, so that a plurality of barcode molecules, each of which comprises the barcode sequence, are amplified in the plurality of cells of the biological sample in situ, and d) detecting the plurality of barcode molecules so that the barcode sequences are located in situ.Claim 2. The method of claim 1, wherein the biological sample is a mammalian tumor tissue.Claim 3. The method of claim 1, wherein the oligonucleotide further comprises a CRISPR gRNA.Claim 4. The method of claim 1, wherein the barcode sequence is operably linked to a promoter.Claim 5. The method of claim 4, wherein the promoter is a bacteriophage T3 promoter, a bacteriophage T7 promoter, or a bacteriophage SP6 promoter.Claim 6. The method of claim 1, wherein the biological sample is obtained by iv. preparing a pooled viral vector library comprising the oligonucleotides; v. transforming the pooled viral vector library to a plurality of cells; and vi. obtaining a biological sample comprising the plurality of transformed cells.Claim 7. The method of claim 6, wherein the pooled viral vector library is delivered to a mammal so that a plurality of mammalian cells are transformed with the pooled viral vector library, and wherein the biological sample is obtained from the mammal.Claim 8. The method of claim 6, wherein the pooled viral vector library is transformed into a plurality of cultured mammalian cells, wherein the transformed cultured mammalian cells are transferred to a mammal, and wherein the biological sample is obtained from the mammal.Claim 9. The method of claim 6, wherein the pooled viral vector library is constructed from a viral vector wherein the viral vector is a retrovirus vector, a lentivirus vector, a gammaretroviral vector, an adenovirus vector, or an adeno-associated virus (AAV) vector.Claim 10. The method of claim 9, wherein the retrovirus vector, the lentivirus vector, or the gammaretroviral vector is a non -integrating viral vector or a self-inactivating retroviral vector.Claim 11. The method of claim 6, wherein the viral vector further comprises a cDNA or a CRISPR gRNA.Claim 12. The method of claim 1, wherein the single barcode sequence comprises 20 to 40 nucleotides.Claim 13. The method of claim 12, wherein the barcode sequence iii. has a GC content of about 30% to about 70%; and iv. has a Tm in a range of about 42°C to about 75°C.Claim 14. The method of claim 13, wherein the Tm is about 60°C or greater.Claim 15. The method of claim 1, wherein detecting the plurality of barcode molecules in situ is achieved by in situ nucleic acid sequencing or fluorescence in situ hybridization.Claim 16. The method of claim 1, wherein step a) further comprises: v. transcribing the barcode sequence to produce an RNA molecule;vi. annealing the RNA molecule to a padlock probe, wherein the padlock probe comprises an imaging barcode flanked by two RNA-binding sequences, and wherein each RNA-binding sequence is complementary to a section of the barcode sequence; vii. ligating the padlock probe to form a single chain circular DNA molecule; and viii. performing rolling circle replication of the single chain circular DNA molecule to amplify the barcode molecule.Claim 17. The method of claim 16, wherein detecting the plurality of barcode molecules is achieved by performing (i) in situ sequencing of the barcode molecule, or (ii) in situ hybridization against the barcode molecule, a branched DNA bound to the barcode molecule, or the imaging barcode.Claim 18. The method of claim 17, wherein the in situ hybridization is fluorescence in situ hybridization (FISH).Claim 19. The method of claim 16, wherein the padlock probe comprises a spacer oligonucleotide placed between the imaging barcode and at least one of the two RNA-binding sequences.Claim 20. The method of claim 16, wherein the imaging barcode has a length of 28 bases, and wherein each of the two RNA-binding sequences has a length of 7-20 bases.Claim 21. The method of claim 16, wherein each of two RNA-binding sequences iv. has a GC content of about 30% to about 70%; v. contains only guanosine (G), cytosine (C), and adenine (A); and vi. has a Tm in a range of about 42°C to about 65°C.Claim 22. The method of claim 16, wherein the padlock probe is designed so that an interaction between the padlock probe and the barcode sequence has a Levenshtein distance greater than 4 and has a Tm greater than 42°C.Claim 23. The method of claim 2, further comprising a step of fixing the mammalian tumor tissue before the step a).Claim 24. The method of claim 23, wherein the fixed mammalian tumor tissue is a formalin-fixed paraffin-embedded (FFPE) tissue.

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