Methods of single-cell barcoding and sequencing

• US 10,392,614 B2
• Filed: 09/29/2017
• Issued: 08/27/2019
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Abvitro LLC (Bristol-Myers Squibb Company)
• Original Assignee: Abvitro LLC (Bristol-Myers Squibb Company)
• Inventors: Vigneault, Francois, Briggs, Adrian Wrangham
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/721,584
• Publication Number:
  US 20180127743A1
• Time in Patent Office: 697 Days
• Field of Search: None
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/54 F(ab')2
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1041 Ribosome/Polysome display, e.g. SPERT, ARM
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays
• CPC Class Codes: G01N 33/6854 : Immunoglobulins

Abstract

Provided herein are methods for immune repertoire sequencing and single cell barcoding. In some aspects, such methods may comprise producing a plurality of cDNAs from a plurality of polynucleotides, wherein at least one polynucleotide encodes a heavy and/or light chain, from a plurality of immune cells from a biological sample. Producing a plurality of cDNAs may comprise reverse transcription and template switching, such as to form a plurality of uniquely barcoded cDNAs. Methods described herein further comprise amplifying the plurality of uniquely barcoded cDNAs, thereby forming a library, and sequencing one or more of the sequences of the library.

Claims

1. A method comprising:

producing a plurality of cDNAs from a plurality of polynucleotides, wherein at least one polynucleotide encodes a heavy and/or light chain, from a plurality of immune cells from a biological sample, the producing comprising;

(i) reverse transcribing a sequence complementary to a sequence downstream of a variable region of the at least one polynucleotide encoding a heavy and/or light chain, using;

(A) a first plurality of primers, and

(B) a reverse transcriptase comprising a non-template terminal transferase activity, wherein 3 or more identical non-template nucleotides are added to a 3′

end of the cDNAs, and

(ii) template switching, using a plurality of template switch polynucleotides, each comprising;

(A) a unique barcode,

(B) a first primer binding site 5′

to the unique barcode, and

(C) a 3′

end region complementary to the 3 or more identical non-template nucleotides,

thereby forming a plurality of uniquely barcoded cDNAs;

wherein the reverse transcribing and the template switching are performed in an emulsion;

wherein the method optionally further comprises;

(a) amplifying the plurality of uniquely barcoded cDNAs, thereby forming a library of uniquely barcoded sequences comprising a variable region of the heavy (V_H) or light (V_L) chain polynucleotides;

and wherein the method further optionally comprises;

(b) sequencing one or more of the sequences of the library.

2. The method of claim 1, further comprising amplifying the plurality of uniquely barcoded cDNAs with:

(i) a first primer comprising

(A) a region that binds to a complement of the same region sequence of the cDNAs, and

(B) a 5′

second primer binding site; and

(ii) a second primer comprising a region that binds to the complement of the first primer binding site.

3. The method of claim 2, wherein the plurality of uniquely barcoded cDNAs are further amplified with:

(i) a third primer comprising a 5′

first universal primer binding site; and

(ii) a fourth primer comprising a 5′

second universal primer binding site.

4. The method of claim 3, further comprising sequencing one or more of the sequences of the library with a primer set comprising a first primer complementary to the first universal primer binding site and a second primer complementary to the second universal primer binding site.

5. The method of claim 3, wherein the third primer comprises a sample barcode sequence 3′

to the first universal primer binding site or the fourth primer comprises a sample barcode sequence 3′

to the second universal primer binding site.

6. The method of claim 4, wherein the sequencing comprises high-throughput sequencing.

7. The method of claim 2, wherein the library comprises a plurality of V_H or V_L sequences representing an immune state of the biological sample.

8. The method of claim 7, further comprising sequencing one or more of the sequences of the library and determining a variance of the V_H or V_L sequences from those of a germ line sequence.

9. The method of claim 7, further comprising sequencing one or more of the sequences of the library and determining:

(i) a total number of unique heavy chain polynucleotides,

(ii) a total number of unique light chain polynucleotides,

(iii) a frequency of a heavy chain polynucleotide, or

(iv) a frequency of a light chain polynucleotide.

10. The method of claim 9, further comprising selecting a therapeutic antibody based on

(i) a total number of unique heavy or light chain polynucleotides,

(ii) a frequency of a heavy or light chain polynucleotide, or

(iii) a variance of a V_H or V_L sequence from that of a germ line sequence.

11. The method of claim 4, further comprising producing a consensus sequence from sequences in the library comprising a same unique barcode.

12. The method of claim 4, wherein sequencing errors are minimized, eliminated, or less than 0.01%.

13. The method of claim 2, wherein amplification bias is minimized, eliminated, or less than 0.01%.

14. The method of claim 4, further comprising comparing the sequences of the library from the biological sample to sequences of a library from a biological sample taken from a same subject at a different time point.

15. The method of claim 1, wherein the biological sample is from a subject with a condition or disease.

16. The method of claim 1, wherein the sequence downstream of the variable region of the polynucleotides encoding a heavy and/or light chain comprises a polyA sequence.

17. The method of claim 1, wherein a region of the first plurality of primers that is complementary to sequence downstream of the variable region of the polynucleotides encoding a heavy and/or light chain comprises a poly-T sequence.

18. The method of claim 1, wherein the sequence downstream of the variable region of the polynucleotides encoding a heavy and/or light chain comprises a heavy or light chain constant region sequence.

19. The method of claim 4, wherein the biological sample is from a subject with a condition or disease and wherein the method further comprises comparing the sequences of the library from the biological sample to sequences of a library from a biological sample taken from a subject without the condition or disease.