Identification of polynucleotides associated with a sample

Patent metadata

Field	Value
Publication number	US-11098302-B2
Application number	US-201213261763-A
Country	US
Kind code	B2
Filing date	Apr 27, 2012
Priority date	Apr 28, 2011
Publication date	Aug 24, 2021
Grant date	Aug 24, 2021

Abstract

Disclosed herein are compositions and methods for sequencing, analyzing, and utilizing samples such as single samples. Also disclosed herein are compositions and methods for matching together two or more sequences from a sample. Also disclosed herein are compositions and methods for expressing and screening molecules of interest.

First claim

The invention claimed is:

1. A polynucleotide library comprising a plurality of compositions, wherein:
each composition comprises:
(i) cDNA molecules derived from a single plasmablast that encode a cognate pair of immunoglobulin heavy and light chain variable regions; and
(ii) a sample identification region attached to the cDNA molecules,
wherein the cDNA molecules derived from the single plasmablast that encode a cognate pair of immunoglobulin heavy and light chain variable regions are coupled to an identical sample identification region and the nucleotide sequence of the sample identification region is unique to the cDNA molecules derived from the single plasmablast and is distinct from the nucleotide sequence of the sample identification regions of the other compositions present in the library, wherein the library comprises an unbiased representation of the antibody repertoire of expressed antibody heavy and light chain variable regions.

2. The library of claim 1 , wherein the cDNA molecules are attached to the sample identification region by an adapter region.

3. The library of claim 2 , wherein the adapter region comprises at least one G nucleotide at its 3′ end and the first strand cDNA molecules comprise a complementary nucleotide C at the 3′ end.

4. The library of claim 1 , wherein each composition further comprises a universal primer region attached to the sample identification region, and wherein the sequence of the universal primer region is substantially identical on each polynucleotide in the library.

5. A polynucleotide library comprising a plurality of compositions, wherein the library comprises cDNAs encoding immunoglobulin heavy and light chain variable regions from the same clonal family and wherein:
each composition comprises:
a plurality of cDNA molecules derived from a single B cell that encode a cognate pair of immunoglobulin heavy and light chain variable regions, each cDNA molecule comprising a sample identification-adapter region comprising a sample identification region coupled to an adapter region, wherein the adapter region comprises the nucleotide G at the 3′ end, where the G nucleotide is complementary to a C nucleotide at the 3′ end of the first strand of the cDNA molecule,
wherein the cDNA molecules derived from the single B cell that encode a cognate pair of immunoglobulin heavy and light chain variable regions are coupled to an identical sample identification region and the nucleotide sequence of the sample identification region is unique to the plurality of cDNA molecules derived from the single B cell and is distinct from the nucleotide sequence of the sample identification region of the other sample identification-adapter regions of other compositions in the library, and
the sample identification-adapter region is covalently attached to cDNA molecules in the composition.

6. The library of claim 5 , wherein each composition further comprises a universal primer region attached to the sample identification region, and wherein the sequence of the universal primer region is substantially identical on each polynucleotide in the library.

7. The library of claim 5 , wherein the single B cell is a single plasmablast.

8. The library of claim 1 , wherein the cDNA molecule and the sample identification region are incorporated into the same DNA strand.

9. The library of claim 1 , wherein at least two of the immunoglobulin heavy chain variable regions or at least two of the immunoglobulin light chain variable regions share at least 80-99% sequence identity to each other.

10. The library of claim 1 , wherein each of the immunoglobulin heavy chain variable regions or each of the immunoglobulin light chain variable regions exhibit at least 80-99% sequence identity to each other.

11. The library of claim 1 , wherein the cDNA molecules in each container are not physically linked to each other.

12. The library of claim 1 , wherein the cDNA molecules encode immunoglobulin heavy chain variable regions and immunoglobulin light chain variable regions comprising a 5′ untranslated region.

13. The library of claim 1 , wherein the cDNA molecules that encode the immunoglobulin heavy chain variable regions comprise a 5′ untranslated region and about 700 bp of contiguous sequence and the cDNA molecules that encode the immunoglobulin light chain variable regions comprise a 5′ untranslated region and about 600 bp of contiguous sequence.

14. The library of claim 1 , wherein the single plasmablast is a CD19 + CD20 − CD27 + CD38 hi plasmablast.

15. The library of claim 1 , wherein the immunoglobulin heavy chain variable region comprises an IgG, IgM, IgD, IgE, or IgA immunoglobulin sequence; a human IgG1, IgG2, IgG3, or IgG4 immunoglobulin sequence; or a mouse IgG1, IgG2a, IgG2b, or IgG3 immunoglobulin sequence.

16. The library of claim 1 , wherein each composition further comprises sequences encoding heavy chain immunoglobulin constant regions alpha, delta, gamma, epsilon, or mu attached to the cDNA molecules.

17. The library of claim 5 , wherein the immunoglobulin heavy chain variable region comprises an IgG, IgM, IgD, IgE, or IgA immunoglobulin sequence; a human IgG1, IgG2, IgG3, or IgG4 immunoglobulin sequence; or a mouse IgG1, IgG2a, IgG2b, or IgG3 immunoglobulin sequence.

18. The library of claim 5 , wherein each composition further comprises sequences encoding heavy chain immunoglobulin constant regions alpha, delta, gamma, epsilon, or mu attached to the cDNA molecules.

19. The library of claim 4 , wherein the 3′ end of the universal primer region is coupled to the 5′ end of the sample identification region, and the 3′ end of the sample identification region is coupled to the 5′ end of an adapter region, and the cDNA molecules are coupled to the 3′ end of the adapter region.

20. A library comprising a plurality of polynucleotide compositions, wherein each composition is present in a separate container;
each composition comprises:
(i) cDNA molecules derived from a single B cell that encode a cognate pair of immunoglobulin heavy and light chain variable regions; and
(ii) a sample identification region attached to the cDNA molecules,
wherein the cDNA molecules derived from the single B cell that encode a cognate pair of immunoglobulin heavy and light chain variable regions are coupled to an identical sample identification region and the nucleotide sequence of the sample identification region is unique to the cDNA molecules derived from the single B cell and is distinct from the nucleotide sequence of the sample identification regions of the other compositions present in each separate container in the library, wherein the library comprises an unbiased representation of the antibody repertoire of expressed antibody heavy and light chain variable regions.

21. The library of claim 1 , wherein the first strand of the cDNA comprises a 3′ end attached to the sample identification region.

22. The library of claim 2 , wherein the 3′ end of the sample identification region is coupled to the 5′ end of the adapter region, and the cDNA molecules are coupled to the 3′ end of the adapter region, wherein the sample identification region is double-stranded.

23. The library of claim 1 , wherein the 3′ end of the first strand cDNA is coupled to the 3′ end of the sample identification region.

24. The library of claim 1 , wherein the sample identification region is double stranded and is attached to the 5′ end of the double-stranded cDNA.

Assignees

• Robinson William H
• Tan Yann Chong
• Sokolove Jeremy
• Univ Leland Stanford Junior
• The Us Gov As Represented By The Department Of Veterans Affair

Inventors

• Robinson William H
• Tan Yann Chong
• Sokolove Jeremy

Classifications

• C40B40/08 Primary

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C12N15/11 Primary

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title

• G01N2458/10

  Oligonucleotides as tagging agents for labelling antibodies · CPC title