ACCURATE MOLECULAR BARCODING

• US 20170314067A1
• Filed: 04/28/2017
• Published: 11/02/2017
• Est. Priority Date: 05/02/2016
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Shum, Eleen, Fu, Glenn
• Granted Patent: US 10,822,643 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

In accordance with some embodiments herein, compositions and methods for accurate barcoding of nucleic acids are described. The compositions and methods can involve a plurality of unique oligonucleotide species comprising unique molecule barcodes. In some embodiments, the molecule barcodes can have a relatively low G content, and can exhibit reduced bias in amplification and analysis.

Claims

1. A composition comprising at least 1000 unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region comprising a molecule barcode comprising at least 7 nucleotides,

wherein the unique oligonucleotide species comprise different nucleic acid sequences in their barcode regions, and

wherein at least one of:

(a) the composition consists essentially of unique oligonucleotide species wherein each molecular barcode has a G content of less than 50%;

or

(b) the molecule barcodes of all of the unique oligonucleotide species in the composition collectively have a G content of no more than 12.5%.

2. The composition of claim 1, wherein the barcode region further comprises a sample barcode comprising at least 3 nucleotides.

3. The composition of claim 1, wherein each unique oligonucleotide species further comprises a uniform region 3′

of the barcode region, the uniform region comprising a target-specific region 3′

of the barcode region, the target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid.

4. The composition claim 1, wherein the composition consists essentially of unique oligonucleotide species wherein each molecular barcode has a G content of less than 50%.

5. The composition of claim 1, wherein the molecule barcodes of all of the unique oligonucleotide species in the composition collectively have a G content of no more than 12.5%.

6. The composition of claim 1, wherein the unique oligonucleotide species are disposed in at least two spatially isolated pools, each pool comprising at least 100 unique oligonucleotides of the unique oligonucleotide species,

wherein unique oligonucleotides in the same pool comprise the same sample barcode sequence, and

wherein different unique oligonucleotides of the same pool comprise a different molecule barcode sequences.

7. The composition of claim 6, wherein the unique oligonucleotide species of each pool are immobilized on a substrate, so that the sample barcodes but not the molecule barcodes are the same for the oligonucleotide species immobilized on each substrate.

8. The composition of claim 1, wherein for at least 95% of the unique oligonucleotide species, any G in the molecule barcode is not adjacent to another G.

9. The composition of claim 1, wherein at least 95% of the molecule barcodes of the unique oligonucleotide species comprise the sequence HNHNHNHN, wherein each “

H”

is any one of A, C, or T, and wherein each “

N”

is any one of A, G, C, or T.

10. The composition of claim 1, wherein each of the unique oligonucleotide species comprises a spacer 3′

of the barcode region and 5′

of the target specific region, said spacer comprising the sequence HHHHHHHH, wherein each “

H”

is any one of A, C, or T.

11. The composition of claim 1, wherein each oligonucleotide species has a length of 24-140 nucleotides.

12. The composition of claim 1, wherein the composition comprises at least two oligonucleotides of the same unique oligonucleotide species.

13. The composition of claim 3, wherein the uniform region comprises a target-specific region comprising a sequence flanking an immune cell receptor or immunoglobulin variable region coding sequence.

14. The composition of claim 13, wherein the immune cell receptor variable region coding sequence is selected from the group consisting of:

a T cell receptor variable region coding sequence, a B cell receptor variable region coding sequence, and a combination thereof.

20. A kit comprising the composition of claim 13, the kit further comprising a primer configured to hybridize on an opposite side of the variable region as the target-specific region, and to hybridize to a complementary strand to a strand hybridized by the target-specific region, and is thereby configured to amplify the variable region in conjunction with the target-specific region.

15. A method of specifically barcoding a plurality of nucleic acids from two or more samples, each sample comprising nucleic acids:

contacting each sample with a pool comprising at least 100 unique oligonucleotide species, wherein each sample is contacted in spatial isolation from the other samples,

each unique oligonucleotide species comprising a barcode region comprising a molecule barcode comprising at least 7 nucleotides,

wherein the unique polynucleotide species of each pool comprise the same sample barcode, and comprise different molecule barcodes, and

wherein at least one of;

(a) the unique oligonucleotide species contacted with the sample consist essentially of unique oligonucleotide species wherein each molecular barcode has a G content of less than 50%;

or

(b) the molecule barcodes of all of the unique oligonucleotide species collectively have a G content of no more than 12.5%;

hybridizing target-specific regions of at least some oligonucleotides of the unique oligonucleotide species to at least some of the nucleic acids of the sample; and

extending the hybridized oligonucleotides, thereby producing strands comprising an oligonucleotide of the unique oligonucleotide species and a sequence complementary to the target, wherein for each sample, the strands comprise the same sample barcode and different molecule barcodes, and wherein for different samples, the molecule barcodes are different.

16. The method of claim 15, wherein the barcode region further comprises a sample barcode comprising at least 3 nucleotides.

17. The method of claim 15, wherein each unique oligonucleotide species further comprises a uniform region 3′

of the barcode region, the uniform region comprising a target-specific region 3′

of the barcode region, the target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid.

18. The method of claim 15, further comprising ascertaining nucleic acid sequences of the strands comprising the oligonucleotides of the unique oligonucleotide species and the sequence complementary to the target.

19. The method of claim 15, wherein the molecule barcodes of the unique oligonucleotide species collectively have a G content of less than 12.5%.