Accurate molecular barcoding
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.
1. A reverse transcription reaction composition comprising;:
mRNA;
a reverse transcriptase; and
at least 1000 unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region and a uniform region, the barcode region comprising a molecule barcode comprising at least 7 nucleotides and having a constraint on G content,
wherein the uniform region is 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,
wherein the unique oligonucleotide species comprise different nucleic acid sequences in their barcode regions,
wherein the constraint on G content comprises:
(a) less than 1% of the unique oligonucleotide species comprising the molecule barcode have having a G content of 50% or more:; and/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%, and
wherein the barcode region further comprises a sample barcode comprising at least 3 nucleotides.
2. The composition of claim 1 , wherein the constraint on G content is less than 1% of the unique oligonucleotide species comprising the molecule barcode have having a G content of 50% or more.
3. The composition of claim 1 , wherein the constraint on G content is the molecule barcodes of all of the unique oligonucleotide species in the composition collectively have a G content of no more than 12.5%.
4. 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.
5. The composition of claim 4 , 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.
6. 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.
7. 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.
8. 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.
9. The composition of claim 1 , wherein each oligonucleotide species has a length of 24-140 nucleotides.
10. The composition of claim 1 , wherein the composition comprises at least two oligonucleotides of the same unique oligonucleotide species.
11. The composition of claim 1 , wherein the uniform region comprises a target-specific region comprising a sequence flanking an immune cell receptor or immunoglobulin variable region coding sequence.
12. The composition of claim 11 , 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.
13. 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 2.5%-10%.
14. The composition of claim 1 , wherein the molecule barcode is 7-9 nucleotides.
15. The composition of claim 1 , comprising at least 6,500 unique oligonucleotide species.
16. A method of specifically barcoding cDNA from two or more samples, each sample comprising nucleic acids, the method comprising:
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 and having a constraint on G content; and
a sample barcode comprising at least 3 nucleotides; and
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,
wherein the unique polynucleotide oligonucleotide species of each pool comprise the same sample barcode, and comprise different molecule barcodes, and wherein the constraint on G content comprises:
(a) less than 1% of the unique oligonucleotide species comprising the molecule barcode have having a G content of 50% or more; and/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, wherein the nucleic acids comprise mRNA; and
extending the hybridized oligonucleotides by reverse transcription, thereby producing extended strands comprising an oligonucleotide of the unique oligonucleotide species and a sequence complementary to the target,
wherein for each sample, the extended strands comprise the same sample barcode and different molecule barcodes, wherein for different samples, the molecule barcodes are different, and
wherein a bias in representation of the unique oligonucleotide species in an amplification product of the extended strands is reduced in comparison to the use of control unique oligonucleotide species comprising control molecule barcodes without the constraint on G content.
17. The method of claim 16 , further comprising ascertaining nucleic acid sequences of the strands comprising the oligonucleotides of the unique oligonucleotide species and the sequence complementary to the target.
18. The method of claim 16 , wherein the constraint on G content is the molecule barcodes of the unique oligonucleotide species collectively having a G content of less than 12.5%.
19. The method of claim 16 , wherein the molecule barcode is 7-9 nucleotides.
20. The method of claim 16 , the pool comprises at least 1000 unique oligonucleotide species.
21. The method of claim 16 , the pool comprises at least 6,500 unique oligonucleotide species.
22. A kit for amplifying barcoded cDNA comprising an immune cell receptor or immunoglobulin variable region coding sequence, comprising;:
a composition comprising: at least 1000 unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region and a uniform region, the barcode region comprising a molecule barcode comprising at least 7 nucleotides and having a constraint on G content,
wherein the uniform region is 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, wherein the unique oligonucleotide species comprise different nucleic acid sequences in their barcode regions,
wherein the constraint on G content comprises:
(a) less than 1% of the unique oligonucleotide species comprising the molecule barcode have having a G content of 50% or more; and/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%, wherein the barcode region further comprises a sample barcode comprising at least 3 nucleotides, and
wherein the uniform region comprises a target-specific region comprising a sequence flanking an immune cell receptor or immunoglobulin variable region coding sequence; and
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.
23. The kit of claim 22 , wherein the molecule barcode is 7-9 nucleotides.
24. The kit of claim 22 , wherein composition comprises at least 6,500 unique oligonucleotide species.
25. A composition comprising:
at least 1000 unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region and a uniform region, the barcode region comprising a molecule barcode comprising at least 7 nucleotides and having a constraint on G content,
wherein the uniform region is 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,
wherein the unique oligonucleotide species comprise different nucleic acid sequences in their barcode regions,
wherein the constraint on G content comprises:
(a) less than 1% of the unique oligonucleotide species comprising the molecule barcode having a G content of 50% or more; and/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%, and
wherein the barcode region further comprises a sample barcode comprising at least 3 nucleotides.
26. The composition of claim 25 , wherein the constraint on G content is less than 1% of the unique oligonucleotide species comprising the molecule barcode having a G content of 50% or more.
27. The composition of claim 25 , wherein the constraint on G content is the molecule barcodes of all of the unique oligonucleotide species in the composition collectively have a G content of no more than 12.5%.
28. The composition of claim 25 , 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.
29. The composition of claim 28 , 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.
30. The composition of claim 25 , wherein for at least 95% of the unique oligonucleotide species, any G in the molecule barcode is not adjacent to another G.
31. The composition of claim 25 , 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.
32. The composition of claim 25 , 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.
33. The composition of claim 25 , wherein each oligonucleotide species has a length of 24-140 nucleotides.
34. The composition of claim 25 , wherein the composition comprises at least two oligonucleotides of the same unique oligonucleotide species.
35. The composition of claim 25 , wherein the uniform region comprises a target-specific region comprising a sequence flanking an immune cell receptor or immunoglobulin variable region coding sequence.
36. The composition of claim 35 , 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.
37. The composition of claim 25 , wherein the molecule barcodes of all of the unique oligonucleotide species in the composition collectively have a G content of 2.5%-10%.
38. The composition of claim 25 , wherein the molecule barcode is 7-9 nucleotides.
39. The composition of claim 25 , comprising at least 6,500 unique oligonucleotide species.
40. The method of claim 16 , wherein the constraint on G content is less than 1% of the unique oligonucleotide species comprising the molecule barcode having a G content of 50% or more.
41. The method of claim 16 , 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.
42. The method of claim 16 , wherein for at least 95% of the unique oligonucleotide species, any G in the molecule barcode is not adjacent to another G.
43. The method of claim 16 , 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.
44. The method of claim 16 , 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.
45. The method of claim 16 , wherein each oligonucleotide species has a length of 24-140 nucleotides.
46. The method of claim 16 , wherein the target-specific region comprises a sequence flanking an immune cell receptor or immunoglobulin variable region coding sequence.
47. The method of claim 46 , 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.
48. The kit of claim 22 , wherein the constraint on G content is the molecule barcodes of the unique oligonucleotide species collectively having a G content of less than 12.5%.
49. The kit of claim 22 , wherein the constraint on G content is less than 1% of the unique oligonucleotide species comprising the molecule barcode having a G content of 50% or more.
50. The kit of claim 22 , 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.
51. The kit of claim 22 , wherein for at least 95% of the unique oligonucleotide species, any G in the molecule barcode is not adjacent to another G.
52. The kit of claim 22 , 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.
53. The kit of claim 22 , 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.
54. The kit of claim 22 , wherein each oligonucleotide species has a length of 24-140 nucleotides.