Polynucleotide barcode generation
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing. In some cases, this disclosure provides methods for the generation of polynucleotide barcode libraries, and for the attachment of such polynucleotides to target polynucleotides.
1. A method of synthesizing a library of polynucleotides comprising barcode sequences, said method comprising:
synthesizing a plurality of polynucleotides comprising barcode sequences;
separating said polynucleotides into a plurality of partitions, thereby generating partitioned polynucleotides, wherein each partition comprising a polynucleotide comprises multiple copies of the same polynucleotide comprising a barcode sequence prior to amplifying said partitioned polynucleotides;
amplifying said partitioned polynucleotides, thereby generating a library of amplified polynucleotides, comprising at least about 100,000 different barcode sequences;
wherein said amplified polynucleotides are attached to a bead; and
isolating partitions comprising amplified polynucleotides.
2. The method of claim 1 , wherein said synthesizing comprises including a mixture of adenine, thymine, guanine, and cytosine in a coupling reaction.
3. The method of claim 1 , wherein said separating comprises performing a limiting dilution, thereby generating diluted polynucleotides.
4. The method of claim 3 , wherein said separating further comprises partitioning said diluted polynucleotides.
5. The method of claim 1 , wherein said amplifying is performed by a method selected from the group consisting of polymerase chain reaction, asymmetric polymerase chain reaction, emulsion PCR (ePCR), ePCR including the use of a bead, ePCR including the use of a hydrogel, multiple annealing and looping-based amplification cycles (MALBAC), single primer isothermal amplification, and combinations thereof.
6. The method of claim 1 , wherein said isolating is performed by flow-assisted sorting.
7. The method of claim 1 , wherein a hairpin structure is formed from a polynucleotide selected from the group consisting of said polynucleotides comprising barcode sequences and said amplified polynucleotides.
8. The method of claim 7 , further comprising cutting said hairpin structure within an unannealed region.
9. The method of claim 1 , further comprising annealing said amplified polynucleotides with a partially complementary sequence.
10. The method of claim 9 , wherein said partially complementary sequence comprises a barcode sequence.
11. The method of claim 1 , further comprising attaching at least one of said amplified polynucleotides to a target sequence.
12. The method of claim 11 , further comprising fragmenting said target sequence prior to said attaching step.
13. The method of claim 12 , wherein fragmenting said target sequence is by a method selected from the group consisting of mechanical shear and treatment with an enzyme.
14. The method of claim 13 , wherein said mechanical shear is induced by ultrasound.
15. The method of claim 13 , wherein said enzyme is selected from the group consisting of a restriction enzyme, a fragmentase, and a transposase.
16. The method of claim 11 , wherein said attaching is performed by a method selected from the group consisting of ligation and amplification.
17. The method of claim 16 , wherein said amplification is a MALBAC amplification performed with MALBAC primers, thereby generating a MALBAC amplification product.
18. The method of claim 17 , wherein said MALBAC primers comprise said amplified polynucleotides.
19. The method of claim 1 , wherein following the separating step, the plurality of partitions comprises, on average, about 0.5 polynucleotides comprising barcode sequences per partition.
20. The method of claim 1 , wherein following the separating step, the plurality of partitions comprises, on average, about 0.1 polynucleotides comprising barcode sequences per partition.
21. The method of claim 1 , wherein said partitions are selected from the group consisting of droplets, capsules, and wells.
22. The method of claim 1 , wherein said polynucleotides comprising barcode sequences further comprise a sequence selected from the group consisting of an immobilization sequence, an annealing sequence for a sequencing primer, and a sequence compatible for ligation with a target polynucleotide.
23. The method of claim 1 , wherein said library comprises at least about 500,000 different barcode sequences.
24. The method of claim 1 , wherein said library comprises at least about 1,000,000 different barcode sequences.
25. The method of claim 1 , wherein said library comprises at least about 2,500,000 different barcode sequences.
26. The method of claim 1 , wherein said library comprises at least about 5,000,000 different barcode sequences.
27. The method of claim 1 , wherein said library comprises at least about 10,000,000 different barcode sequences.
28. The method of claim 1 , wherein said library comprises at least about 25,000,000 different barcode sequences.
29. The method of claim 1 , wherein said library comprises at least about 50,000,000 different barcode sequences.
30. The method of claim 1 , wherein said library comprises at least about 100,000,000 different barcode sequences.
31. A method of synthesizing a library of polynucleotides comprising barcode sequences, said method comprising:
synthesizing a plurality of polynucleotides comprising barcode sequences;
separating said polynucleotides into a plurality of partitions, thereby generating partitioned polynucleotides;
amplifying said partitioned polynucleotides, using an RNA primer thereby generating amplified polynucleotides;
optionally exposing said amplified polynucleotides to RNase H; and
isolating partitions comprising amplified polynucleotides.
32. A method of synthesizing a library of polynucleotides comprising barcode sequences, said method comprising:
synthesizing a plurality of MALBAC primer polynucleotides comprising barcode sequences;
separating said polynucleotides into a plurality of partitions, thereby generating partitioned polynucleotides;
amplifying said partitioned polynucleotides, thereby generating amplified polynucleotides; and
isolating partitions comprising amplified polynucleotides.
33. The method of claim 31 , wherein said library comprises at least about 500,000 different barcode sequences.
34. The method of claim 31 , wherein said library comprises at least about 1,000,000 different barcode sequences.
35. The method of claim 31 , wherein said library comprises at least about 2,500,000 different barcode sequences.
36. The method of claim 31 , wherein said library comprises at least about 5,000,000 different barcode sequences.
37. The method of claim 32 , wherein said library comprises at least about 500,000 different barcode sequences.
38. The method of claim 32 , wherein said library comprises at least about 1,000,000 different barcode sequences.
39. The method of claim 32 , wherein said library comprises at least about 2,500,000 different barcode sequences.
40. The method of claim 32 , wherein said library comprises at least about 5,000,000 different barcode sequences.