Labeling and sample preparation for sequencing
View Patent ↗The invention provides methods for sequencing and sample preparation for sequencing and amplification.
1. A method for preparing nucleic acid for analysis, the method comprising the steps of:
fragmenting nucleic acid molecules from a sample into long fragments comprising a length in a range of 5 kilobases-100 kilobases and short fragments comprising a length in a range of 100 bases-1000 bases;
attaching an adaptor to each of 3′ and 5′ ends of the short fragments;
hybridizing a plurality of the short fragments from the sample to individual long fragments from the sample to form a plurality of fragment complexes;
compartmentalizing the fragment complexes in droplets with a primer pair species complementary to adaptors attached to the short fragments, wherein each droplet comprises one fragment complex and the primer pair species comprises a barcode; and
amplifying the short fragments from the fragment complex in each droplet using the primer pair species.
2. The method according to claim 1 , wherein removing comprises conducting a gel purification assay that separates the hybridized fragments from the unhybridized short fragments.
3. The method according to claim 1 , wherein prior to the hybridizing step, the method further comprises attaching a first member of a binding pair to the long fragments.
4. The method according to claim 3 , further comprising the step of removing unhybridized short fragments that comprises exposing the fragment complexes and the unhybridized short fragments to a solid support comprising a second member of the binding pair such that the fragment complexes bind to the solid support via the binding pair and are separated from the unhybridized short fragments that do not bind the support.
5. The method according to claim 4 , wherein the binding pair is selected from the group consisting of: biotin/streptavidin; carbohydrate/lectin; and antibody/antigen.
6. The method according to claim 1 , wherein compartmentalizing comprises:
encapsulating the primer pair species into the droplets; and
introducing the fragment complexes to the droplets.
7. The method according to claim 1 , wherein the droplets comprise an aqueous fluid surrounded by an immiscible fluid.
8. The method according to claim 7 , wherein compartmentalizing comprises partitioning the aqueous fluid as it is flowing through a channel with the immiscible fluid.
9. The method according to claim 8 , wherein the immiscible fluid is oil.
10. The method according to claim 9 , wherein the oil is a fluorinated oil.
11. The method according to claim 9 , wherein the oil comprises a surfactant.
12. The method according to claim 11 , wherein the surfactant is a fluorosurfactant.
13. The method according to claim 1 , wherein compartmentalizing comprises merging the droplet that comprises the fragment complex with a droplet comprising the primer pair species.
14. The method according to claim 13 , wherein merging occurs in the presence of an electric field.
15. The method according to claim 1 , wherein compartmentalizing comprises merging a portion of a fluid stream comprising the fragment complex with a droplet comprising the barcoded primers.
16. The method according to claim 15 , wherein merging occurs in the presence of an electric field.
17. The method according to claim 1 , wherein amplifying is by polymerase chain reaction.
18. The method according to claim 1 , further comprising sequencing the amplified short fragments and aligning obtained sequence reads.
19. The method according to claim 1 , wherein the primer pair species further comprises a universal priming site.
20. The method according to claim 19 , wherein the universal priming site is used for sequencing.
21. The method according to claim 1 , wherein the barcode identifies the droplet.