Systems and methods for barcoding nucleic acids
The present invention generally relates to microfluidics and labeled nucleic acids. For example, certain aspects are generally directed to systems and methods for labeling nucleic acids within microfluidic droplets. In one set of embodiments, the nucleic acids may include “barcodes” or unique sequences that can be used to distinguish nucleic acids in a droplet from those in another droplet, for instance, even after the nucleic acids are pooled together. In some cases, the unique sequences may be incorporated into individual droplets using particles and attached to nucleic acids contained within the droplets (for example, released from lysed cells). In some cases, the barcodes may be used to distinguish tens, hundreds, or even thousands of nucleic acids, e.g., arising from different cells or other sources.
1. A method, comprising:
i) providing a plurality of at least 10,000 microfluidic droplets containing cells, at least about 90% of the plurality of droplets containing only one cell or no cell, and an oligonucleotide tag attached to a particle;
ii) lysing the cells within the plurality of microfluidic droplets to release nucleic acid from the cells;
iii) bonding the released nucleic acid to the oligonucleotide tags without first amplifying the released nucleic acid, and
iv) releasing at least some of the oligonucleotide tags from the particles by applying light,
a) wherein the oligonucleotide tag uniquely identifies the released nucleic acids from nucleic acids released from other cells by a barcode sequence and the tag additionally comprises a primer sequence; and
b) wherein for at least about 90% of the droplets, the oligonucleotide tag within the droplet is distinguishable from oligonucleotide tags within other droplets of the plurality of droplets, and
wherein the plurality of droplets is provided by use of a microfluidic device with four inlets for i) oligonucleotide tags, ii) cells, iii) nucleic acid amplification reagents and iv) carrier oil, and one outlet port for droplet collection.
2. A method according to claim 1 , wherein the oligonucleotide tags comprises a poly-T sequence.
3. A method according to claim 1 , wherein the nucleic acid is an RNA.
4. A method according to claim 1 , wherein the RNA is reverse transcribed within the droplet.
5. A method according to claim 4 , wherein after the reverse transcription a barcoded cDNA is created.
6. A method according to claim 5 , wherein the cDNA is subsequently amplified.
7. A method according to claim 1 , wherein the oligonucleotide tags is attached to a bead or particle in the droplet.
8. A method according to claim 1 , wherein the plurality of microfluidic droplets have a volume of less than about 10 nl but more than 3 nl.
9. A method according to claim 1 , wherein the bead or particle is a hydrogel bead or particle, polymeric bead or particle, a microparticle or comprises polyacrylamide, agarose, polystyrene, poly-N-isopropylacrylamide, and/or wherein the bead or particle is magnetic.
10. A method according to claim 1 , wherein the oligonucleotide tag is covalently attached via an acrylic phosphoramidite linkage or an amino linkage.
11. A method according to claim 1 , wherein at least some of the oligonucleotide tag comprises a cleavable linker.
12. The method of claim 11 , wherein the linker is photocleavable.
13. The method of claim 11 , wherein the linker is chemically cleavable.
14. A method according to claim 1 , further comprising sequencing the amplified nucleic acid.