Barcoding systems and methods for gene sequencing and other applications
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 or other compartments, for instance, arising from a cell. In one set of embodiments, particles may be prepared containing oligonucleotides that can be used to determine target nucleic acids, e.g., attached to the surface of the particles. The oligonucleotides 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 or removed from the droplets. Certain embodiments of the invention are generally directed to systems and methods for attaching additional or arbitrary sequences to the nucleic acids within microfluidic droplets or other compartments, e.g., recognition sequences that can be used to selectively determine or amplify a desired sequence suspected of being present within a droplet. Such systems may be useful, for example, for selective amplification in various applications, such as high-throughput sequencing applications.
1 . A method, comprising:
(a) providing a plurality of droplets comprising particles such that at least about 90% of the droplets contains one particle or no particle, the particles comprising oligonucleotides, the oligonucleotides comprising an adapter sequence that is identical for all of the plurality of droplets and particles, and a first barcode selected from a pre-defined pool of first barcodes, and a second barcode selected from a pre-defined pool of second barcodes, such that substantially each of the particles comprises distinguishable barcode sequences;
(b) encapsulating cells within the plurality of droplets at a density of less than 1 cell/droplet;
(c) lysing at least some of the cells within the plurality of droplets to release nucleic acid from the cells;
(d) attaching a released nucleic acid sequence to the oligonucleotides via covalent bonding after lysing at least some of the cells; and
(e) selectively amplifying the attached released nucleic acid sequence by:
(i) exposing the adapter sequence to a gene-specific inner forward primer comprising a sequence complementary to the adapter sequence and a sequence complementary to the released nucleic acid sequence; and
(ii) exposing the gene-specific inner forward primer and a gene-specific reverse primer to the released nucleic acid sequence, wherein the concentration of the gene-specific inner forward primer is lower than the concentration of the gene-specific reverse primer;
thereby generating an amplicon, wherein the amplicon is further amplified using the oligonucleotide and the gene-specific reverse primer to produce an oligonucleotide comprising the first barcode, the second barcode, and the amplicon.
2 . The method according to claim 1 , wherein covalent bonding occurs by enzymatic attachment or ligation.
3 . The method according to claim 1 , wherein, prior to (d), the oligonucleotides and the released nucleic acid sequence both have sticky ends.
4 . The method according to claim 3 , wherein the released nucleic acid sequence comprises an overhang region and the oligonucleotides comprise a complementary overhang region, and wherein the overhang region and the complementary overhang region contain a single nucleotide, or a random sequence of nucleotides, or a restriction site.
5 . The method according to claim 1 , wherein the pre-defined pool of first barcodes comprises at least about 300 distinguishable barcodes.
6 . The method according to claim 1 , wherein the pre-defined pool of second barcodes comprises at least about 300 distinguishable barcodes.
7 . The method according to claim 1 , wherein the distinguishable barcode sequences comprise at least 10,000 distinguishable barcode sequences.
8 . The method according to claim 1 , wherein at least some of the oligonucleotides are attached to the surface of the particles.
9 . The method according to claim 1 , wherein at least some of the oligonucleotides are covalently bonded to the particles.
10 . The method according to claim 1 , wherein at least some of the oligonucleotides comprise a cleavable linker.
11 . The method according to claim 1 , further comprising releasing at least some of the oligonucleotides from the particles prior to lysing at least some of the cells.
12 . The method according to claim 1 , wherein the particles are contained within the droplets at no more than about 1 particle/droplet.
13 . The method according to claim 1 , wherein the plurality of droplets further comprise DNA-tagged antibodies.