Compositions and methods for molecular labeling
The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.
1. A method for analyzing a nucleic acid, the method comprising:
isolating a cell within a droplet of a plurality of droplets;
lysing the cell within the droplet to release nucleic acid;
exposing the nucleic acid to transposase enzymes and barcoded oligonucleotides in the droplet, wherein the barcoded oligonucleotides comprise a universal priming site and a barcode specific to the droplet;
conducting a transposition reaction with the transposase enzymes in the droplet to produce barcoded fragments of the nucleic acid;
breaking the plurality of droplets; and
amplifying the barcoded fragments to yield barcoded amplicons, wherein the amplifying step incorporates sequencing adaptors into the barcoded amplicons.
2. The method of claim 1 , wherein the nucleic acid comprises genomic DNA.
3. The method of claim 1 , wherein the lysing step includes exposing the cell to heat and/or detergent in the droplet.
4. The method of claim 3 , further comprising sequencing the amplified fragments.
5. The method of claim 3 , wherein each of the plurality of droplets comprises a different set of barcoded fragments.
6. The method of claim 1 , wherein the amplifying step comprises PCR.
7. The method of claim 6 , wherein breaking the droplets comprises releasing the barcoded fragments into an aqueous phase and pooling barcoded products from the plurality of droplets.
8. The method of claim 1 , wherein the barcoded fragments are not attached to beads.
9. The method of claim 7 , further comprising sequencing the barcoded amplicons.
10. A method for analyzing a nucleic acid, the method comprising:
isolating a single cell within one of a plurality of aqueous partitions;
lysing the single cell to release nucleic acid from the single cell;
exposing the nucleic acid to transposase enzymes and oligonucleotides in the one aqueous partition, wherein the oligonucleotides include a primer-binding site and a barcode specific to the one aqueous partition;
conducting a transposition reaction with the transposase enzymes in the partition to produce barcoded segments of the nucleic acid;
releasing contents of partitions; and
amplifying the barcoded segments using primers that include sequencing adaptors and that anneal to the primer-binding site to form barcoded amplicons that include the sequencing adaptors.
11. The method of claim 10 , further comprising sequencing the barcoded amplicons.