Detection of sequence elements in nucleic acid molecules
Provided herein are methods and systems for establishing the presence of a sequence element in nucleic acid molecules. The sequence element may comprise a fused gene, a reporter gene, or another useful sequence for cell and tissue engineering, such as those used for labeling cells, identifying successfully transfected or transduced cells, etc. A method provided herein may additionally allow for barcoding of nucleic acid molecules and analysis of libraries of barcoded nucleic acid molecules.
1. A method for establishing a presence of a sequence element in a nucleic acid molecule, said method comprising
(a) performing (i) a first amplification of a target nucleic acid molecule sequence with aid of a first primer annealed to said sequence element in said target nucleic acid molecule sequence, to generate a first amplification product, and (ii) a second amplification of said first amplification product with aid of a second primer annealed to said sequence element, to generate a second amplification product, wherein said first primer is annealed to said sequence element at a first position different than a second position to which said second primer is annealed, wherein said first primer and said second primer are annealed to a same strand of said target nucleic acid molecule sequence, wherein said target nucleic acid molecule sequence comprises a first sequence fused to a sequence encoding an optically-active agent, and wherein said sequence element comprises said sequence encoding said optically-active agent;
(b) fragmenting said second amplification product, thereby generating a nucleic acid fragment; and
(c) detecting said nucleic acid fragment, or a derivative thereof, wherein said detecting establishes said presence of said sequence element in said target nucleic acid molecule sequence of (a).
2. The method of claim 1 , wherein said optically-active agent is green fluorescent protein (GFP).
3. The method of claim 1 , wherein said nucleic acid fragment comprises a barcode indicative of a cell from which said target nucleic acid molecule sequence is derived.
4. The method of claim 1 , wherein said nucleic acid fragment comprises a unique molecular identifier.
5. The method of claim 1 , wherein said performing said first amplification occurs in a presence of a pool of nucleic acid molecules derived from multiple cells.
6. The method of claim 5 , wherein members of said pool of nucleic acid molecules comprise a barcode indicative of a cell from which a nucleic acid molecule was derived.
7. The method of claim 1 , further comprising, following (b) and before (c), performing end-repair on said nucleic acid fragment.
8. The method of claim 7 , further comprising ligating an adaptor to said nucleic acid fragment following said performing said end-repair.
9. The method of claim 8 , further comprising amplifying said nucleic acid fragment, wherein said amplifying said nucleic acid fragment introduces a sample index to said nucleic acid fragment, thereby generating said derivative thereof.
10. The method of claim 9 , wherein said detecting comprises sequencing said nucleic acid fragment or derivative thereof.
11. The method of claim 1 , wherein said detecting comprises detecting said first sequence.
12. The method of claim 1 , wherein said target nucleic acid molecule sequence is derived from a single cell, wherein said nucleic acid fragment, or derivative thereof, comprises a barcode indicative of a single cell from which said target nucleic acid molecule sequence is derived, and further comprising comparing said barcode to a plurality of barcodes in a plurality of nucleic acid library members, wherein each barcode of said plurality of barcodes identifies a cell from which a member of said plurality of nucleic acid library members is derived, wherein said comparing identifies a cell as comprising said sequence element.
13. The method of claim 1 , wherein said fragmenting comprises contacting said second amplification product with a DNase.
14. The method of claim 1 , further comprising generating said target nucleic acid molecule sequence by (I) annealing an oligonucleotide comprising a poly T sequence, a barcode sequence, and a unique molecular identifier to a messenger RNA comprising a poly A tail; (II) extending said oligonucleotide, wherein said extending adds a terminal string of deoxycytosine triphosphate (dCTP) residues to an end of said extension product; (III) annealing a template switch oligonucleotide to said terminal string of dCTP residues; (IV) extending a 3′ end of said string of dCTP residues using said template switch oligonucleotide as a template, thereby generating an extension product; and (V) amplifying said extension product, thereby generating said target nucleic acid molecule sequence.
15. The method of claim 14 , wherein (I)-(IV) occur in a partition.
16. The method of claim 15 , wherein said partition is a droplet.