Molecular indexing of internal sequences
The present disclosure relates to compositions, methods and kits for labeling an internal sequence of a target nucleic acid molecule with molecular barcodes. In some embodiments, the methods comprise intramolecular circulation of a labeled target nucleic acid molecule. Further provided methods for generating sequencing libraries comprising overlapping fragments covering the full length of a target nucleic acid molecule, sequencing the libraries using the methods disclosed herein, and methods of analyzing sequencing results therefrom.
1. A method of labeling a target nucleic acid sequence in a sample with a molecular barcode, comprising:
hybridizing an oligonucleotide comprising a molecular barcode with a first nucleic acid molecule comprising the target nucleic acid sequence;
extending the oligonucleotide to generate a second nucleic acid molecule comprising the molecular barcode and the target nucleic acid sequence;
circularizing the second nucleic acid molecule or complement thereof to generate a circularized nucleic acid molecule comprising the molecular barcode in close proximity to the target nucleic acid sequence; and
amplifying the circularized nucleic acid molecule to generate a plurality of amplicons comprising the molecular barcode in close proximity to the target nucleic acid sequence, wherein one or more amplification reactions use at least one primer comprising a binding site for a sequencing primer, wherein the sequencing primer does not comprise a sequence homologous to the sequence of the first nucleic acid molecule.
2. The method of claim 1 , further comprising synthesizing a complementary strand of the second nucleic acid molecule to generate a double-stranded nucleic acid molecule.
3. The method of claim 2 , wherein the circularizing comprises circularizing the double-stranded nucleic acid molecule.
4. The method of claim 1 , further comprising amplifying the second nucleic acid molecule or complement thereof to generate a copy of the second nucleic acid molecule or complement thereof.
5. The method of claim 4 , wherein the circularizing comprises circularizing a copy of the second nucleic acid molecule or complement thereof.
6. The method of claim 1 , further comprising sequencing the plurality of amplicons.
7. The method of claim 1 , wherein the first nucleic acid is an mRNA.
8. The method of claim 1 , wherein the oligonucleotide specifically binds to a binding site on the first nucleic acid molecule.
9. The method of claim 8 , wherein the binding site is a gene-specific sequence.
10. The method of claim 8 , wherein the binding site is a poly-A sequence.
11. The method of claim 1 , wherein the target nucleic acid sequence comprises 20 nt to 30 nt.
12. The method of claim 1 , wherein the target nucleic acid sequence comprises 30 nt to 40 nt.
13. The method of claim 1 , wherein the target nucleic acid sequence comprises 40 nt to 50 nt.
14. The method of claim 8 , wherein the binding site is at least 200 nt away from the target nucleic acid sequence on the first nucleic acid molecule.
15. The method of claim 8 , wherein the binding site is at least 500 nt away from the target nucleic acid sequence on the first nucleic acid molecule.
16. The method of claim 8 , wherein the binding site is at least 1,000 nt away from the target nucleic acid sequence on the first nucleic acid molecule.
17. The method of claim 8 , wherein the binding site is at least 2,000 nt away from the target nucleic acid sequence on the first nucleic acid molecule.
18. The method of claim 1 , wherein the molecular barcode comprises a sample label, a cellular label, a molecular label, or a combination thereof.
19. The method of claim 1 , wherein the molecular barcode comprises a binding site for a primer.
20. The method of claim 19 , wherein the primer is a universal primer.
21. The method of claim 1 , wherein the sequencing primer is a sequence associated with a high-throughput sequencing platform.