Methods and compositions for forming ligation products
In some aspects, the present disclosure provides methods for forming ligation products comprising single-stranded polynucleotides. Ligation products formed by various aspects of the present disclosure can be useful for various applications, including but not limited to sequence analysis. In some embodiments, the ligation products comprise cell-free polynucleotides. In some aspects, the present disclosure provides reaction mixtures, kits and complexes consistent with the methods herein.
1. A method of conducting a targeted rolling circle amplification, comprising:
(a) providing a circular polynucleotide, wherein the circular polynucleotide is formed by: (i) forming a first polynucleotide complex by mixing a plurality of cell-free DNA polynucleotides and a single-stranded adaptor with a capture probe, wherein a first segment of the capture probe hybridizes with a target polynucleotide sequence from the plurality of cell-free DNA polynucleotides via sequence complementarity and a second segment of the capture probe hybridizes with the single-stranded adaptor via sequence complementarity, and 3′ end of single-stranded adaptor and 5′ end of the target polynucleotide sequence from the plurality of cell-free DNA polynucleotides of the first polynucleotide complex are located adjacent from each other on the capture probe and 5′ end of the target polynucleotide sequence from the plurality of cell-free polynucleotides of the first polynucleotide complex lacks a sequence complementarity to the capture probe and forms a flap structure;
(ii) contacting the first polynucleotide complex with a reaction mixture comprising a flap endonuclease under conditions such that the flap structure located on 5′ end of the target polynucleotide sequence from the plurality of cell-free polynucleotides of the first polynucleotide complex is cleaved and producing a second polynucleotide complex comprising a cleaved target polynucleotide sequence, the single-stranded adaptor and the capture probe, wherein the flap endonuclease acts as a structure specific endonuclease for cleaving the flap structure located on 5′ end of the target polynucleotide sequence of the first polynucleotide complex;
(iii) ligating the cleaved target polynucleotide sequence to the single-stranded adaptor of the second polynucleotide complex, thereby forming a ligation product; and
(iv) producing the circular polynucleotide by circularizing the ligation product;
(b) generate a plurality of amplification products comprising concatemers of the ligation product by subjecting an amplification reaction mixture to multiple cycles of a rolling circle amplification, wherein the amplification reaction mixture comprises (i) a polymerase having strand displacement activity, (ii) the circular polynucleotide obtained from step (a), and (iii) at least a first primer that hybridizes to a sequence of at least a segment of the single-stranded adaptor.
2. The method of claim 1 , further comprising degrading or selectively removing the capture probe from the second polynucleotide complex prior to step (iv).
3. The method of claim 1 , wherein the single stranded adaptor comprises a tag.
4. The method of claim 3 , further comprising isolating the first polynucleotide complex or the second polynucleotide complex by immobilizing it directly or indirectly to a support comprising a selective binding agent that specifically binds the tag.
5. The method of claim 1 , wherein the method further comprises sequencing the plurality of amplification products to identify a sequence variant of the target polynucleotide sequence comprising at least one of a single nucleotide polymorphism, a single nucleotide variation, an insertion, a deletion, a duplication, an inversion, a translocation, a copy number variation, and a gene fusion.
6. The method of claim 1 , further comprising contacting the ligation product with bisulfite to modify unmethylated cytosines in the ligation product to uridines.
7. The method of claim 1 , wherein the plurality of amplification products comprising the concatemers of the ligation product is formed via extension of at least the first primer hybridized to the circular polynucleotide in step (b).
8. The method of claim 1 , further comprising generating a plurality of extension products, wherein an individual extension product of the plurality of extension products is formed via extension of a second primer that hybridizes to the concatemers of the ligation product of the plurality of amplification products via sequence complementarity.
9. The method of claim 1 , wherein at least the first primer also hybridizes to a sequence of at least a segment of the target polynucleotide sequence from the plurality of cell-free DNA polynucleotides.
10. The method of claim 1 , wherein at least the first primer comprises a barcode sequence.