Methods and compositions for enrichment of amplification products
In some aspects, the present disclosure provides methods for enriching amplicons, or amplification products, comprising a concatemer of at least two or more copies of a target polynucleotide. In some embodiments, a method comprises sequencing the amplicons comprising at least two or more copies of a target polynucleotide. In some embodiments, the target polynucleotides comprise sequences resulting from chromosome rearrangement, including but not limited to point mutations, single nucleotide polymorphisms, insertions, deletions, and translocations including fusion genes. In some aspects, the present disclosure provides compositions and reaction mixtures useful in the described methods.
1. A method of nucleic acid processing, comprising:
(a) providing a reaction mixture comprising: (i) a polymerase, (ii) a circular polynucleotide; and (iii) a plurality of primers;
(b) in said reaction mixture, subjecting said circular polynucleotide to more than one cycle of amplification using said polymerase and said plurality of primers to generate a plurality of amplification products each amplification product of said plurality of amplification products comprising more than one copy of a sequence of said circular polynucleotide, wherein each cycle of amplification comprises:
annealing a primer of said plurality of primers to said circular polynucleotide at an annealing temperature;
performing a primer elongation reaction at an elongation temperature; and
subjecting said reaction mixture or derivative thereof to denaturation at a denaturation temperature; and
(c) subjecting said plurality of amplification products to nucleic acid sequencing to identify said sequence of said circular polynucleotide.
2. The method of claim 1 , wherein said circular polynucleotide is a circularized cell-free nucleic acid.
3. The method of claim 1 , wherein said circular polynucleotide is a circularized ribonucleic acid (RNA).
4. The method of claim 3 , wherein said circularized RNA comprises messenger RNA (mRNA) or micro-RNA (miRNA).
5. The method of claim 1 , wherein said circular polynucleotide is a circularized deoxyribonucleic acid (DNA).
6. The method of claim 1 , wherein said circular polynucleotide is derived from a biological sample from a subject.
7. The method of claim 6 , wherein said biological sample is a cell-free biological sample.
8. The method of claim 6 , wherein said biological sample is serum, plasma, blood, perspiration, saliva, urine, stool, semen, mucosal excretions, spinal fluid, amniotic fluid, or lymph fluid.
9. The method of claim 1 , wherein said circular polynucleotide is single stranded.
10. The method of claim 1 , wherein said denaturation temperature is different from said annealing temperature and said elongation temperature.
11. The method of claim 10 , wherein said denaturation temperature is between about 75° C. and about 95° C.; said annealing temperature is between about 45° C. and about 65° C.; and said elongation temperature is between about 65° C. and about 75° C.
12. The method of claim 1 , wherein said primer comprises a random sequence.
13. The method of claim 1 , wherein said primer comprises a gene specific sequence.
14. The method of claim 1 , wherein said primer comprises a barcode sequence.
15. The method of claim 1 , wherein said primer comprises an adaptor sequence.
16. The method of claim 1 , further comprising processing said sequence of said circular polynucleotide to identify a variant in said sequence.
17. The method of claim 16 , wherein said variant comprises at least one of a single nucleotide polymorphism, a deletion, an insertion, a copy number variant, a duplication, a translocation, a fusion, or a epigenetic variant.
18. The method of claim 16 , wherein said variant is associated with a disease in a subject.
19. The method of claim 18 , wherein said disease is cancer.
20. The method of claim 16 , further comprising processing said sequence variant to select a treatment for a disease.
21. The method of claim 16 , further comprising processing said sequence variant to monitor disease progression in a subject.
22. The method of claim 1 , wherein said polymerase has strand displacement activity.