Methods and compositions for preparing polynucleotides
Provided herein are methods, compositions, and kits for forming amplification products. In various embodiments provided herein, transposomes comprising transposases are used in forming tagged polynucleotides for downstream amplification and polynucleotide processing steps.
1. A method of forming amplification products of a target polynucleotide, comprising:
(a) contacting a target polynucleotide present in a polynucleotide sample with transposomes to yield a plurality of tagged fragments, wherein individual transposomes comprise a transposase complexed with a transposon sequence having a transposon element, wherein a given tagged fragment of the plurality comprises the transposon sequence joined to the 5′ end of a segment of the target polynucleotide;
(b) subjecting said plurality of tagged fragments to an extension reaction using extension primers to yield extension products, wherein individual extension primers have a segment at a 3′ end exhibiting sequence complementarity to a tagged fragment and a segment at a 5′ end lacking sequence complementarity to the tagged fragment, and wherein a given extension product comprises (i) a sequence of the given tagged fragment and a complement of an extension primer sequence, or (ii) a complement of the given tagged fragment sequence and the extension primer sequence, wherein the transposon sequence or the extension primer comprises a methylated cytosine, wherein the methylated cytosine stabilizes a primer-dimer formed by the extension primers; and
(c) amplifying the extension products using a primer pair to yield amplification products, wherein the primer pair includes a first primer comprising the transposon sequence or a portion thereof and a second primer comprising the sequence of the segment at the 5′ end of the extension primer or a portion thereof, wherein individual amplification products comprise a single copy of the transposon sequence or a complement thereof.
2. The method of claim 1 , wherein the segment at the 3′ end of individual extension primers lacks sequence complementarity to the transposon sequence.
3. The method of claim 1 , wherein the segment at the 3′ end of individual extension primers comprises a gene specific sequence.
4. The method of claim 3 , wherein the extension primers comprise a mixture of gene specific extension primers.
5. The method of claim 4 , wherein the extension primers share an identical segment at the 5′ end.
6. The method of claim 1 , wherein the extension products comprise hemi-methylated double-stranded DNA.
7. The method of claim 6 , further comprising, subsequent to (b), subjecting said extension products comprising hemi-methylated double-stranded DNA to a methylation reaction to yield extension products comprising fully methylated double-stranded DNA.
8. The method of claim 7 , wherein methylation is effected by methyl transferase activity.
9. The method of claim 7 , wherein methylation is effected by a DNA methyltransferase enzyme.
10. The method of claim 9 , wherein the DNA methyltransferase enzyme is DNA (cytosine-5)-methyltransferase 1 (DNMT1).
11. The method of claim 10 , further comprising, prior to (c), subjecting said extension products comprising fully methylated double-stranded DNA to cytosine deamination to convert unmethylated cytosines to uracil.
12. The method of claim 11 , wherein cytosine deamination is effected by bisulfite or apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC).
13. The method of claim 1 , wherein at least one of the first and second primers of the primer pair comprises a barcode sequence, an amplification primer binding sequence, a sequencing primer binding sequence, or combinations thereof.
14. The method of claim 1 , wherein the polynucleotide is a cell-free polynucleotide.
15. The method of claim 1 , wherein the polynucleotide is a genomic polynucleotide.
16. The method of claim 1 , wherein the polynucleotide sample is obtained from a formalin-fixed paraffin-embedded (FFPE) tissue sample.
17. The method of claim 1 , wherein the polynucleotide sample is obtained from a frozen tissue sample.
18. The method of claim 1 , wherein the polynucleotide sample is obtained from a biological fluid.
19. The method of claim 1 , wherein the transposon sequence comprises methylated cytosines.
20. The method of claim 1 , wherein the transposase is a Tn transposase, an MuA transposase, or a Vibhar transposase.
21. The method of claim 20 , wherein the transposase is a Tn transposase selected from Tn3, Tn5, Tn7, and Tn10.
22. The method of claim 1 , wherein individual transposomes comprise a dimer of monomers, which monomers comprise a transposase complexed with a transposon sequence.
23. The method of claim 1 , wherein the amplifying of (c) is effected by a Hot-Start enzyme.
24. The method of claim 23 , wherein the Hot-Start enzyme is a Hot-Start polymerase.