Methods for estimating cluster numbers
The present invention relates to methods of imaging template hybridisation for estimating cluster numbers prior to solid phase amplification and sequencing. More particularly, an initial round of imaging is carried out at the single molecule template hybridisation stage which allows a general estimation of cluster numbers prior to clusters being formed. Amplification of the signal allows single molecule imaging to be carried out using standard sequencing imaging apparatus.
1. A method of predicting cluster numbers or cluster densities on a first solid support resulting from cluster amplification of template strands of nucleic acids on the first solid support prior to the cluster amplification of said template strands of nucleic acids, comprising:
(a) providing the first solid support, wherein a plurality of capture primers is immobilised on the first solid support;
(b) hybridising said template strands of nucleic acids to at least some of said capture primers, thereby forming hybridized template strands;
(c) obtaining labelled strands by labelling the hybridized template strands;
(d) detecting signals produced from a label on the labelled strands, and determining the number of hybridized template strands present on the first solid support by counting images on the first solid support generated by the signals produced from the label on the labelled strands; and
(e) correlating the number of hybridized template strands present on the first solid support to a number of hybridized reference template strands present in a reference, wherein the reference is a standard curve produced by plotting different numbers of hybridized reference template strands present on a second solid support versus different densities of clusters on the second solid support generated by cluster amplification of the different numbers of the hybridized reference template strands present on the second solid support,
thereby predicting the cluster numbers or cluster densities on the first solid support resulting from the cluster amplification of said template strands of nucleic acids on the first solid support prior to the cluster amplification of said template strands of nucleic acids.
2. The method of claim 1 , wherein said labelling the hybridized template strands comprises hybridising a labelled oligonucleotide to each of the hybridized template strands and exposing complexes formed by the labelled oligonucleotide and the hybridized template strands to a dye labelled streptavidin, the labelled oligonucleotide is attached to biotin and the label is the dye labelled streptavidin.
3. The method of claim 2 , further comprising contacting complexes formed by the labelled oligonucleotide, the hybridized template strands, and the dye labelled streptavidin with an anti-streptavidin antibody.
4. The method of claim 2 , wherein the dye labelled streptavidin comprises a fluorescent dye.
5. The method of claim 4 , wherein the detecting step is carried out with a fluorescence microscope.
6. The method of claim 1 , wherein each of said hybridized template strands comprise a label binding site.
7. The method of claim 6 , wherein the label binding site is a sequencing primer site.
8. The method of claim 7 , wherein the sequencing primer site comprises a SBS3 or SBS12 sequence.
9. The method of claim 6 , wherein said labelling the hybridized template strands comprises hybridizing a labelled oligonucleotide to the label binding site.
10. The method of claim 9 , wherein the method includes extending the capture primers on said hybridized template strands to produce complements of the template strands of nucleic acids.
11. The method of claim 1 , wherein the solid support is a flow cell.
12. The method of claim 1 , wherein the capture primers comprise P5 and P7 oligonucleotides.
13. The method of claim 1 , wherein the template strands of nucleic acids comprise adapter sequences.
14. The method of claim 13 , wherein said adapter sequences are complementary to at least one of the capture primers and hybridise thereto under appropriate conditions.