Methods of equalizing representation levels of nucleic acid targets
View Patent ↗The disclosure provides methods of reducing the range of representation levels of nucleic acid targets. The methods are particularly useful for multi-target analyses benefiting from a low variance of target representations, such as, e.g., single molecule sequencing and/or heterozygous genotyping, and pathogen diagnosis. Two general methods are provided. In Method 1, starting concentrations of probes are adjusted. In Method 2, target-specific probes are “binned,” i.e., several subsets of probes are selected based on similar representation levels. Thereafter, each subset of corresponding targets is extracted, with or without amplification, using a separate portion of the sample (i.e., separate vessels).
1. A method of equalizing amounts of nucleic acid targets in a sample, the method comprising:
a) contacting a plurality of probes with a sample comprising a plurality of targets;
b) capturing the plurality of nucleic acid targets with the plurality of probes;
c) optionally, amplifying the captured targets using the plurality of probes;
d) determining an initial range of amounts of different captured nucleic acid targets; and
e) repeating steps a), b), and c) if performed, at starting concentrations of the probes adjusted to reduce said initial range, thereby producing a final sample.
2. The method of claim 1 , wherein the step of amplifying the captured targets is required.
3. The method of claim 1 , wherein the targets are amplified by multiplex PCR.
4. The method of claim 1 , further comprising f) analyzing the targets obtained in the final sample.
5. The method of claim 4 , wherein the targets are analyzed by sequencing.
6. The method of claim 5 , wherein the sequencing is sequencing by synthesis or ligation.
7. The method of claim 6 , wherein the sequencing by synthesis is performed at a single molecule resolution.
8. The method of claim 1 , wherein the capturing step comprises circularizing probes annealed to targets and removing non-circularized nucleic acids.
9. The method of claim 1 , wherein the range of representation levels is reduced by at least two-fold.
10. The method of claim 1 , wherein the initial range of representation levels is greater than 100× and/or the reduced range of representation levels is less than 50×.
11. The method of claim 1 , wherein the starting concentrations of at least five probes are adjusted at least two-fold up or down.
12. A method equalizing amounts of nucleic acid targets, the method comprising:
a) introducing a plurality of probes to a sample comprising a plurality of targets;
b) capturing the plurality of nucleic acid targets with the plurality of probes;
c) optionally, amplifying the captured targets using the plurality of probes;
d) determining an initial range of amounts of the captured nucleic acid targets;
e) selecting two or more subsets of targets/probes so that each of the subsets contains targets of a range of amounts that is reduced relative to the initial range; and
f) repeating steps a), b), and c) if performed, using separate samples so that each sample contains only targets/probes of different selected subsets.
13. The method of claim 12 , wherein the method further comprises g) combining the samples produced in step f) or portions thereof to produce a final sample.
14. The method of claim 12 , wherein the step of amplifying the captured targets is required.
15. The method of claim 12 , wherein the targets are amplified by multiplex PCR.
16. The method of claim 15 , wherein in two or more multiplex amplification reactions at least five targets are amplified in each.
17. The method of claim 12 , further comprising h) analyzing targets in the combined sample.
18. The method of claim 17 , wherein the targets are analyzed by sequencing.
19. The method of claim 18 , wherein the sequencing is sequencing by synthesis or ligation.
20. The method of claim 19 , wherein the sequencing by synthesis is performed at a single molecule resolution.
21. The method of claim 12 , wherein the capturing step comprises circularizing probes annealed to respective targets and removing non-circularized nucleic acids.
22. The method of claim 12 , wherein the range of representation levels is reduced by at least two-fold.
23. The method of claim 12 , wherein the initial range of representation levels is greater than 100× and/or the reduced range of representation levels is less than 50×.
24. The method of claim 12 , wherein five or more subsets of probes are selected.
25. The method of claim 1 , wherein two or more subsets of probes comprise at least five probes each.