Methods of equalizing representation levels of nucleic acid targets

Abstract

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 representation levels of nucleic acid targets, 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 representation levels of the captured nucleic acid targets; and   e) repeating steps a), b), and c) if performed, at starting concentrations of the probes adjusted to reduce the range of representation levels of captured targets relative to the 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 , wherein the methods further comprises
 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 representation levels of nucleic acid targets, 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 representation levels 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 representation levels which 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 14 , 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.