Methods and systems for construction of normalized nucleic acid libraries

A method for normalizing multiplexed amplification of target nucleic acids on a droplet actuator, comprising:

• a) providing a plurality of input samples comprising target nucleic acids;
• b) loading the plurality of input samples onto a droplet operations surface of the droplet actuator having droplet operations electrodes arranged thereon;
• c) dispensing a normalization reagent droplet to each of the loaded input samples to obtain a plurality of reaction droplets, wherein the normalization reagent droplet comprises a plurality of first amplification primers and a plurality of second amplification primers, wherein:
• i. the plurality of first amplification primers is immobilized on a solid support, the first amplification primers being capable of specifically hybridizing to a first sequence of the target nucleic acids, wherein the plurality of first amplification primers is provided in an amount which limits the yield of amplification products comprising the first amplification primers, wherein the amount of the first amplification primers is substantially the same in each normalization reagent droplet;
• and
• ii. the plurality of second amplification primers is in solution, the plurality of second primers being capable of specifically hybridizing to a second sequence of the target nucleic acids, wherein the plurality of second amplification primers is provided in an amount that exceeds the amount of the first amplification primers;
• d) amplifying the target nucleic acids of the reaction droplets under isothermal conditions, wherein amplification reagents except the first amplification primers are provided in excess amounts such that substantially all of the first amplification primers are incorporated into amplification products, thereby obtaining a plurality of output samples each comprising substantially the same amount of amplified target nucleic acids, and thereby normalizing the multiplexed amplification to the amount of the first amplification primers.

A method for normalizing a plurality of nucleic acid amplification reactions comprising:

• providing a first set of target nucleic acids;
• performing a first amplification reaction, comprising:
• a) providing first amplification primers which bind to a first sequence of the target nucleic acids, are immobilized on a solid support, and are provided in an amount which limits the yield of amplification products, and
• b) providing second amplification primers which bind to a second sequence of the target nucleic acid, are in solution, and are provided in an amount that exceeds the amount of the first amplification primers;
• and
• c) amplifying the target nucleic acids with the first and second amplification primers under isothermal conditions such that substantially all of the first amplification primers are incorporated into amplification products to yield a first limited amount of amplification products;
• and
• performing a second amplification reaction by repeating steps (a)-(c) with a second, different, target nucleic acid sample, thereby obtaining a second limited amount of amplification products that is substantially the same as the first limited amount of amplification products.