Multi-primer amplification method for barcoding of target nucleic acids

A method for amplifying a plurality of target nucleic acids in a plurality of samples, the method comprising:

• preparing an amplification mixture for each target nucleic acid, said amplification mixture comprising:
• a forward primer comprising a target-specific sequence;
• and
• a reverse primer comprising a target-specific sequence;
• subjecting each amplification mixture to amplification to produce a plurality of target nucleotide sequences;
• tagging the target nucleotide sequences to produce a plurality of target amplicons, each comprising first and/or second nucleotide tags flanking the target nucleotide sequence;
• wherein at least 50 percent of the target amplicons are present at greater than 50 percent of the average number of copies of target amplicons and less than 2-fold the average number of copies of target amplicons.

A microfluidic device comprising:

• a plurality of first input lines;
• a plurality of second input lines;
• a plurality of sets of first chambers, wherein each set of first chambers is in fluid communication with one of the plurality of first input lines;
• a plurality of sets of second chambers, wherein each set of second chambers is in fluid communication with one of the plurality of second input lines;
• a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines;
• and
• a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.

A method of operating a microfluidic device having an assay chamber, a sample chamber, and a harvesting port, the method comprising:

• closing a fluid line between the assay chamber and the sample chamber;
• flowing a sample into the sample chamber via a sample input line;
• flowing an assay into the assay chamber via an assay input line;
• opening the fluid line between the assay chamber and the sample chamber;
• combining at least a portion of the sample and at least a portion of the assay to form a mixture;
• reacting the mixture to form a reaction product;
• closing the fluid line between the assay chamber and the sample chamber;
• flowing a harvesting reagent from the harvesting port to the sample chamber;
• and
• removing the reaction product from the microfluidic device.

A method of preparing reaction products, the method comprising:

• providing M samples;
• providing N assays;
• mixing the M samples and N assays to form M×N pairwise combinations, each of the M×N pairwise combinations being contained in a closed volume;
• forming M×N reaction products from the M×N pairwise combinations;
• and
• recovering the M×N reaction products.