Methods and compositions for addressing inefficiencies in amplification reactions

A method for assembling an oligonucleotide composition provided herein, comprising:

• a) providing an initial primer pool including a plurality of primers wherein each primer includes a target sequence;
• b) amplifying target polynucleotides from a sample using the initial primer pool;
• c) identifying a subgroup of primers in the initial primer pool wherein the products of amplification result in an inaccurate allelic ratio or increased formation of primer dimers;
• d) modifying one or more primers in the subgroup of primers wherein modifying comprises:
• i) modifying one or more primers to include a quality control sequence (QCS);
• ii) modifying one or more QCS to include one or more extension sequences (ES);
• e) repeating steps b-d with modified primers until the products of amplification result in an accurate allelic ratio or decreased formation of primer dimers;
• thereby producing an optimized primer pool.

A computer-implemented method of determining a nucleotide spacer sequence for disrupting primer dimer formation, comprising:

• receiving a set of primer sequences;
• determining, using at least one microprocessor, a plurality of candidate spacers between an adapter sequence and a gene-specific portion of the primer sequence, the determined plurality of candidate spacers comprises sequences that disrupt stable interactions between sequences of the set of primer sequences;
• computing, using at least one microprocessor, a set of candidate spacers that meet a predetermined threshold value of stable interactions in the extension sequences;
• and
• outputting a set of the ranked spacers that meet the predetermined threshold.