Methods and primer sets for high throughput PCR sequencing

A method for reducing cross-contamination between a plurality of nucleic acid amplification reactions for amplifying a target nucleic acid sequence tC-tV-tC′ comprised within a sequence tract tn-tC-tV-tC′-tn′, said method comprising conducting a plurality of amplification reactions, each reaction comprising

• a first amplification step, whereby said target nucleic acid sequence is amplified using
• a left (forward) initial PCR primer having a sequence ma-K-pC and
• a right (reverse) initial primer having a sequence and ma-K′-pC′,
• yielding a first amplificate,
• wherein the sequences of K and K′ are different in each reaction within the plurality of amplification reactions;
• and
• a second amplification step, whereby said first amplificate is amplified using
• a left (forward) adaptor PCR primer having a sequence aL-aP-aK and
• a right (reverse) adaptor PCR primer having a sequence aL′-aP′-aK′,
• yielding a second amplificate only if aK will hybridize to K, and aK′ will hybridize to K′, and thereby reducing cross-contamination between each reaction within the plurality of reactions,
• wherein the primer pairs used in the first and second amplification steps of each reaction within the plurality of reactions are different from the primer pairs used in every other reaction within the plurality of reactions, wherein
• tV is a variable region within said target nucleic acid sequence,
• pC is the same sequence as sequence element tC and pC′ is the reverse complementary sequence to tC′,
• K comprises a sequence element k1 and a 3′-terminal sequence element s, and K′ comprises a sequence element k1′ and a 3′-terminal sequence element s′, wherein
• k1 and k1′ each independently from one another are a sequence 2 to 9 nucleotides in length,
• s and s′ are mismatch sequences selected not to form a continuous hybrid sequence with sequence element tn and tn′, and s and s′ are each independently 1, 2, 3, 4 or 5 nucleotides in length,
• and wherein in each amplification reaction of the plurality of reactions, k1 and/or k1′ is different from k1 and/or k1′ in every other amplification reaction within the plurality of reactions,
• ak is the same sequence as sequence element k1 and aK′ is the same sequence as sequence element k1′,
• aP-aK hybridizes to a contiguous sequence on ma-K and aP′-aK′ hybridizes to a contiguous sequence on ma-K′,
• pC, pC′, ma-K and ma-K′ each independently from one another are a sequence 10 to 40 nucleotides in length, and aL and aL,′ independently from one another can be any sequence.