Methods and compositions for multiplex PCR
The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.
1. A method for amplifying a plurality of different target sequences within a sample, comprising:
a) amplifying within a single amplification reaction mixture between one hundred and twelve thousand different target sequences from a sample including a plurality of different target sequences, wherein the amplifying includes contacting at least some portion of the sample with a plurality of target-specific primers and a polymerase under amplification conditions, thereby producing a plurality of different amplified target sequences, wherein a plurality of the target-specific primers include a cleavable group and at least one of the plurality of amplified target sequences includes a primer-derived sequence containing the cleavable group;
b) cleaving the cleavable group of the primer-derived sequence of the at least one amplified target sequence, thereby forming a cleaved end of the at least one amplified target sequence; and
c) ligating at least one double-stranded adapter to the cleaved end of the at least one amplified target sequence in a blunt-ended ligation reaction, thereby producing one or more double-stranded adapter-ligated amplified target sequences.
2. The method of claim 1 , further comprising reamplifying at least one of the double-stranded adapter-ligated amplified target sequences.
3. The method of claim 2 , wherein the reamplifying includes contacting the at least one double-stranded adapter-ligated amplified target sequence with one or more primers including a sequence that is complementary to at least one of the double-stranded adapters, and a polymerase under amplification conditions, thereby producing at least one reamplified adapter-ligated amplified target sequence.
4. The method of claim 1 , wherein at least one target-specific primer is at least 85% identical to at least a portion of a corresponding target sequence in the sample.
5. The method of claim 1 , wherein a double-stranded adapter that is ligated to the cleaved end of the at least one amplified target sequence is susceptible to exonuclease digestion.
6. The method of claim 1 , wherein a double-stranded adapter that is ligated to the cleaved end of the at least one amplified target sequence does not include a protecting group.
7. The method of claim 1 , wherein the ligating includes contacting at least one amplified target sequence having a 3′ end and a 5′end with a ligation reaction mixture including one or more double-stranded adapters and a ligase under ligation conditions, wherein none of the double-stranded adapters in the ligation reaction mixture includes, prior to the ligating, a target-specific sequence.
8. The method of claim 7 , wherein the ligating includes contacting at least one amplified target sequence with a ligation reaction mixture including one or more double-stranded adapters and a ligase under ligation conditions, wherein the ligation reaction mixture does not include one or more additional oligonucleotide adapters prior to ligating the one or more double-stranded adapters to at least one amplified target sequence.
9. The method of claim 1 , wherein the amplifying further includes a digestion step prior to the ligating, thereby producing a plurality of blunt-end double-stranded amplified target sequences possessing a 5′ phosphate group.
10. The method of claim 1 , wherein at least 75% of the at least 100 different amplified target sequences differ in size by no more than 50 nucleotides.
11. The method of claim 1 , wherein no individual amplified target sequence is over-represented in the at least 100 different amplified sequences by more than 50% as compared to any other different amplified target sequence.