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 preparing a library of different target sequences from a sample, comprising:
amplifying within a single amplification reaction mixture a multiplex of 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 multiplex of different amplified target sequences, wherein at least one of the plurality of target-specific primers and at least one of the produced multiplex of different amplified target sequences includes a cleavable group;
cleaving the cleavable group of at least one of the multiplex of different amplified target sequences and forming a cleaved end; and
ligating at least one adapter to a cleaved end of at least one of the multiplex of different amplified target sequences, thereby producing one or more adapter-ligated amplified target sequences;
thereby preparing a library of different adapter-ligated target sequences, wherein the number of different target-specific sequences amplified during the single multiplex amplification reaction is about 12-plex to about 10000 different target sequences, and
wherein none of the adapters in the ligation reaction hybridizes under high stringency conditions to any one of the multiplex of different amplified target sequences.
2. The method of claim 1 , wherein one or more of the at least one adapter is not completely complementary to at least one amplified target sequence.
3. The method of claim 1 , wherein at least one of the one or more adapters is less than 15% complementary along its length to at least one amplified target sequence.
4. The method of claim 1 , wherein each of the plurality of target-specific primers has any one or more of the following criteria: (1) includes two or more modified nucleotides within the primer sequence, at least one of which is included near or at the termini of the primer and at least one of which is included at, or about the center nucleotide position of the primer sequence; (2) length is about 15 to about 40 bases in length; (3) T m is from about 60° C. to about 70° C.; (4) has low cross-reactivity with non-target sequences present in the sample; (5) at least the first four nucleotides (going from 3′ to 5′ direction) are non-complementary to any sequence within any other primer present in the reaction; and (6) are non-complementary to any consecutive stretch of at least 5 nucleotides within any other produced amplified target sequence.
5. The method of claim 1 , wherein a adapter that is ligated to at least one of the plurality of different amplified target sequences is susceptible to exonuclease digestion.
6. The method of claim 1 , wherein a adapter that is ligated to at least one of the plurality of different amplified target sequences does not include a protecting group.
7. The method of claim 1 , wherein prior to the ligating at least one of the multiplex of different amplified target sequences is phosphorylated at the 5′ end.
8. The method of claim 1 , wherein the ligating includes contacting at least one of the multiplex of different amplified target sequences with a ligation reaction mixture including one or more adapters and a ligase under ligation conditions, wherein the ligation reaction mixture does not include one or more additional oligonucleotides prior to ligating the one or more adapters to at least one of the plurality of different amplified target sequences.
9. The method of claim 1 , wherein the method further includes a phosphorylating step prior to the ligating, thereby producing at least one-different amplified target sequence possessing a 5′ phosphate group.
10. The method of claim 1 , wherein the ligating includes an isothermal ligation reaction.
11. The method of claim 1 , wherein the ligation reaction includes no more than two different adapters.
12. The method of claim 1 , wherein the plurality of different target sequences is no less than twenty four different target sequences.
13. The method of claim 1 , wherein the plurality of different target sequences is no greater than 6144 different target sequences.
14. The method of claim 2 , wherein the at least one adapter does not include a sequence that is completely complementary to the 3′ end or the 5′ end of the plurality of different amplified target sequences.
15. The method of claim 1 , wherein the 3′ end or the 5′ end of the plurality of different amplified target sequences includes a cleavable group within about 15 terminal nucleotides.
16. The method of claim 1 , wherein the method further includes reamplifying at least one of the adapter-ligated amplified target sequences.
17. The method of claim 1 , further comprising combining two or more libraries each separately prepared in a single reaction, thereby preparing a library of different adapter-ligated target sequences.
18. The method of claim 1 , wherein the steps of amplifying, cleaving, ligating and preparing the library are carried out in a singles reaction vessel.