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) producing a plurality of different amplified target sequences by amplifying a plurality of different target sequences within a single amplification reaction mixture by contacting the plurality of different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions, at least one of the plurality of target-specific primers and at least one of the amplified target sequences including a cleavable group,
wherein each of the plurality of target specific primers have the following criteria:
(1) includes two or more modified cleavable 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;
b) cleaving a cleavable group of at least one amplified target sequence;
c) producing one or more adapter-ligated amplified target sequences by ligating at least one adapter to at least one amplified target sequence in a ligation reaction; and
d) wherein the method includes no more than one round of target-specific selection to produce any one adapter-ligated amplified target sequence from any target sequence, wherein the one round of target-specific selection comprises hybridizing a first target-specific primer and a second target-specific primer to at least one of the target sequences to be amplified and performing an amplification reaction having a plurality of cycles.
2. The method of claim 1 , wherein one or more of the at least one adapter is less than 15% complementary along its length to at least one amplified target sequence.
3. The method of claim 1 , wherein at least one of the one or more adapters is not complementary to about 15 nucleotides of the 3′ end or the 5′ end of the at least one amplified target sequence.
4. The method of claim 1 , wherein at least one target-specific primer is at least 75% complementary along its length to at least a portion of a corresponding target sequence in the sample.
5. The method of claim 1 , wherein an adapter that is ligated to at least one of the amplified target sequences is susceptible to exonuclease digestion.
6. The method of claim 1 , wherein an adapter that is ligated to at least one of the amplified target sequences 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 adapters and a ligase under ligation conditions, wherein none of the 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 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 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 amplified target sequences possessing a 5′ phosphate group.
10. A method for amplifying a plurality of different target sequences within a sample, comprising:
a) producing a plurality of amplified target sequences by amplifying within a single amplification reaction mixture a plurality 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 completely complementary target-specific primers and a polymerase under amplification conditions,
wherein each of the plurality of target specific primers have the following criteria:
(1) includes two or more modified cleavable 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; and
b) producing at least one adapter-ligated amplified target sequence by ligating at least one single-stranded adapter to each strand of at least one amplified target sequence through a ligation reaction.
11. The method of claim 9 , wherein at least one member of the group consisting of: an amplified target sequence, a target-specific primer, and an adapter, includes a cleavable group.
12. The method of claim 11 , wherein the cleavable group includes uracil.
13. The method of claim 12 , further including performing a cleavage step on at least one member of the group consisting of: an amplified target sequence, a target-specific prime, and an adapter, prior to the ligating.
14. The method of claim 13 , further including performing a cleavage step on at least one amplified target sequence, prior to the ligating.
15. The method of claim 1 , wherein the sample is selected from the group consisting of a single cell, cell-free circulating DNA, FFPE DNA, genomic DNA or blood.
16. The method of claim 1 , wherein the sample is obtained from a maternal subject, fetal subject or newborn.
17. The method of claim 1 , wherein the method further includes nucleic acid sequencing.
18. The method of claim 1 , wherein the amplifying includes no more than 30 cycles of amplification.
19. The method of claim 1 , wherein the ligation reaction does not include thermal cycling.
20. The method of claim 1 , wherein no less than two different target sequences are amplified.
21. The method of claim 1 , wherein no more than 100,000 different targets sequences are amplified.
22. The method of claim 1 , wherein no less than two and no more than 100,000 different target sequences are amplified.
23. The method of claim 1 , further including reamplifying at least one of the adapter-ligated amplified target sequences using primers.
24. The method of claim 1 , wherein the ligation reaction comprises a pair of universal adapters.
25. The method of claim 10 , wherein at least one of the plurality of completely complementary target-specific primers has no more than a 5 nucleotide sequence overlap with at least one other target-specific primer in the amplification reaction.
26. The method of claim 23 , wherein the reamplifying includes contacting the at least one adapter-ligated amplified target sequence with one or more primers including a sequence that is complementary to at least one of the adapters and a polymerase under amplification conditions, thereby producing at least one reamplified adapter-ligated amplified target sequence.