Methods and compositions for multiplex PCR
The present invention provides methods, compositions, kits, systems and apparatus that are useful for determining copy number variation of one or more nucleic acids present in a sample. In some aspects, the method includes various target-specific primers that allow for the selective amplification of one or more target nucleic acids in the sample. In yet another aspect, the invention relates to determining copy number variation with respect to gene or chromosome representation of a nucleic acid in the sample. In some aspects, the method for determining copy number variation of different target nucleic acids in a sample using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including diagnosis, predictive therapeutic regimes or other therapeutic purposes.
1. A method for determining copy number variation, comprising amplifying a plurality of different target sequences in a sample, comprising:
a) producing a plurality of different amplified target sequences within a single multiplex amplification reaction mixture by contacting the plurality of different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions, where at least one of the plurality of target-specific primers and at least one of the amplified target sequences includes a cleavable group, thereby producing a multiplex of different amplified target sequences, 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
wherein the plurality of different target-specific sequences amplified during the single multiplex amplification reaction is about 12-plex to about 10000-plex different target sequences;
b) cleaving a cleavable group of at least one of the amplified target sequences;
c) producing one or more adapter-ligated amplified target sequences, by ligating at least one adapter to the cleaved end of at least one of the amplified target sequences, wherein the ligating excludes patch oligonucleotides, and wherein the adapters in the ligation reaction do not hybridize under high stringency conditions to the multiplex of different amplified target sequences;
d) reamplifying at least one of the one or more adapter-ligated amplified target sequences using universal primers;
e) sequencing at least one of the one or more amplified adaptor-ligated target sequences;
f) calculating the number of sequencing reads for at least one of the one or more amplified adaptor-ligated target sequence; and
g) determining copy number variation of at least one of the one or more amplified adaptor-ligated target sequences.
2. The method of claim 1 , wherein determining copy number variation of the at least one amplified adaptor-ligated target sequence includes a copy number variation associated with an aneuploidy.
3. The method of claim 2 , wherein the aneuploidy is a sex chromosome aneuploidy.
4. The method of claim 3 , wherein the aneuploidy is selected from the group consisting of XO, XXX, XXXX, XXXXX, XXY, XXYY, XXXY, XXYYY, XXXYY, XXXXY, XYY, XYYY or XYYYY.
5. The method of claim 1 , further comprising calculating the sequencing depth for at least one of the one or more amplified adaptor-ligated target sequences.