METHODS FOR SIMULTANEOUS AMPLIFICATION OF TARGET LOCI
The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.
1 . A method for amplifying and sequencing DNA, comprising:
amplifying isolated cell-free DNA or DNA derived therefrom one or more times to generate amplification products, wherein the isolated cell-free DNA is isolated from a blood sample collected from a subject who is not a pregnant women, wherein one of the amplification steps comprises targeted amplification of a plurality of single nucleotide polymorphism or variant loci in a single reaction volume;
tagging the amplification products with one or more universal tail adaptors to generate tagged products, and amplifying the tagged products using adaptor-specific primers to generate final amplification products, wherein the final amplification products comprise a barcode and one or more sequencing tags; and
sequencing the plurality of single nucleotide polymorphism or variant loci on the cell free DNA by conducting massively parallel sequencing on the final amplification products, wherein the plurality of single nucleotide polymorphism or variant loci comprises 25-2,000 loci associated with cancer.
2 . The method of claim 1 , wherein tagging the amplification products comprises ligating the one or more universal tail adaptors to the amplification products.
3 . The method of claim 2 , wherein the one or more universal tail adaptors each comprise a first strand and a second strand, wherein a first end of each of the universal tail adaptors comprises a double-stranded section comprising the 5′ portion of the first strand and the 3′ portion of the second strand, wherein the first end is ligated to the amplification products.
4 . The method of claim 1 , wherein the one or more universal tail adaptors comprise the barcode.
5 . The method of claim 1 , wherein the one or more universal tail adaptors comprise the one or more sequencing tags.
6 . The method of claim 1 , wherein the one or more universal tail adaptors comprise a first universal tail adaptor and a second universal tail adaptor.
7 . The method of claim 6 , wherein amplifying the tagged products comprises a single amplifying step.
8 . The method of claim 7 , wherein amplifying the tagged products comprises using a first adaptor-specific primer and a second adaptor-specific primer, wherein the first adaptor-specific primer comprises a first sequencing tag and wherein the second adaptor-specific primer comprises a second sequencing tag.
9 . The method of claim 1 , wherein at least one amplicon in the amplification products comprises two or more nearby single nucleotide polymorphisms or variants loci.
10 . The method of claim 1 , wherein the isolated cell-free DNA is isolated from a plasma portion of the blood sample.