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 preparing a population of amplicons from a nucleic sample derived from a subject useful for analyzing a plurality of polymorphic loci associated with cancer, comprising:
generating tagged products by tagging nucleic acids with one or more universal adaptors, wherein the nucleic acids are derived from cell-free DNA, wherein the cell-free DNA is obtained from a biological sample derived from a human subject;
producing a population of amplicons each containing at least one polymorphic locus associated with cancer, a barcode, and one or more sequencing tags by amplifying a plurality of the tagged products one or more times, wherein the amplifying includes performing multiplex amplification of 20-2,000 polymorphic loci associated with cancer in a single reaction volume; and
sequencing one or more of the polymorphic loci in the amplicons by conducting massively parallel sequencing of a plurality of the amplicons.
2 . The method of claim 1 wherein the polymorphic loci comprise single nucleotide polymorphisms or single nucleotide variants or both.
3 . The method of claim 1 , wherein tagging nucleic acids comprises ligating one or more universal tail adaptors to the nucleic acids.
4 . The method of claim 3 , 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 nucleic acids.
5 . The method of claim 4 , wherein amplifying the tagged products comprises a first amplifying step and a second amplifying step, wherein the first amplifying step comprises using a first target-specific primer that specifically anneals to a target sequence and a first adaptor primer having a nucleotide sequence identical to a first portion of the first strand to generate a first amplification product.
6 . The method of claim 5 , wherein the second amplifying step comprises using a second target-specific primer that specifically anneals to the first amplification product and a second adaptor primer having a nucleotide sequence identical to a second portion of the first strand to generate the final amplification product.
7 . The method of claim 6 , wherein the second adaptor primer is nested relative to the first adaptor primer.
8 . The method of claim 7 , wherein the second target-specific primer comprises an index tag.
9 . The method of claim 8 , wherein the second amplifying step further comprises using an index primer comprising a sequence complementary to the index tag.
10 . The method of claim 9 , wherein the index primer comprises the barcode and a first sequencing tag.
11 . The method of claim 3 , wherein the one or more universal tail adaptors comprise a second barcode.
12 . The method of claim 3 , wherein the one or more universal tail adaptors comprise a second sequencing tag.
13 . The method of claim 3 , wherein the one or more universal tail adaptors comprise a first universal tail adaptor and a second universal tail adaptor.
14 . The method of claim 1 , wherein tagging nucleic acids comprises amplifying the nucleic acids with a first primer comprising a first universal tail adaptor and a second primer comprising a second universal tail adaptor.
15 . The method of claim 1 , wherein amplifying the tagged products comprises a single amplifying step.
16 . The method of claim 15 , wherein amplifying the tagged products comprises using a third primer and a fourth primer, wherein the third primer comprises a first sequencing tag and wherein the fourth primer comprises a second sequencing tag.