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 of nucleic acid preparation comprising:
tagging isolated cell free DNA with one or more universal tail adaptors to generate tagged products,
wherein the isolated cell-free DNA is isolated from a blood sample collected from a subject who is not a pregnant woman;
amplifying the tagged products one or more times to generate final amplification products,
wherein one of the amplifications comprises targeted amplification of a plurality of single nucleotide variants (SNVs) in a single reaction volume,
wherein one of the amplifications introduces a barcode and one or more sequencing tags; and
subjecting the final amplification products to massively parallel sequencing;
wherein the plurality of SNVs comprise 25-2,000 loci associated with cancer.
2 . The method of claim 1 , wherein tagging the cell free DNA comprises ligating the one or more universal tail adaptors to the cell free DNA.
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 cell free DNA.
4 . The method of claim 3 , wherein amplifying the tagged products comprises a first amplification and a second amplification, wherein the first amplification 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.
5 . The method of claim 4 , wherein the second amplification 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.
6 . The method of claim 5 , wherein the second adaptor primer is nested relative to the first adaptor primer.
7 . The method of claim 6 , wherein the second target-specific primer comprises an index tag.
8 . The method of claim 7 , wherein the second amplification further comprises using an index primer comprising a sequence complementary to the index tag.
9 . The method of claim 8 , wherein the index primer comprises the barcode and a first sequencing tag.
10 . The method of claim 1 , wherein the one or more universal tail adaptors comprise a second barcode.
11 . The method of claim 1 , wherein the one or more universal tail adaptors comprise a second sequencing tag.
12 . 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.
13 . The method of claim 12 , wherein tagging the cell free DNA comprises amplifying the cell free DNA with a first primer comprising the first universal tail adaptor and a second primer comprising the second universal tail adaptor.
14 . The method of claim 12 , wherein amplifying the tagged products comprises a single amplification.
15 . The method of claim 14 , 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.