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 DNA fraction from a biological sample of a subject useful for analyzing genetic or epigenetic features involved with cancer, comprising:
(a) extracting cell-free DNA from the biological sample;
(b) producing an enriched fraction of DNA by: (1) introducing at least one adaptor containing a universal priming sequence to the extracted cell-free DNA or their derivatives and producing a plurality of adapted DNA sequences containing the universal priming sequence, (2) performing universal amplification on the plurality of adapted DNA sequences using the universal priming sequence, thereby producing a plurality of amplified adapted DNA sequences, and (3) selectively enriching for a subset of the plurality of amplified adapted DNA sequences or their derivatives that contain one or more preselected loci, thereby producing enriched DNA sequences; and
(c) performing massively parallel sequencing on the enriched DNA sequences or their derivatives and obtaining sequence reads containing at least a portion of one or more of the preselected loci, and obtaining an identification of one or more genetic or epigenetic features involved with cancer.
2 . The method of claim 1 , wherein the biological sample is a blood, plasma, serum, or urine sample.
3 . The method of claim 1 , wherein the genetic or epigenetic features involved with cancer comprises single nucleotide polymorphism or variant, copy number variation, insertion, deletion, or differential methylation.
4 . The method of claim 1 , wherein step (b) comprises selectively enriching for 1,000-500,000 preselected loci.
5 . The method of claim 1 , wherein step (b) comprises selectively enriching for 10,000-200,000 preselected loci.
6 . The method of claim 1 , wherein the selectively enriching comprises targeted multiplex amplification.
7 . The method of claim 1 , wherein the selectively enriching comprises capturing some of the plurality of amplified adapted DNA sequences or their derivatives that contain one or more preselected loci using hybrid capture probes.
8 . The method of claim 1 , wherein the adaptor further comprises a molecular barcode, wherein sequence reads derived from the same original cell-free DNA molecule are identified using the molecular barcode.
9 . The method of claim 1 , wherein the universal amplification introduces a sample-specific barcode, and wherein the enriched DNA sequences of multiple samples are pooled together and sequenced in the same sequencing run.
10 . The method of claim 1 , wherein the cell-free DNA comprises cancer DNA, and wherein the method further comprises estimating the fraction of cancer DNA in the cell-free DNA based on the sequence reads.