DETECTING MUTATIONS AND PLOIDY IN CHROMOSOMAL SEGMENTS
The invention provides methods, systems, and computer readable medium for detecting ploidy of chromosome segments or entire chromosomes, for detecting single nucleotide variants and for detecting both ploidy of chromosome segments and single nucleotide variants. In some aspects, the invention provides methods, systems, and computer readable medium for detecting cancer or a chromosomal abnormality in a gestating fetus.
1 . A method for preparing a DNA fraction from a biological sample collection of a subject useful for analyzing epigenetic and genetic changes involved with cancer, comprising:
(a) extracting cell-free DNA from a first portion of the biological sample collection;
(b) producing a first enriched DNA fraction by: (i) treating a first portion of the extracted cell-free DNA or DNA derived therefrom with an agent that discriminates between methylated and unmethylated cytosines, wherein the extracted DNA or its derivative or the treated DNA or its derivative is further tagged with at least one adaptor containing a universal priming sequence, thereby producing adapted treated DNA, (ii) performing universal amplification on at least some of the adapted treated DNA using the universal priming sequence and producing amplified DNA, and (iii) selectively enriching for a subset of the amplified DNA or its derivative that contain one or more preselected target loci and producing enriched DNA that contain one or more preselected target loci, wherein the enrichment comprises selectively enriching for at least 1,000 different preselected target loci in a single reaction volume using at least 1,000 target-specific oligonucleotides;
(c) performing massively parallel sequencing on the first enriched DNA fraction or its derivative and obtaining sequence reads, identifying a presence or absence of one or more methylation patterns involved with cancer from the sequence reads; and
(d) further identifying a presence or absence of one or more genetic mutations involved with cancer in cell-free DNA from the biological sample collection.
2 . The method of claim 1 , wherein the biological sample collection is a blood, plasma, serum, or urine sample collection.
3 . The method of claim 1 , wherein the one or more methylation patterns involved with cancer comprise methylation of at least a portion of one or more CpG islands in promoter region of one or more tumor-suppressor genes.
4 . The method of claim 3 , wherein the tumor-suppressor genes comprise p16, APC, RASSF1A, GSTP1, DAPK, or a combination thereof.
5 . The method of claim 1 , wherein the one or more genetic mutations comprise single nucleotide variants (SNVs).
6 . The method of claim 1 , wherein step (b) comprises selectively enriching for 1,000-500,000 preselected target loci.
7 . The method of claim 1 , wherein step (b) comprises selectively enriching for 10,000-200,000 preselected target loci.
8 . The method of claim 1 , wherein the selectively enriching comprises targeted multiplex amplification.
9 . The method of claim 1 , wherein the selectively enriching comprises capturing at least some of the amplified DNA comprising one or more preselected target loci using hybrid capture probes.
10 . The method of claim 1 , wherein the universal amplification introduces a sample-specific barcode, and wherein the enriched DNA of multiple samples are pooled together and sequenced in the same sequencing run.
11 . The method of claim 1 , wherein the cell-free DNA comprises a mixture of cancer DNA and host DNA, and wherein the method further comprises estimating the fraction of cancer DNA in the cell-free DNA based on the sequence reads.
12 . The method of claim 1 , wherein the agent is bisulfite.
13 . The method of claim 1 , wherein the agent is an enzyme.
14 . The method of claim 13 , wherein the enzyme is one or more methylation sensitive restriction enzymes.
15 . The method of claim 1 , wherein the extracted DNA or its derivative is tagged with the at least one adaptor before being treated with the agent.
16 . The method of claim 1 , wherein the extracted DNA or its derivative is treated with the agent before being tagged with the at least one adaptor.
17 . The method of claim 1 , wherein the identifying a presence or absence of one or more genetic mutations involved with cancer comprises performing massively parallel sequencing on the first enriched DNA fraction or its derivative.
18 . The method of claim 1 , wherein the identifying a presence or absence of one or more genetic mutations involved with cancer comprises performing massively parallel sequencing on a second enriched DNA fraction or its derivative.
19 . The method of claim 1 , wherein the identifying a presence or absence of one or more genetic mutations involved with cancer comprises selectively enriching for DNA containing one or more preselected target loci each encompassing one of the one or more genetic mutations, and wherein the enrichment comprises selectively enriching for at least 10 different preselected target loci in a single reaction volume using at least 10 target-specific oligonucleotides.
20 . A method for preparing a DNA fraction from a biological sample collection of a subject useful for analyzing methylation patterns and/or somatic mutations involved with cancer, comprising:
(a) extracting cell-free DNA from the biological sample collection;
(b) producing an enriched DNA fraction by: (i) partitioning the extracted cell-free DNA or its derivative into a plurality of fractions, (ii) tagging the partitioned DNA or its derivative with at least one adaptor comprising a universal priming sequence and a barcode and producing barcoded DNA, wherein the barcoded DNA in different fractions are tagged with different barcodes, (iii) treating at least one of the fractions with an agent that discriminates between methylated and unmethylated cytosines, (iv) performing universal amplification using the universal priming sequence and producing amplified DNA, and (v) selectively enriching for a subset of the amplified DNA or its derivative that contain one or more preselected target loci and producing enriched DNA that contain one or more preselected target loci, wherein the enrichment comprises selectively enriching for at least 1,000 different preselected target loci in a single reaction volume using at least 1,000 target-specific oligonucleotides; and
(c) performing massively parallel sequencing on the enriched DNA or its derivative and obtaining sequence reads, and identifying from the sequence reads a presence or absence of one or more cancer-specific methylation patterns and one or more cancer-specific genetic mutations.
21 . The method of claim 20 , wherein the biological sample collection is a blood, plasma, serum, or urine sample collection.
22 . The method of claim 20 , wherein the one or more cancer-specific methylation patterns comprise methylation of at least a portion of one or more CpG islands in promoter region of one or more tumor-suppressor genes.
23 . The method of claim 20 , wherein the one or more cancer-specific genetic mutations comprise one or more single nucleotide variants (SNVs).
24 . The method of claim 20 , wherein step (b) comprises selectively enriching for 1,000-500,000 preselected target loci or 10,000-200,000 preselected target loci.
25 . The method of claim 20 , wherein the selectively enriching comprises targeted multiplex amplification.
26 . The method of claim 20 , wherein the selectively enriching comprises capturing at least some of the amplified DNA comprising one or more preselected target loci using hybrid capture probes.
27 . The method of claim 20 , wherein the barcoded DNA of the plurality of fractions are pooled together for universal amplification, selectively enriching, and/or sequencing.
28 . The method of claim 20 , wherein the agent comprises a bisulfite or an enzyme.
29 . The method of claim 20 , wherein the enzyme comprises a methylation sensitive restriction enzyme, wherein the methylation sensitive restriction enzyme cleaves unmethylated sequences, thereby allowing methylated sequences to be identified in the sequence reads.
30 . The method of claim 20 , wherein the cell-free DNA comprises a mixture of cancer DNA and host DNA, and wherein the method further comprises estimating the fraction of cancer DNA in the cell-free DNA based on the sequence reads.