Optimization of multigene analysis of tumor samples

1. A method of analyzing nucleic acids from a human subject for identifying somatic mutations, the method comprising:

(a) providing DNA fragments from the human subject;

(b) attaching adapters to the DNA fragments to generate adapter-ligated DNA, wherein the adapters comprise:

(1) a primer binding site for amplification, sequencing, or both, and

(2) a barcode sequence;

(c) hybridizing, in solution, a plurality of bait molecules with the adapter-ligated DNA of step (b), thereby producing nucleic acid hybrids in a hybridization mixture;

wherein the bait molecules each comprise:

(1) a binding entity that allows for the separation of the nucleic acid hybrids of step (c); and

(2) a sequence configured to capture a target sequence;

wherein each nucleic acid hybrid comprises a bait molecule and a captured adapter-ligated DNA; and

wherein the plurality of bait molecules is capable of capturing a plurality of target sequences, and wherein the plurality of target sequences comprises at least two of the following types of somatic mutations in the DNA fragments from the human subject:

(i) point mutation;

(ii) in-frame deletion of one or more codons;

(iii) intragenic deletion;

(iv) intragenic insertion;

(v) deletion of a full gene;

(vi) inversion;

(vii) interchromosomal translocation;

(viii) tandem duplication;

(ix) gene fusion;

(x) genomic rearrangement that comprises an intron sequence; or

(xi) gene amplification or duplication;

(d) separating the nucleic acid hybrids from the hybridization mixture; and

(e) sequencing the captured adapter-ligated DNA, wherein the plurality of bait molecules is configured to provide a first sequencing depth for a first target sequence and a second sequencing depth for a second target sequence, and wherein the first sequencing depth differs from the second sequencing depth by at least 2-fold.

2. The method of claim 1 , wherein the captured adapter-ligated DNA is sequenced using next generation sequencing.

3. The method of claim 1 , wherein the somatic mutations are associated with a cancerous phenotype.

4. The method of claim 3 , wherein the cancerous phenotype comprises one or more of cancer risk, cancer progression, cancer treatment, or resistance to cancer treatment.

5. The method of claim 1 , wherein step (a) comprises providing less than 5 micrograms of DNA fragments from the human subject.

6. The method of claim 1 , wherein the size of the DNA fragments is 600 bp or less.

7. The method of claim 1 , wherein the size of the bait molecules is about 100-300 bases.

8. The method of claim 1 , wherein the target sequence is in a gene, and wherein the gene is ABL1, AKT1, AKT2, AKT3, ALK, APC, AR, BRAF, CCND1, CDK4, CDKN2A, CEBPA, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FLT3, HRAS, JAK2, KIT, KRAS, MAP2K1, MAP2K2, MET, MLL, MYC, NF1, NOTCH1, NPM1, NRAS, NTRK3, PDGFRA, PIK3CA, PIK3CG, PIK3R1, PTCH1, PTCH2, PTEN, RB1, RET, SMO, STK11, SUFU, or TP53.

9. The method of claim 1 , wherein the target sequence is in a gene, and wherein the gene is ABL2, ARAF, ARFRP1, ARID1A, ATM, ATR, AURKA, AURKB, BAP1, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL6, BRCA1, BRCA2, CBL, CARD11, CBL, CCND2, CCND3, CCNE1, CD79A, CD79B, CDH1, CDH2, CDH20, CDH5, CDK6, CDK8, CDKN2B, CDKN2C, CHEK1, CHEK2, CRKL, CRLF2, DNMT3A, DOT1L, EPHA3, EPHA5, EPHA6, EPHA7, EPHB1, EPHB4, EPHB6, ERBB3, ERBB4, ERG, ETV1, ETV4, ETV5, ETV6, EWSR1, EZH2, FANCA, FBXW7, FGFR4, FLT1, FLT4, FOXP4, GATA1, GNA11, GNAQ, GNAS, GPR124, GUCY1A2, HOXA3, HSP90AA1, IDH1, IDH2, IGF1R, IGF2R, IKBKE, IKZF1, INHBA, IRS2, JAK1, JAK3, JUN, KDM6A, KDR, LRP1B, LRP6, LTK, MAP2K4, MCL1, MDM2, MDM4, MEN1, MITF, MLH1, MPL, MRE11A, MSH2, MSH6, MTOR, MUTYH, MYCL1, MYCN, NF2, NKX2-1, NTRK1, NTRK2, PAK3, PAX5, PDGFRB, PKHD1, PLCG1, PRKDC, PTPN11, PTPRD, RAF1, RARA, RICTOR, RPTOR, RUNX1, SMAD2, SMAD3, SMAD4, SMARCA4, SMARCB1, SOX10, SOX2, SRC, TBX22, TET2, TGFBR2, TMPRSS2, TNFAIP3, TNK, TNKS2, TOP1, TSC1, TSC2, USP9X, VHL, or WT1.

10. The method of claim 1 , wherein the bait molecules further comprise a primer binding site for amplification.

11. The method of claim 1 , wherein the barcode sequence allows for identification of a sample from the subject or identification of the DNA fragments.

12. The method of claim 1 , wherein the plurality of bait molecules is capable of capturing at least three types of somatic mutations (i)-(xi) in step (c) in the nucleic acids from the human subject.

13. The method of claim 1 , further comprising amplifying the captured target sequences.

14. The method of claim 1 , wherein the adapter-ligated DNA of step (b) is amplified before hybridization in step (c).

15. The method of claim 1 , further comprising:

analyzing sequencing reads generated by sequencing the captured adapter-ligated DNA, the analyzing comprising:

(1) distinguishing between sequencing errors and real sequence variations by identifying differences between at least three duplicate reads of a DNA molecule from the human subject, wherein any difference between duplicate reads is a sequencing error; and

(2) generating a consensus sequence from the at least three duplicate reads of the DNA molecule;

wherein the duplicate reads of the DNA molecule are identified by (i) the barcode sequence of step (b), (ii) the possession of the same starting or ending sequences, or (iii) both (i) and (ii).

16. The method of claim 15 , wherein step (a) comprises providing less than 100 ng of DNA fragments from the human subject.

17. A method of analyzing nucleic acids from a human subject for identifying somatic mutations, the method comprising:

(a) providing nucleic acid fragments from the human subject;

(b) attaching adapters to the nucleic acid fragments to generate adapter-ligated nucleic acids, wherein the adapters comprise:

(1) primer binding sites for amplification, sequencing, or both and

(2) a barcode sequence;

(c) hybridizing, in solution, a plurality of bait molecules with the adapter-ligated nucleic acids of step (b), thereby producing nucleic acid hybrids in a hybridization mixture;

wherein the bait molecules each comprise:

(1) a binding entity that allows for the separation of the nucleic acid hybrids of step (c); and

(2) a sequence configured to capture a target sequence;

wherein each nucleic acid hybrid comprises a bait molecule and a captured adapter-ligated nucleic acid; and

wherein the plurality of bait molecules is capable of capturing a plurality of target sequences, and wherein the plurality of target sequences comprises at least two of the following types of somatic mutations in the nucleic acid fragments from the human subject:

(i) point mutation;

(ii) in-frame deletion of one or more codons;

(iii) intragenic deletion;

(iv) intragenic insertion;

(v) deletion of a full gene;

(vi) inversion;

(vii) interchromosomal translocation;

(viii) tandem duplication;

(ix) gene fusion;

(x) genomic rearrangement that comprises an intron sequence; or

(xi) gene amplification or duplication;

(d) separating the nucleic acid hybrids from the hybridization mixture; and

(e) sequencing the captured adapter-ligated nucleic acids, wherein the plurality of bait molecules is configured to provide a first sequencing depth for a first target sequence and a second sequencing depth for a second target sequence, and wherein the first sequencing depth differs from the second sequencing depth by at least 2-fold.

18. The method of claim 17 , wherein the nucleic acid fragments comprise genomic DNA or cDNA.

19. A method of analyzing nucleic acids from a human subject for identifying somatic mutations, the method comprising:

(a) providing DNA fragments from the human subject;

(b) attaching adapters to the DNA fragments to generate adapter-ligated DNA, wherein the adapters comprise:

(1) a primer binding site for amplification, sequencing, or both, and

(2) a barcode sequence;

(c) hybridizing a plurality of bait molecules with the adapter-ligated DNA of step (b), thereby producing nucleic acid hybrids in a hybridization mixture;

wherein the bait molecules each comprise:

(1) a binding entity that allows for the separation of the nucleic acid hybrids of step (c); and

(2) a sequence configured to capture a target sequence;

wherein each nucleic acid hybrid comprises a bait molecule and a captured adapter-ligated DNA; and

wherein the plurality of bait molecules is capable of capturing a plurality of target sequences, and wherein the plurality of target sequences comprises at least two of the following types of somatic mutations associated with a cancerous phenotype in the DNA fragments from the human subject:

(i) point mutation;

(ii) in-frame deletion of one or more codons;

(iii) intragenic deletion;

(iv) intragenic insertion;

(v) deletion of a full gene;

(vi) inversion;

(vii) interchromosomal translocation;

(viii) tandem duplication;

(ix) gene fusion;

(x) genomic rearrangement that comprises an intron sequence; or

(xi) gene amplification or duplication;

(d) separating the nucleic acid hybrids from the hybridization mixture; and

(e) sequencing the captured adapter-ligated DNA, wherein the plurality of bait molecules is configured to provide a first sequencing depth for a first target sequence and a second sequencing depth for a second target sequence, and wherein the first sequencing depth differs from the second sequencing depth by at least 2-fold.

20. The method of claim 19 , wherein in step (c) the plurality of bait molecules is hybridized with the adapter-ligated DNA of step (b) in solution or on solid support.