Methods for sequencing samples
Personalized medicine involves the use of a patient's molecular markers to guide treatment regimens for the patient. The scientific literature provides multiple examples of correlations between drug treatment efficacy and the presence or absence of molecular markers in a patient sample. Methods are provided herein that permit efficient dissemination of scientific findings regarding treatment efficacy and molecular markers found in patient tumors to health care providers.
1. A method of determining a sequence of more than one gene from a formalin-fixed paraffin embedded biopsy from a subject, said method comprising:
(a) obtaining a formalin-fixed paraffin embedded (FFPE) biopsy, wherein said FFPE biopsy comprises nucleic acid;
(b) preparing a whole genome library of nucleic acids from said nucleic acid from said FFPE biopsy; and
(c) determining a sequence of more than one gene in said whole genome library of nucleic acids, wherein said determining comprises next-generation DNA sequencing of said whole genome library of nucleic acids, wherein said next-generation DNA sequencing comprises reversibly terminating nucleotides.
2. The method of claim 1 , wherein said FFPE biopsy comprises a tumor cell.
3. The method of claim 1 , wherein said nucleic acid comprises DNA.
4. The method of claim 1 , further comprising amplifying said whole genome library of nucleic acids from said FFPE biopsy.
5. The method of claim 1 , further comprising determining a presence or absence of a point mutation, a single nucleotide polymorphism, a copy number variation, a substitution, an insertion, a deletion, a rearrangement, a de novo mutation, a nonsense mutation, a missense mutation, a silent mutation, a frameshift mutation, an amplification, a chromosomal translocation, an interstitial deletion, a chromosomal inversion, a loss of heterozygosity, a loss of function mutation, a gain of function mutation, a dominant negative mutation, or a lethal mutation.
6. The method of claim 1 , further comprising determining a presence or absence of a substitution, an insertion, a deletion, a rearrangement, or an amplification.
7. The method of claim 1 , further comprising determining a presence or absence of a copy number variation.
8. The method of claim 1 , further comprising determining a presence or absence of a point mutation.
9. The method of claim 1 , further comprising determining a presence or absence of a single nucleotide polymorphism.
10. The method of claim 1 , further comprising determining a presence or absence of an amplification.
11. The method of claim 1 , wherein said next-generation DNA sequencing comprises attaching said nucleic acid to a solid surface.
12. The method of claim 1 , wherein said next-generation DNA sequencing comprises bridge amplification.
13. The method of claim 1 , wherein said more than one gene comprises KIT, BRAF, Hsp90AA1, EGFR, KRAS, ESR1, TOPOI, PTEN, PIK3CA, NFE2L2, and ERBB2.
14. The method of claim 1 , wherein said more than one gene comprises KIT, BRAF, EGFR, ERBB2, ESR1, KRAS, and PIK3CA.
15. The method of claim 1 , wherein said more than one gene comprises KRAS, EGFR, BRAF, ERBB2, and KIT.
16. The method of claim 1 , wherein said more than one gene comprises KRAS, EGFR, BRAF, and KIT.
17. The method of claim 1 , wherein said more than one gene comprises KRAS.
18. The method of claim 1 , wherein said more than one gene comprises PIK3CA.
19. The method of claim 1 , wherein said FFPE biopsy comprises a tumor cell from a colon cancer, a bone cancer, a breast cancer, a central nervous system cancer, a gastric cancer, a cervical cancer, a blood cancer, an esophageal cancer, a head and neck cancer, a kidney cancer, a skin cancer, a lung cancer, or a carcinoma.
20. The method of claim 1 , wherein said FFPE biopsy comprises a tumor cell from a colon cancer.
21. The method of claim 1 , wherein said FFPE biopsy comprises a tumor cell from blood.
22. The method of claim 1 , wherein said determining said sequence of more than one gene comprises hybridizing a probe nucleic acid to nucleic acids corresponding to said more than one gene from a tumor cell.
23. The method of claim 1 , wherein said next generation DNA sequencing comprises using a laser to excite a fluorophore.
24. The method of claim 1 , wherein preparing said whole genome library further comprises ligating an adaptor to said nucleic acid.
25. The method of claim 1 , wherein said subject is a human.
26. The method of claim 1 , wherein said obtaining comprises receiving said sample through a mail or delivery service.
27. The method of claim 1 , further comprising obtaining an additional sample after obtaining said FFPE biopsy from said subject, wherein said additional sample comprises a second FFPE biopsy comprising DNA from a tumor cell.
28. The method of claim 1 , wherein said next-generation DNA sequencing occurs in a Clinical Laboratory Improvement Amendment (CLIA) certified laboratory.