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 comprising:
(a) determining a status of one or more molecular markers in a historic sample from a subject, wherein the historic sample is a formalin-fixed paraffin embedded (FFPE) tumor biopsy, wherein the determining comprises de novo DNA sequencing,
(b) determining a status of one or more molecular markers in a second sample from the subject;
(c) comparing the status of the one or more molecular markers from the historic sample to the status of the one or more molecular markers from the second sample; and
(d) recommending a treatment option for the subject based on the status of the one or more molecular markers in the historic sample.
2 . The method of claim 1 , wherein the second sample is a fine needle aspiration biopsy or a cell-free sample.
3 . The method of claim 2 , wherein the second sample is a cell-free sample, wherein the cell-free sample is blood.
4 . The method of claim 1 , wherein the determining a status of one or more molecular markers in a second sample comprises de novo DNA sequencing.
5 . The method of claim 1 , wherein the one or more molecular markers in the historic sample comprise nucleic acid.
6 . The method of claim 5 , wherein the nucleic acid comprises genomic DNA.
7 . The method of claim 1 , wherein the de novo DNA sequencing comprises use of reversibly terminating nucleotides.
8 . A method of determining a status of more than one gene from a formalin-fixed paraffin embedded biopsy, the method comprising:
(a) obtaining a formalin-fixed paraffin embedded (FFPE) biopsy, wherein the FFPE biopsy comprises nucleic acid; and
(b) determining a status of more than one gene in the nucleic acid, wherein the determining comprises de novo DNA sequencing of the nucleic acid.
9 . The method of claim 8 , wherein the FFPE biopsy comprises a cell from a tumor.
10 . The method of claim 8 , wherein the nucleic acid comprises genomic DNA.
11 . The method of claim 8 , wherein the de novo DNA sequencing comprises attaching the nucleic acid to a solid surface.
12 . The method of claim 8 , wherein the de novo DNA sequencing comprises bridge amplification.
13 . The method of claim 8 , wherein the de novo DNA sequencing comprises use of reversibly terminating nucleotides.
14 . The method of claim 8 , wherein the de novo DNA sequencing does not comprise pyrosequencing.
15 . The method of claim 8 , further comprising amplifying the nucleic acid from the FFPE biopsy.
16 . The method of claim 15 , wherein the amplifying comprises polymerase chain reaction.
17 . The method of claim 8 , wherein the determining the status comprises determining a presence or absence of a copy number variation.
18 . A method of determining a status of a gene from a fine needle aspiration biopsy, the method comprising:
(a) obtaining a fine needle aspiration biopsy, wherein the fine needle aspiration biopsy comprises nucleic acid; and
(b) determining a status of a gene in the nucleic acid, wherein the determining comprises de novo DNA sequencing of the nucleic acid.
19 . The method of claim 18 , wherein the fine needle aspiration biopsy comprises a cell from a tumor.
20 . The method of claim 18 , wherein the fine needle aspiration biopsy comprises greater than 15% tumor.
21 . The method of claim 18 , wherein the nucleic acid comprises genomic DNA.
22 . The method of claim 18 , wherein the de novo DNA sequencing comprises attaching the nucleic acid to a solid surface.
23 . The method of claim 18 , wherein the de novo DNA sequencing comprises bridge amplification.
24 . The method of claim 18 , wherein the de novo DNA sequencing comprises use of reversibly terminating nucleotides.
25 . The method of claim 18 , further comprising amplifying the nucleic acid from the fine needle aspiration biopsy.
26 . A method of determining a status of a gene from a tumor cell in a cell-free sample, the method comprising:
(a) obtaining a cell-free sample from a subject, wherein the cell-free sample comprises cell-free genomic DNA from a tumor cell; and
(b) determining a status of a gene from a tumor cell in the genomic DNA, wherein the determining comprises de novo DNA sequencing of the genomic DNA, wherein the determining comprises determining a presence or absence of a point mutation or single nucleotide polymorphism.
27 . The method of claim 26 , wherein the cell-free sample is blood.
28 . The method of claim 26 , further comprising determining a status of more than one gene from a tumor cell in the genomic DNA, wherein the determining the status of more than one gene comprises de novo DNA sequencing of the genomic DNA, wherein the determining the status of more than one gene comprises determining a presence or absence of a point mutation or a single nucleotide polymorphism.
29 . The method of claim 26 , wherein the de novo DNA sequencing comprises use of reversibly terminating nucleotides.
30 . The method of claim 26 , further comprising amplifying the cell-free genomic DNA from the cell-free sample.