Deep sequencing profiling of tumors
In one aspect of the present disclosure is a targeted sequencing workflow where an input sample comprising a sufficient quantity of genomic material is provided such minimal or no amplification cycles are utilized prior to sequencing.
1 . A method of measuring allele frequency and/or copy number variation without introducing bias during sequencing comprising: (i) isolating at least about 10 micrograms of genomic material from a homogenized sample, and wherein no amplification cycles are performed prior to enrichment, wherein the homogenized sample is prepared by mechanically shearing an obtained tumor sample with a blender or an ultra sonicator, wherein the obtained tumor sample is derived from a tumor of a human subject, and wherein the obtained tumor sample comprises at least 40% of the tumor derived from the human subject, wherein any heterogeneity of cells within the obtained tumor sample is substantially distributed within the homogenized sample, and wherein any portion of the homogenized sample expresses the heterogeneity of the obtained tumor sample; (ii) capturing one or more target nucleic acid molecules from the isolated genomic material, wherein an amount of captured one or more target nucleic acid molecules is at least about 90 ng; (iii) sequencing the captured one or more target nucleic acid molecules; and (iv) measuring allele frequency and/or copy number variation.
2 . The method of claim 1 , wherein the captured one or more target nucleic acid molecules are sequenced without performing post-capture amplification.
3 . The method of claim 1 , wherein the capturing of the one or more target nucleic acid molecules comprises: (i) introducing one or more probes to the isolated genomic material; (ii) removing non-target nucleic acids; and (iii) releasing the target nucleic acid molecules from the capture probes.
4 . The method of claim 1 , wherein the sequencing comprises next generation sequencing.
5 . The method of claim 1 , wherein the human subject was previously diagnosed with cancer.
6 . The method of claim 1 , wherein the whole obtained tumor sample is homogenized.
7 . The method of claim 1 , wherein the whole obtained tumor is homogenized except for one or more biopsy samples used for conducting diagnostic tests.
8 . The method of claim 1 , wherein the sample is homogenized through mechanical shearing.