Systems and methods for analyzing circulating tumor dna

Abstract

The invention provides oncogenomic methods for detecting tumors by identifying circulating tumor DNA. A patient-specific reference directed acyclic graph (DAG) represents known human genomic sequences and non-tumor DNA from the patient as well as known tumor-associated mutations. Sequence reads from cell-free plasma DNA from the patient are mapped to the patient-specific genomic reference graph. Any of the known tumor-associated mutations found in the reads and any de novo mutations found in the reads are reported as the patient’s tumor mutation burden.

What is claimed: 
     
         1 - 20 . (canceled) 
     
     
         21 . A method for monitoring a tumor of a patient over time, the method comprising:
 using at least one computer hardware processor to perform:
 creating, in at least one non-transitory computer-readable storage medium, a patient-specific genomic reference graph that represents at least one non-tumor sequence previously obtained by sequencing non-tumor DNA from the patient, the patient-specific genomic reference graph comprising a directed acyclic graph having nodes and edges connecting the nodes, the nodes including a first node and a second node, wherein:
 the first node is stored as a first object in the at least one non-transitory computer-readable storage medium, 
 the second node is stored as a second object in the at least one non-transitory computer-readable storage medium, and 
 a first edge of the edges is stored as a pointer from the first object to the second object in the at least one non-transitory computer-readable storage medium; 
 
 aligning a first set of sequence reads, previously obtained by sequencing a first sample containing cell-free plasma DNA from the patient, to the patient-specific genomic reference graph to identify a first population of one or more mutations in the cell-free plasma DNA relative to the at least one non-tumor sequence from the patient; 
 aligning a second set of sequence reads, previously obtained by sequencing a second sample containing the cell-free plasma DNA from the patient, to the patient-specific genomic reference graph to identify a second population of one or more mutations in the cell-free plasma DNA relative to the at least one non-tumor sequence from the patient, wherein the first sample was obtained at a first time and the second sample was obtained at a second time after the first time; and 
 generating a report indicating a comparison between the first population of one or more mutations and the second population of one or more mutations. 
   
     
     
         22 . The method of  claim 21 , wherein creating the patient-specific genomic reference graph comprises:
 aligning the at least one non-tumor sequence to an initial genomic reference graph representing a plurality of known human genomic sequences;   identifying mutations of the at least one non-tumor sequence relative to the initial genomic reference graph; and   incorporating the identified mutations into the initial genomic reference graph to create the patient-specific genomic reference graph.   
     
     
         23 . The method of  claim 21 , wherein:
 a first path through the patient-specific genomic reference graph represents a tumor-associated mutation, and   the method further comprises:
 determining, based on the aligning of the first set of sequence reads to the patient-specific genomic reference graph, a proportion of the first set of sequence reads aligned to the first path. 
   
     
     
         24 . The method of  claim 23 , further comprising:
 determining, based on the aligning of the second set of sequence reads to the patient-specific genomic reference graph, a proportion of the second set of sequence reads aligned to the first path,   wherein the report further indicates a comparison between the proportion of the first set of sequence reads aligned to the first path and the proportion of the second set of sequence reads aligned to the first path.   
     
     
         25 . The method of  claim 21 , further comprising, prior to aligning the second set of sequence reads to the patient-specific genomic reference graph, incorporating the first population of one or more mutations into the patient-specific genomic reference graph. 
     
     
         26 . The method of  claim 21 , wherein the report further indicates a proportion of sequence reads aligned to one or more tumor-associated mutations, the one or more tumor-associated mutations comprising one or more of: the first population of one or more mutations, the second population of one or more mutations, and at least one known tumor-associated mutation. 
     
     
         27 . The method of  claim 21 , wherein the second sample was obtained from the patient after administration of a treatment, and wherein the method further comprises determining an effectiveness of the treatment based on the comparison between the first population of one or more mutations and the second population of one or more mutations. 
     
     
         28 . The method of  claim 21 , further comprising determining a presence and/or a progression of the tumor based on the comparison between the first population of one or more mutations and the second population of one or more mutations. 
     
     
         29 . At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one computer hardware processor, cause the at least one computer hardware processor to perform a method for monitoring a tumor of a patient over time, the method comprising:
 creating, in the at least one non-transitory computer-readable storage medium, a patient-specific genomic reference graph that represents at least one non-tumor sequence previously obtained by sequencing non-tumor DNA from the patient, the patient-specific genomic reference graph comprising a directed acyclic graph having nodes and edges connecting the nodes, the nodes including a first node and a second node, wherein:
 the first node is stored as a first object in the at least one non-transitory computer-readable storage medium, 
 the second node is stored as a second object in the at least one non-transitory computer-readable storage medium, and 
 a first edge of the edges is stored as a pointer from the first object to the second object in the at least one non-transitory computer-readable storage medium; 
   aligning a first set of sequence reads, previously obtained by sequencing a first sample containing cell-free plasma DNA from the patient, to the patient-specific genomic reference graph to identify a first population of one or more mutations in the cell-free plasma DNA relative to the at least one non-tumor sequence from the patient;   aligning a second set of sequence reads, previously obtained by sequencing a second sample containing the cell-free plasma DNA from the patient, to the patient-specific genomic reference graph to identify a second population of one or more mutations in the cell-free plasma DNA relative to the at least one non-tumor sequence from the patient, wherein the first sample was obtained at a first time and the second sample was obtained at a second time after the first time;   generating a report indicating a comparison between the first population of one or more mutations and the second population of one or more mutations.   
     
     
         30 . The at least one non-transitory computer-readable storage medium of  claim 29 , wherein creating the patient-specific genomic reference graph comprises:
 aligning the at least one non-tumor sequence to an initial genomic reference graph representing a plurality of known human genomic sequences;   identifying mutations of the at least one non-tumor sequence relative to the initial genomic reference graph; and   incorporating the identified mutations into the initial genomic reference graph to create the patient-specific genomic reference graph.   
     
     
         31 . The at least one non-transitory computer-readable storage medium of  claim 29 , wherein:
 a first path through the patient-specific genomic reference graph represents a tumor-associated mutation, and   the method further comprises:
 determining, based on the aligning of the first set of sequence reads to the patient-specific genomic reference graph, a proportion of the first set of sequence reads aligned to the first path. 
   
     
     
         32 . The at least one non-transitory computer-readable storage medium of  claim 31 , further comprising:
 determining, based on the aligning of the second set of sequence reads to the patient-specific genomic reference graph, a proportion of the second set of sequence reads aligned to the first path,   wherein the report further indicates a comparison between the proportion of the first set of sequence reads aligned to the first path and the proportion of the second set of sequence reads aligned to the first path.   
     
     
         33 . The at least one non-transitory computer-readable storage medium of  claim 29 , further comprising, prior to aligning the second set of sequence reads to the patient-specific genomic reference graph, incorporating the first population of one or more mutations into the patient-specific genomic reference graph. 
     
     
         34 . The at least one non-transitory computer-readable storage medium of  claim 29 , wherein the report further indicates a proportion of sequence reads aligned to one or more tumor-associated mutations, the one or more tumor-associated mutations comprising one or more of: the first population of one or more mutations, the second population of one or more mutations, and at least one known tumor-associated mutation. 
     
     
         35 . The at least one non-transitory computer-readable storage medium of  claim 29 , wherein the second sample was obtained from the patient after administration of a treatment, and wherein the method further comprises determining an effectiveness of the treatment based on the comparison between the first population of one or more mutations and the second population of one or more mutations. 
     
     
         36 . A system, comprising:
 at least one computer hardware processor; and   at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one computer hardware processor, cause the at least one computer hardware processor to perform a method for monitoring a tumor of a patient over time, the method comprising:
 creating, in the at least one non-transitory computer-readable storage medium, a patient-specific genomic reference graph that represents at least one non-tumor sequence previously obtained by sequencing non-tumor DNA from the patient, the patient-specific genomic reference graph comprising a directed acyclic graph having nodes and edges connecting the nodes, the nodes including a first node and a second node, wherein:
 the first node is stored as a first object in the at least one non-transitory computer-readable storage medium, 
 the second node is stored as a second object in the at least one non-transitory computer-readable storage medium, and 
 a first edge of the edges is stored as a pointer from the first object to the second object in the at least one non-transitory computer-readable storage medium; 
 
 aligning a first set of sequence reads, previously obtained by sequencing a first sample containing cell-free plasma DNA from the patient, to the patient-specific genomic reference graph to identify a first population of one or more mutations in the cell-free plasma DNA relative to the at least one non-tumor sequence from the patient; 
 aligning a second set of sequence reads, previously obtained by sequencing a second sample containing the cell-free plasma DNA from the patient, to the patient-specific genomic reference graph to identify a second population of one or more mutations in the cell-free plasma DNA relative to the at least one non-tumor sequence from the patient, wherein the first sample was obtained at a first time and the second sample was obtained at a second time after the first time; 
 generating a report indicating a comparison between the first population of one or more mutations and the second population of one or more mutations. 
   
     
     
         37 . The system of  claim 36 , wherein creating the patient-specific genomic reference graph comprises:
 aligning the at least one non-tumor sequence to an initial genomic reference graph representing a plurality of known human genomic sequences;   identifying mutations of the at least one non-tumor sequence relative to the initial genomic reference graph; and   incorporating the identified mutations into the initial genomic reference graph to create the patient-specific genomic reference graph.   
     
     
         38 . The system of claim  16 , wherein:
 a first path through the patient-specific genomic reference graph represents a tumor-associated mutation, and   the method further comprises:
 determining, based on the aligning of the first set of sequence reads to the patient-specific genomic reference graph, a proportion of the first set of sequence reads aligned to the first path. 
   
     
     
         39 . The system of  claim 38 , further comprising:
 determining, based on the aligning of the second set of sequence reads to the patient-specific genomic reference graph, a proportion of the second set of sequence reads aligned to the first path,   wherein the report further indicates a comparison between the proportion of the first set of sequence reads aligned to the first path and the proportion of the second set of sequence reads aligned to the first path.   
     
     
         40 . The system of  claim 36 , further comprising, prior to aligning the second set of sequence reads to the patient-specific genomic reference graph, incorporating the first population of one or more mutations into the patient-specific genomic reference graph.