Methods and Systems for Analyzing Nucleic Acid Molecules

Abstract

Processes and materials to detect cancer from a biopsy are described. In some cases, cell-free nucleic acids can be sequenced, and the sequencing result can be utilized to detect sequences derived from a neoplasm. Detection of somatic variants occurring in phase can indicate the presence of cancer in a diagnostic scan and a clinical intervention can be performed.

1 . A method comprising:
 (a) obtaining, by a computer system, sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject;   (b) processing, by the computer system, the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence, wherein at least about 10% of the one or more cell-free nucleic acid molecules comprises a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants that are separated by at least one nucleotide; and   (c) analyzing, by the computer system, the identified one or more cell-free nucleic acid molecules to determine a condition of the subject.   
     
     
         2 .- 115 . (canceled) 
     
     
         116 . The method of  claim 1 , further comprising separating, in silico, (i) at least a portion of the identified one or more cell-free nucleic acid molecules from (ii) one or more other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that are not identified to comprise the plurality of phased variants. 
     
     
         117 . The method of  claim 116 , further comprising analyzing, by the computer system, (i) and (ii) as different variables. 
     
     
         118 . The method of  claim 1 , wherein the analyzing of the identified one or more cell-free nucleic acid molecules is not based on other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that are not identified to comprise the plurality of phased variants. 
     
     
         119 . The method of  claim 1 , wherein the sequencing data is obtained without in silico removal or suppression of (i) background error or (ii) sequencing error. 
     
     
         120 . The method of  claim 1 , wherein a number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. 
     
     
         121 . The method of  claim 120 , wherein a ratio of (i) the number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules and (ii) a number of single nucleotide variants (SNVs) from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. 
     
     
         122 . The method of  claim 1 , wherein a frequency of the plurality of phased variants in the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. 
     
     
         123 . The method of  claim 1 , wherein the at least about 10% of the one or more cell-free nucleic acid molecules comprises at least about 50% of the one or more cell-free nucleic acid molecules. 
     
     
         124 . The method of  claim 123 , wherein the at least about 10% of the one or more cell-free nucleic acid molecules comprises at least about 100% of the one or more cell-free nucleic acid molecules. 
     
     
         125 . The method of  claim 1 , wherein the first and second phased variants are separated by at least 2 nucleotides. 
     
     
         126 . The method of  claim 1 , wherein the first phased variant and the second phased variant are separated by at most about 160 nucleotides. 
     
     
         127 . The method of  claim 1 , wherein the reference genome sequence comprises at least a portion of hg19 human genome, hg18 genome, hg17 genome, hg16 genome, or hg38 genome. 
     
     
         128 . The method of  claim 1 , wherein the reference genomic sequence is derived from a sample of the subject. 
     
     
         129 . The method of  claim 1 , wherein the reference genomic sequence is derived from a healthy cell of the subject. 
     
     
         130 . The method of  claim 1 , wherein the plurality of cell-free nucleic acid molecules comprise a plurality of cell-free deoxyribonucleic acid (DNA) molecules. 
     
     
         131 . The method of  claim 1 , wherein the condition comprises neoplasm, cancer, or tumor. 
     
     
         132 . The method of  claim 1 , wherein the condition comprises a solid tumor. 
     
     
         133 . The method of  claim 1 , wherein the condition comprises a lymphoma. 
     
     
         134 . The method of  claim 1 , further comprising determining, by the computer, that the identified one or more cell-free nucleic acid molecules are derived from a sample associated with the condition, based on performing a statistical model analysis of the identified one or more cell-free nucleic acid molecules. 
     
     
         135 . A method comprising:
 (a) providing a mixture comprising (1) a set of nucleic acid probes and (2) a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject,
 wherein an individual nucleic acid probe of the set of nucleic acid probes is designed to hybridize to at least a portion of a target cell-free nucleic acid molecule comprising a plurality of phased variants relative to a reference genomic sequence that are separated by at least one nucleotide, and 
 wherein the individual nucleic acid probe comprises an activatable reporter agent, activation of the activatable reporter agent being selected from the group consisting of: (i) hybridization of the individual nucleic acid probe to the plurality of phased variants and (ii) dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants; 
   (b) detecting the activatable reporter agent that is activated, to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises the plurality of phased variants; and   (c) analyzing the identified one or more cell-free nucleic acid molecules to determine a condition of the subject.   
     
     
         136 . The method of  claim 135 , wherein the activatable reporter agent is activated upon hybridization of the individual nucleic acid probe to the plurality of phased variants. 
     
     
         137 . The method of  claim 135 , wherein the activatable reporter agent is activated upon dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants. 
     
     
         138 . The method of  claim 135 , wherein the activatable reporter agent is a fluorophore. 
     
     
         139 . The method of  claim 135 , a number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. 
     
     
         140 . A method comprising:
 (a) obtaining sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject;   (b) processing the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules with a limit of detection of less than about 1 out of 50,000 observations from the sequencing data; and   (c) analyzing the identified one or more cell-free nucleic acid molecules to determine a condition of the subject,   wherein each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence.   
     
     
         141 . The method of  claim 140 , wherein the limit of detection of the identification step is less than about 1 out of 300,000 observations from the sequencing data. 
     
     
         142 . The method of  claim 141 , wherein the limit of detection of the identification step is less than about 1 out of 1,000,000 observations from the sequencing data. 
     
     
         143 . The method of  claim 140 , wherein a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants are separated by at least one nucleotide. 
     
     
         144 . A composition comprising a bait set comprising a set of nucleic acid probes designed to capture cell-free DNA molecules derived from at least about 5% of genomic regions set forth in (i) the genomic regions identified in Table 1, (ii) the genomic regions identified in Table 3, or (iii) the genomic regions identified to have a plurality of phased variants in Table 3.