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.

What is claimed is: 
     
         1 . A method for identifying a subset of phased variant-containing nucleic acids molecules from a subject, the method comprising:
 (a) obtaining, by a computer system, sequencing data derived from a plurality of nucleic acid molecules from a subject, wherein the plurality nucleic acid molecules from the subject are cell-free nucleic acid molecules or nucleic acids molecules from tumor tissue; and   (b) processing, by the computer system, the sequencing data to identify a subset of phased-variant containing nucleic acid molecules from the plurality of nucleic acid molecules, wherein each nucleic acid molecule of the subset of phased-variant containing nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence, wherein each of the plurality of phased variants are separated from each other by no more than 170 nucleotides, and wherein at least 10% of the subset of 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, wherein processing the sequence data comprises:
 (i) aligning the sequencing data to the reference genomic sequence, wherein aligning the sequencing data to the reference genomic sequence comprises aligning at least 1000 sequences to the reference genomic sequence; 
 (ii) identifying phased variant-containing nucleic acid molecules from the plurality of nucleic acid molecules based on alignment of the sequencing data to the reference genomic sequence; and 
 (iii) filtering out phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a subject-matched non-tumor sample or a population of non-tumor samples that are not from the subject. 
   
     
     
         2 . The method of  claim 1 , wherein filtering out phased variant-containing nucleic acid molecules comprises filtering out phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a subject-matched non-tumor sample. 
     
     
         3 . The method of  claim 2 , wherein the subject-matched non-tumor sample comprises peripheral blood cells. 
     
     
         4 . The method of  claim 2 , wherein filtering out phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a subject-matched non-tumor sample comprises filtering out phased variant-containing nucleic acid molecules having a non-reference variant with a variant allele fraction of at least 40% upon sequencing to a depth of at least 10×. 
     
     
         5 . The method of  claim 2 , wherein filtering out phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a subject-matched non-tumor sample comprises filtering out phased variant-containing nucleic acid molecules having a non-reference variant with a variant allele fraction of at least 0.25% upon sequencing to a depth of at least 100×. 
     
     
         6 . The method of  claim 4 , wherein filtering out phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a subject-matched non-tumor sample comprises filtering our phased variant-containing nucleic acid molecules having a non-reference variant with a variant allele fraction of at least 0.25% upon sequencing to a depth of at least 100×. 
     
     
         7 . The method of  claim 1 , wherein filtering out phased variant-containing nucleic acid molecules comprises filtering out phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a population of non-tumor samples that are not from the subject. 
     
     
         8 . The method of  claim 6 , wherein filtering out phased variant-containing nucleic acid molecules comprises filtering out phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a population of non-tumor samples that are not from the subject. 
     
     
         9 . The method of  claim 7 , wherein filtering out phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a population of non-tumor samples that are not from the subject comprises filtering out phased variant-containing nucleic acid molecules in which the plurality of phased variants were identified in a paired-end read from at least one of the non-tumor samples that are not from the subject. 
     
     
         10 . The method of  claim 8 , wherein filtering out phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a population of non-tumor samples that are not from the subject comprises filtering out phased variant-containing nucleic acid molecules in which the plurality of phased variants were identified in a paired-end read from at least one of the non-tumor samples that are not from the subject. 
     
     
         11 . The method of  claim 1 , wherein filtering out phased variant-containing nucleic acid molecules comprises filtering out both (1) phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a subject-matched non-tumor sample and (2) phased variant-containing nucleic acid molecules in which at least one variant of the plurality of phased variants is observed in a population of non-tumor samples that are not from the subject. 
     
     
         12 . The method of  claim 1 , wherein the plurality of nucleic acid molecules from the subject are cell-free nucleic acid molecules. 
     
     
         13 . The method of  claim 12 , wherein the cell-free nucleic acid molecules are obtained or derived from a plasma sample of the subject. 
     
     
         14 . The method of  claim 1 , wherein the plurality of nucleic acid molecules from the subject are nucleic acid molecules from tumor tissue. 
     
     
         15 . The method of  claim 1 , wherein the method does not comprise performing barcode-mediated error suppression of the sequencing data. 
     
     
         16 . The method of  claim 1 , further comprising (c) analyzing the subset of phased variant-containing nucleic acid molecules to determine a condition of the subject. 
     
     
         17 . The method of  claim 1 , wherein the reference genomic sequence is at least a portion of a human genome.