Detecting mutations for cancer screening

Abstract

Embodiments are related to the accurate detection of somatic mutations in the plasma (or other samples containing cell-free DNA) of cancer patients and for subjects being screened for cancer. The detection of these molecular markers would be useful for the screening, detection, monitoring, management, and prognostication of cancer patients. For example, a mutational load can be determined from the identified somatic mutations, and the mutational load can be used to screen for any or various types of cancers, where no prior knowledge about a tumor or possible cancer of the subject may be required. Embodiments can be useful for guiding the use of therapies (e.g. targeted therapy, immunotherapy, genome editing, surgery, chemotherapy, embolization therapy, anti-angiogenesis therapy) for cancers. Embodiments are also directed to identifying de novo mutations in a fetus by analyzing a maternal sample having cell-free DNA from the fetus.

What is claimed is: 
     
         1 . A method for identifying tumor-derived mutations in a human subject, the method comprising:
 obtaining template DNA fragments from a biological sample of the human subject comprising cell-free DNA fragments, wherein the template DNA fragments comprise at least some of the cell-free DNA fragments;   preparing a sequencing library of analyzable DNA molecules using the template DNA fragments, wherein a duplication rate of the sequencing library from the template DNA fragments is less than 5%;   sequencing the sequencing library of analyzable DNA molecules to obtain first sequence reads, the first sequence reads comprised of first sequences;   obtaining second sequence reads for DNA molecules from a plurality of blood cells of the human subject, the second sequence reads comprised of second sequences; and   detecting the tumor-derived mutations in the cell-free DNA fragments by filtering out at least some of the first sequences of the cell-free DNA fragments that are also present in the second sequences.   
     
     
         2 . The method of  claim 1 , wherein the blood cells comprise white blood cells. 
     
     
         3 . The method of  claim 1 , wherein sequencing the sequencing library of analyzable DNA molecules comprises paired-end sequencing. 
     
     
         4 . The method of  claim 1 , wherein sequencing the sequencing library of the analyzable DNA molecules comprises random sequencing. 
     
     
         5 . The method of  claim 1 , wherein the sequencing the sequencing library of the analyzable DNA molecules comprises massively parallel sequencing. 
     
     
         6 . The method of  claim 1 , further comprising sequencing the DNA molecules from the plurality of blood cells of the human subject to obtain the second sequence reads. 
     
     
         7 . A method for identifying tumor-derived mutations in a human subject, the method comprising:
 (a) preparing a sequencing library of analyzable DNA molecules based on template DNA fragments from a biological sample of the human subject comprising cell-free DNA fragments, wherein the template DNA fragments comprise at least some of the cell-free DNA fragments, and wherein a duplication rate of the sequencing library from the template DNA fragments is less than 5%;   (b) sequencing the sequencing library of analyzable DNA molecules to obtain sequence reads;   (c) receiving sequence reads of DNA molecules from a buffy coat sample of the human subject; and   (d) analyzing the sequence reads of (b) and (c) to identify single-nucleotide variants present in the cell-free DNA fragments and not present in the DNA molecules from the buffy coat sample of the human subject, as being tumor-derived.   
     
     
         8 . The method of  claim 7 , wherein analyzing the sequence reads of (b) and (c) to determine single-nucleotide variants comprises comparing the sequence reads of (b) to the sequence reads of (c) to detect a plurality of loci having single-nucleotide variants, and
 wherein, at each of the plurality of loci, a number of the sequence reads of (b) having a single-nucleotide sequence variant relative to the sequence reads of (c) is above a cutoff value greater than one.   
     
     
         9 . The method of  claim 7 , further comprising identifying, by a computer system, the human subject as having a cancer based on the single-nucleotide variants present in the cell-free DNA fragments. 
     
     
         10 . The method of  claim 7 , further comprising identifying a level of cancer in the human subject based, at least in part, on the single-nucleotide variants present in the cell-free DNA fragments. 
     
     
         11 . The method of  claim 7 , further comprising determining a fractional concentration of tumor DNA based, at least in part, on the single-nucleotide variants present in the cell-free DNA fragments. 
     
     
         12 . A method for identifying one or more tumor-derived mutations in a human subject, the method comprising:
 obtaining template DNA fragments from a biological sample of the human subject comprising cell-free DNA fragments, wherein the template DNA fragments comprise at least some of the cell-free DNA fragments;   preparing a sequencing library of analyzable DNA molecules using template DNA fragments, wherein a duplication rate of the sequencing library from the template DNA fragments is less than 5%;   sequencing the sequencing library of analyzable DNA molecules to obtain first sequence reads;   determining a consensus sequence from second sequence reads obtained from sequencing DNA molecules from another sample from the human subject;   identifying one or more potential sequence variants, wherein identifying the one or more potential sequence variants comprises:
 determining genomic positions for the first sequence reads; and 
 detecting the one or more potential sequence variants in one or more first sequence reads positioned at one or more loci; and 
   identifying the one or more tumor-derived mutations in the human subject based on a comparison of the one or more first sequence reads of the one or more potential sequence variants to the consensus sequence at the one or more loci.   
     
     
         13 . The method of  claim 12 , wherein, at each of the one or more loci, a number of the one or more first sequence reads having a sequence variant relative to the consensus sequence is above a cutoff value greater than one. 
     
     
         14 . The method of  claim 12 , wherein the one or more potential sequence variants at the one or more loci are detected in the one or more first sequence reads relative to a reference genome. 
     
     
         15 . The method of  claim 14 , wherein the one or more potential sequence variants comprise single-nucleotide variants. 
     
     
         16 . The method of  claim 12 , further comprising detecting one or more variants, wherein identifying the one or more variants comprises:
 determining genomic positions for the second sequence reads obtained from the other sample; and   detecting one or more variants at one or more loci relative to a reference genome.   
     
     
         17 . The method of  claim 12 , further comprising identifying the one or more tumor-derived mutations as associated with a non-synonymous change. 
     
     
         18 . The method of  claim 12 , wherein the consensus sequence is part of a human genome. 
     
     
         19 . The method of  claim 12 , wherein identifying the one or more tumor-derived mutations in the human subject based on the comparison of the one or more potential sequence variants to the consensus sequence comprises removing variants of the consensus sequence from the one or more potential sequence variants. 
     
     
         20 . The method of  claim 12 , wherein determining the consensus sequence from the second sequence reads obtained from sequencing DNA molecules from the other sample of the human subject comprises determining a consensus sequence that includes a determination of a homozygous locus or a heterozygous locus having two alleles. 
     
     
         21 . The method of  claim 12 , wherein the other sample is a constitutional sample.