Systems and methods to detect rare mutations and copy number variation

Abstract

The present disclosure provides a system and method for the detection of rare mutations and copy number variations in cell free polynucleotides. Generally, the systems and methods comprise sample preparation, or the extraction and isolation of cell free polynucleotide sequences from a bodily fluid; subsequent sequencing of cell free polynucleotides by techniques known in the art; and application of bioinformatics tools to detect rare mutations and copy number variations as compared to a reference. The systems and methods also may contain a database or collection of different rare mutations or copy number variation profiles of different diseases, to be used as additional references in aiding detection of rare mutations, copy number variation profiling or general genetic profiling of a disease.

What is claimed is: 
     
         1 . A method comprising:
 (a) providing a sample comprising initial DNA molecules;   (b) incubating said sample with at least a 40× molar excess of polynucleotide adapters comprising barcodes as compared to said initial DNA molecules, wherein said polynucleotide adapters are bidirectional adapters; and   (c) without performing tailing with forked adapters on said initial DNA molecules, ligating said polynucleotide adapters to one or more ends of said initial DNA molecules, thereby providing tagged DNA molecules,   wherein said initial DNA molecules are converted into said tagged DNA molecules with a conversion efficiency of at least 20%.   
     
     
         2 . The method of  claim 1 , wherein said conversion efficiency is at least 30%. 
     
     
         3 . The method of  claim 2 , wherein said conversion efficiency is at least 40%. 
     
     
         4 . The method of  claim 3 , wherein said conversion efficiency is at least 50%. 
     
     
         5 . The method of  claim 4 , wherein said conversion efficiency is at least 60%. 
     
     
         6 . The method of  claim 1 , wherein (b) comprises incubating said sample with at least 60× molar excess of polynucleotide adapters comprising barcodes. 
     
     
         7 . The method of  claim 6 , wherein (b) comprises incubating said sample with at least 80× molar excess of polynucleotide adapters comprising barcodes. 
     
     
         8 . The method of  claim 7 , wherein (b) comprises incubating said sample with at least 100× molar excess of polynucleotide adapters comprising barcodes. 
     
     
         9 . The method of  claim 1 , wherein said initial DNA molecules are uniquely tagged. 
     
     
         10 . The method of  claim 1 , wherein said initial DNA molecules are non-uniquely tagged. 
     
     
         11 . The method of  claim 1 , wherein said initial DNA molecules are cell-free DNA. 
     
     
         12 . The method of  claim 11 , wherein said cell-free DNA has a distribution of fragments with a peak at about 160 nucleotides. 
     
     
         13 . The method of  claim 11 , wherein said cell-free DNA has a distribution of fragments including sizes ranging from 140 nucleotides to 180 nucleotides. 
     
     
         14 . The method of  claim 1 , further comprising (i) sequencing at least a portion of said tagged DNA molecules, thereby providing a set of sequencing reads, and (ii) analyzing said set of sequencing reads to detect rare variants in said initial DNA molecules. 
     
     
         15 . The method of  claim 14 , wherein no more than 1% of said initial DNA molecules are derived from a genome comprising said rare variants. 
     
     
         16 . The method of  claim 14 , wherein no more than 0.5% of said initial DNA molecules are derived from a genome comprising said rare variants. 
     
     
         17 . The method of  claim 14 , wherein no more than 0.1% of said initial DNA molecules are derived from a genome comprising said rare variants. 
     
     
         18 . The method of  claim 14 , wherein said analyzing detects said rare variants with a lower sensitivity threshold of about 1% of said initial DNA molecules from a given locus comprising a variant within a population of said initial DNA molecules from said given locus that do not comprise said variant. 
     
     
         19 . The method of  claim 14 , wherein said analyzing detects said rare variants with a lower sensitivity threshold of about 0.5% of said initial DNA molecules from a given locus comprising a variant within a population of said initial DNA molecules from said given locus that do not comprise said variant. 
     
     
         20 . The method of  claim 14 , wherein said analyzing detects said rare variants with a lower sensitivity threshold of about 0.1% of said initial DNA molecules from a given locus comprising a variant within a population of said initial DNA molecules from said given locus that do not comprise said variant. 
     
     
         21 . The method of  claim 14 , further comprising:
 a. prior to said sequencing, amplifying at least a portion said tagged DNA molecules to produce a corresponding set of amplified progeny polynucleotides;   b. sequencing a subset of said set of amplified progeny polynucleotides to produce said set of sequencing reads; and   c. collapsing said set of sequencing reads to generate a set of consensus sequences, each consensus sequence corresponding to a unique polynucleotide among said set of tagged DNA molecules.   
     
     
         22 . The method of  claim 21 , wherein said generation of consensus sequences is based on information from a tag of said tagged DNA molecules and/or at least one of (i) sequence information at the beginning (start) region of a sequencing read of said set of sequencing reads, (ii) the end (stop) regions of the sequencing read and (iii) the length of the sequencing read. 
     
     
         23 . The method of  claim 21 , wherein said collapsing comprises:
 (i) grouping sequences reads sequenced from amplified progeny polynucleotides into families, each family amplified from the same tagged parent polynucleotide; and   (ii) determining a consensus sequence based on sequence reads in a family.   
     
     
         24 . The method of  claim 21 , wherein said subset of said set of amplified progeny polynucleotides that is sequenced is of sufficient size such that a nucleotide sequence represented in said tagged DNA molecules, at a same percentage as a percentage per-base sequencing error rate of a sequencing platform used, has at least a 95% chance of being represented among the set of consensus sequences. 
     
     
         25 . The method of  claim 24 , wherein said nucleotide sequence represented in said tagged DNA molecules has at least a 98% chance of being represented among said set of consensus sequences. 
     
     
         26 . The method of  claim 24 , wherein said nucleotide sequence represented in said tagged DNA molecules has at least a 99% chance of being represented among said set of consensus sequences. 
     
     
         27 . The method of  claim 24 , wherein said nucleotide sequence represented in said tagged DNA molecules has at least a 99.9% chance of being represented among said set of consensus sequences. 
     
     
         28 . The method of  claim 24 , wherein said percentage per-base sequencing error rate is between 0.5 and 1%. 
     
     
         29 . The method of  claim 24 , wherein said percentage per-base sequencing error rate is between 2% and 3%. 
     
     
         30 . The method of  claim 23 , further comprising determining a quantitative measure of unique families; and based on (i) the quantitative measure of unique families and (ii) a quantitative measure of sequence reads in each family, inferring a measure of unique tagged parent polynucleotides in the set.