SYSTEMS AND METHODS TO DETECT RARE MUTATIONS AND COPY NUMBER VARIATION
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.
1 . A method for detecting copy number variation comprising:
a. sequencing extracellular polynucleotides from a bodily sample from a subject, wherein each of the extracellular polynucleotide generate a plurality of sequencing reads;
b. filtering out reads that fail to meet a set threshold;
c. mapping the sequence reads obtained from step (a) to a reference sequence;
d. quantifying or enumerating mapped reads in two or more predefined regions of the reference sequence;
e. determining copy number variation in one or more of the predefined regions by:
i. normalizing number of reads in the predefined regions to each other and/or the number of unique sequence reads in the predefined regions to one other;
ii. comparing the normalized numbers obtained in step (i) to normalized numbers obtained from a control sample.
2 . A method for detecting a rare mutation in a cell-free or substantially cell free sample obtained from a subject comprising:
a. sequencing extracellular polynucleotides from a bodily sample from a subject, wherein each of the extracellular polynucleotide generate a plurality of sequencing reads; sequencing extracellular polynucleotides from a bodily sample from a subject, wherein each of the extracellular polynucleotide generate a plurality of sequencing reads;
b. performing multiplex sequencing on regions or whole-genome sequencing if enrichment is not performed;
c. filtering out reads that fail to meet a set threshold;
d. mapping sequence reads derived from the sequencing onto a reference sequence;
e. identifying a subset of mapped sequence reads that align with a variant of the reference sequence at each mappable base position;
f. for each mappable base position, calculating a ratio of (a) a number of mapped sequence reads that include a variant as compared to the reference sequence, to (b) a number of total sequence reads for each mappable base position;
g. normalizing the ratios or frequency of variance for each mappable base position and determining potential rare variant(s) or mutation(s);
h. and comparing the resulting number for each of the regions with potential rare variant(s) or mutation(s) to similarly derived numbers from a reference sample.
3 . A method of characterizing the heterogeneity of an abnormal condition in a subject, the method comprising generating a genetic profile of extracellular polynucleotides in the subject, wherein the genetic profile comprises a plurality of data resulting from copy number variation and rare mutation analyses.
4 - 49 . (canceled)
50 . A method comprising: determining copy number variation or rare mutation analysis in a cell-free or substantially cell free sample obtained from a subject using multiplex sequencing.
51 - 69 . (canceled)
70 . A system comprising a computer readable medium for performing the following steps: selecting predefined regions in a genome; enumerating number of sequence reads in the predefined regions; normalizing the number of sequence reads across the predefined regions; and determining percent of copy number variation in the predefined regions.
71 - 76 . (canceled)
77 . A method comprising:
a. providing at least one set of tagged parent polynucleotides, and for each set of tagged parent polynucleotides;
b. amplifying the tagged parent polynucleotides in the set to produce a corresponding set of amplified progeny polynucleotides;
c. sequencing a subset (including a proper subset) of the set of amplified progeny polynucleotides, to produce a set of sequencing reads; and
d. collapsing the set of sequencing reads to generate a set of consensus sequences, each consensus sequence corresponding to a unique polynucleotide among the set of tagged parent polynucleotides.
78 . The method of claim 77 wherein each polynucleotide in a set is mappable to a reference sequence.
79 . The method of claim 77 comprising providing a plurality of sets of tagged parent polynucleotides, wherein each set is mappable to a different reference sequence.
80 . The method of claim 77 further comprising: e. analyzing the set of consensus sequences for each set of tagged parent molecules.
81 . The method of claim 77 further comprising converting initial starting genetic material into the tagged parent polynucleotides.
82 . The method of claim 81 wherein the initial starting genetic material comprises no more than 100 ng of polynucleotides.
83 . The method of claim 81 comprising bottlenecking the initial starting genetic material prior to converting.
84 . The method of claim 81 comprising converting the initial starting genetic material into tagged parent polynucleotides with a conversion efficiency of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 80% or at least 90%.
85 . The method of claim 81 wherein converting comprises any of blunt-end ligation, sticky end ligation, molecular inversion probes, PCR, ligation-based PCR, single strand ligation and single strand circularization.
86 . The method of claim 81 wherein the initial starting genetic material is cell-free nucleic acid.
87 . The method of claim 86 wherein a plurality of the reference sequences are from the same genome.
88 . The method of claim 77 wherein each tagged parent polynucleotide in the set is uniquely tagged.
89 - 103 . (canceled)
104 . A method comprising detecting genetic variation in initial starting genetic material with a sensitivity of at least 5%, at least 1%, at least 0.5%, at least 0.1% or at least 0.05%.
105 . (canceled)
106 . A system comprising a computer readable medium for performing the following steps:
a. providing at least one set of tagged parent polynucleotides, and for each set of tagged parent polynucleotides;
b. amplifying the tagged parent polynucleotides in the set to produce a corresponding set of amplified progeny polynucleotides;
c. sequencing a subset (including a proper subset) of the set of amplified progeny polynucleotides, to produce a set of sequencing reads;
d. collapsing the set of sequencing reads to generate a set of consensus sequences, each consensus sequence corresponding to a unique polynucleotide among the set of tagged parent polynucleotides and, optionally,
e. analyzing the set of consensus sequences for each set of tagged parent molecules.
107 . A method comprising detecting the presence or absence of genetic alteration and/or amount of genetic variation in an individual, wherein the detecting is performed with the aid of sequencing of cell-free nucleic acid, wherein at least 10% of the individual's genome is sequenced.
108 - 133 . (canceled)