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 a presence or an absence of one or more somatic genetic variants, the method comprising:
(a) non-uniquely tagging a plurality of cell-free deoxyribonucleic acid (cfDNA) molecules from a human subject with adapters comprising molecular barcodes to produce non-uniquely tagged parent polynucleotides, wherein the adapters are ligated to both ends of molecules of cfDNA from the plurality of cfDNA molecules, wherein the plurality of cfDNA molecules comprises a subset of cfDNA molecules that map to a mappable base position of a human reference genome, and wherein the plurality of cfDNA molecules is tagged with a number of different molecular barcodes ranging from at least 2 to fewer than a number of cfDNA molecules of the subset of cfDNA molecules that map to the mappable base position;
(b) amplifying a plurality of the non-uniquely tagged parent polynucleotides to produce progeny polynucleotides with associated molecular barcodes;
(c) sequencing a plurality of the progeny polynucleotides to produce sequencing reads of the progeny polynucleotides with associated molecular barcodes; and
(d) detecting, from among a plurality of the sequencing reads, the presence or the absence of the one or more somatic genetic variants, based at least in part on the associated molecular barcodes of the sequencing reads.
2. The method of claim 1 , wherein the human subject has a cancer or is suspected of having a cancer.
3. The method of claim 1 , wherein the non-uniquely tagging comprises blunt-end ligation or sticky-end ligation.
4. The method of claim 1 , wherein the molecular barcodes are from a set of molecular barcodes having 5 to 1,000 different nucleotide sequences that are 5 to 20 nucleotides in length.
5. The method of claim 1 , further comprising selectively enriching the progeny polynucleotides for target regions associated with cancer.
6. The method of claim 1 , further comprising filtering out one or more sequencing reads from among the sequencing reads that fail to meet a quality score threshold.
7. The method of claim 1 , further comprising mapping a plurality of the sequencing reads to a human reference sequence to produce mapped sequencing reads.
8. The method of claim 7 , further comprising grouping a plurality of the mapped sequencing reads into families based on sequence information of the molecular barcodes, a start base position of a given mapped sequencing read from among the mapped sequencing reads at which the given mapped sequencing read is determined to start mapping to a human reference sequence, and a stop base position of the given mapped sequencing read at which the given mapped sequencing read is determined to stop mapping to the human reference sequence.
9. The method of claim 8 , further comprising generating a set of consensus sequences from among the mapped sequencing reads in the families.
10. The method of claim 9 , further comprising detecting the presence or the absence of the one or more somatic genetic variants in the set of consensus sequences as compared to the human reference sequence.
11. The method of claim 10 , further comprising quantifying a number of consensus sequences from the set of consensus sequences that comprise one or more somatic genetic variants as compared to the human reference sequence.
12. The method of claim 8 , wherein the detecting comprises generating a base call at a genetic locus of the human reference sequence for a family from among the families.
13. The method of claim 8 , further comprising quantifying a number of the families.
14. The method of claim 1 , wherein the one or more somatic genetic variants comprise a single nucleotide variant (SNV), a copy number variation (CNV), an insertion or deletion (indel), a gene fusion, or any combination thereof.
15. The method of claim 1 , wherein the detecting further comprises detecting cancer in the human subject when the presence of the one or more somatic genetic variants is detected.
16. The method of claim 1 , further comprising generating a tumor mutation profile of the human subject based on the detected presence or absence of the one or more somatic genetic variants.
17. The method of claim 16 , wherein the tumor mutation profile is generated based on a presence or an absence of one or more somatic genetic variants detected from different samples obtained from the human subject at different time points.
18. The method of claim 1 , further comprising performing (a) to (d) in combination with immune repertoire profiling.
19. The method of claim 1 , wherein the one or more somatic genetic variants are located in a microsatellite region.
20. The method of claim 19 , wherein the one or more somatic genetic variants comprise an indel.
21. A method for detecting a presence or an absence of one or more somatic genetic variants, the method comprising:
(a) non-uniquely tagging a plurality of cell-free deoxyribonucleic acid (cfDNA) molecules from a human subject with molecular barcodes to produce non-uniquely tagged parent polynucleotides, wherein the molecular barcodes are ligated to both ends of molecules of cfDNA from the plurality of cfDNA molecules, wherein the plurality of cfDNA molecules comprises a subset of cfDNA molecules that map to a mappable base position of a human reference genome, and wherein the plurality of cfDNA molecules is tagged with a number of different molecular barcodes ranging from at least 2 to fewer than a number of cfDNA molecules of the subset of cfDNA molecules that map to the mappable base position;
(b) amplifying a plurality of the non-uniquely tagged parent polynucleotides to produce progeny polynucleotides with associated molecular barcodes;
(c) selectively enriching the progeny polynucleotides for target regions associated with cancer, to produce enriched progeny polynucleotides;
(d) sequencing a plurality of the enriched progeny polynucleotides to produce sequencing reads of the plurality of the enriched progeny polynucleotides with associated molecular barcodes;
(e) mapping a plurality of the sequencing reads to a human reference sequence to produce mapped sequencing reads;
(f) grouping a plurality of the mapped sequencing reads into families based on sequence information of the molecular barcodes, a start base position of a given mapped sequencing read from among the mapped sequencing reads at which the given mapped sequencing read is determined to start mapping to the human reference sequence, and a stop base position of the given mapped sequencing read at which the given mapped sequencing read is determined to stop mapping to the human reference sequence; and
(g) detecting, from among a plurality of the families, the presence or the absence of the one or more somatic genetic variants comprising a single nucleotide variant (SNV), a copy number variation (CNV), an insertion or deletion (indel), a gene fusion, or any combination thereof.
22. The method of claim 21 , wherein the plurality of cfDNA molecules comprises between 1 nanogram (ng) and 100 ng of cfDNA molecules.
23. The method of claim 21 , wherein the molecular barcodes are from a set of molecular barcodes having 5 to 1,000 different nucleotide sequences that are 5 to 20 nucleotides in length.
24. The method of claim 21 , wherein the molecular barcodes are part of adapter sequences.
25. The method of claim 21 , further comprising filtering out one or more sequencing reads from among the sequencing reads or the mapped sequencing reads that fail to meet a set accuracy, quality score, or mapping score threshold.
26. The method of claim 21 , further comprising generating a set of consensus sequences from among the mapped sequencing reads in the families.
27. The method of claim 26 , further comprising quantifying a number of consensus sequences from the set of consensus sequences that comprise one or more somatic genetic variants as compared to the human reference sequence.
28. The method of claim 21 , further comprising quantifying a number of the families.
29. The method of claim 21 , wherein 0.1% to 1.0% of the cfDNA molecules in the plurality of cfDNA molecules are from one or more cancer genomes.
30. The method of claim 21 , further comprising detecting cancer in the human subject when the presence of the one or more somatic genetic variants is detected.