SYSTEMS AND METHODS TO DETECT RARE MUTATIONS AND COPY NUMBER VARIATION

• US 20160251704A1
• Filed: 03/21/2016
• Published: 09/01/2016
• Est. Priority Date: 09/04/2012
• Status: Active Grant

• Current Assignee: Guardant Health Inc.
• Original Assignee: Guardant Health Inc.
• Inventors: Talasaz, AmirAli, Eltoukhy, Helmy
• Granted Patent: US 9,902,992 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

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.

Claims

1. A method for detecting genetic aberrations in cfDNA (“

cfDNA”

) from a subject, comprising;

a) providing cfDNA molecules obtained from a bodily sample of the subject;

b) attaching to the cfDNA molecules, tags comprising barcodes having distinct barcode sequences to generate tagged parent polynucleotides, wherein at least 10% of the cfDNA is tagged by the attaching step;

c) amplifying the tagged parent polynucleotides to produce tagged progeny polynucleotides;

d) sequencing the tagged progeny polynucleotides to produce sequence reads, wherein each sequence read comprises a barcode sequence and a sequence derived from a cfDNA molecule;

e) grouping the sequence reads into families based at least on the barcode sequence;

f) comparing the sequence reads grouped within each family to determine consensus sequences for each family, wherein each of the consensus sequences corresponds to a unique polynucleotide among the tagged parent polynucleotides; and

g) detecting, at one or more genetic loci, a plurality of genetic aberrations, wherein the genetic aberrations are differently selected from;

a single base substitution, a copy number variation, an indel and a gene fusion.

2. The method of claim 1, comprising providing less than 100 ng of the cfDNA.

3. The method of claim 1, comprising providing less than 10 ng of the cfDNA.

4. The method of claim 1, comprising providing between 100 and 100,000 human haploid genome equivalents of cfDNA, wherein the cfDNA molecules are tagged with between 2 and 1,000,000 unique identifiers.

5. The method of claim 1, comprising providing between 1000 and 50,000 human haploid genome equivalents of cfDNA, wherein the cfDNA molecules are tagged with between 2 and 1,000 unique identifiers.

6. The method of claim 1, wherein each barcode sequence is at least 5 nucleotides in length.

7. The method of claim 1, wherein the attaching comprises tagging the cfDNA molecules with at least 10 and at most 1000 distinct barcodes.

8. The method of claim 1, wherein the attaching comprises uniquely tagging the cfDNA molecules.

9. The method of claim 1, wherein the attaching comprises performing blunt-end ligation or sticky end ligation.

10. The method of claim 1, wherein the attaching comprises using more than 10×

molar excess of barcode oligonucleotides as compared to the provided cfDNA molecules.

11. The method of claim 1, wherein the attaching comprises ligating adaptors comprising barcodes to both ends of the cfDNA molecules.

12. The method of claim 1, wherein the attaching comprises non-uniquely tagging the cfDNA molecules such that no more than 5% of the parent polynucleotides are uniquely tagged.

13. The method of claim 1, wherein at least 30% of the cfDNA molecules are tagged by the attaching step.

14. The method of claim 1, wherein at least 50% of the cfDNA molecules are tagged by the attaching step.

15. The method of claim 1 further comprising selectively enriching sequences of interest prior to the sequencing.

16. The method of claim 15, wherein the enriching comprises enriching the following genes:

KRAS, APC and TP53.

17. The method of claim 1, wherein sequencing comprises massively parallel sequencing.

18. The method of claim 1, wherein the amplified progeny polynucleotides are sequenced to produce an average of 5 to 10 sequence reads for each family.

19. The method of claim 1, wherein the consensus sequence possesses an error rate below 0.0001%.

20. The method of claim 1, wherein each base of the tagged parent polynucleotides has at least 99% chance of being represented by at least one sequence read.

21. The method of claim 4, wherein grouping the sequence reads is further based on one or more of:

sequence information at a beginning of the sequence derived from the cfDNA molecule, sequence information at an end of the sequence derived from the cfDNA molecule, and length of the sequence read.

22. The method of claim 4, wherein grouping the sequence reads is further based on a plurality of:

sequence information at a beginning of the sequence derived from the cfDNA molecule, sequence information at an end of the sequence derived from the cfDNA molecule, and length of the sequence read.

23. The method of claim 1, wherein at least one single base substitution is detected.

24. The method of claim 1, wherein at least one copy number variation is detected.

25. The method of claim 1, wherein at least one indel is detected.

26. The method of claim 1, wherein at least one gene fusion is detected.

27. The method of claim 1, wherein at least one single base substitution and at least one copy number variation is detected.

28. The method of claim 1, further comprising detecting, at one or more genetic loci, one or more genetic aberrations selected from:

a transversion, a translocation, an inversion, a deletion, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, chromosome fusions, a gene truncation, a gene amplification, a gene duplication, a chromosomal lesion, a DNA lesion, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns and abnormal changes in nucleic acid methylation.

29. The method of claim 23, wherein the single base substitution is detected with a sensitivity of at least 1%.

30. The method of claim 23, wherein the single base substitution is detected with a sensitivity of at least 0.1%.