SYSTEMS AND METHODS TO DETECT RARE MUTATIONS AND COPY NUMBER VARIATION

• US 20180327862A1
• Filed: 05/14/2018
• Published: 11/15/2018
• 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 10,501,808 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• 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 system comprising:

a communication interface that receives, over a communication network, sequencing reads comprising cfDNA molecule sequences and non-unique molecular barcode sequences generated by a nucleic acid sequencer; and

a computer in communication with the communication interface, wherein the computer comprises one or more computer processors and a computer readable medium comprising machine-executable code that, upon execution by the one or more computer processors, implements a method comprising;

(i) receiving, over the communication network, the sequencing reads generated by the nucleic acid sequencer;

(ii) mapping the sequence reads to one or more reference sequences from a human genome;

(iii) grouping the sequence reads into families, each of the families comprising sequence reads comprising identical barcode sequences and having the same start and stop positions, wherein each of the families comprises sequencing reads corresponding to a same original cell free deoxyribonucleic acid (cfDNA) molecule;

(iv) at each genetic locus of a plurality of genetic loci in the one or more reference sequences, collapsing sequence reads in each family to yield a base call for each family at the genetic locus; and

(v) measuring a frequency of one or more bases called at the locus from among the families.

2. The system of claim 1, wherein the nucleic acid sequencer sequences a sequencing library generated from cfDNA molecules derived from a subject, wherein the sequencing library comprises the cfDNA molecules and adapters comprising the non-unique molecular barcode sequences.

3. The system of claim 1, wherein the nucleic acid sequencer performs sequencing-by-synthesis on the sequencing library to generate the sequencing reads.

4. The system of claim 1, wherein the nucleic acid sequencer performs pyrosequencing, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation or sequencing-by-hybridization on the sequencing library to generate the sequencing reads.

5. The system of claim 1, wherein the nucleic acid sequencer uses a clonal single molecule array derived from the sequencing library to generate the sequencing reads.

6. The system of claim 1, wherein the nucleic acid sequencer comprises a chip having an array of microwells for sequencing the sequencing library to generate the sequencing reads.

7. The system of claim 1, wherein the computer readable medium comprises a memory, a hard drive or a computer server.

8. The system of claim 1, wherein the communication network comprises a telecommunication network, an internet, an extranet, or an intranet.

9. The system of claim 1, wherein the communication network includes one or more computer servers capable of distributed computing.

10. The system of claim 9, wherein distributed computing is cloud computing.

11. The system of claim 1, wherein the computer is located on a computer server that is remotely located from the nucleic acid sequencer.

12. The system of claim 2, wherein the sequencing library further comprises sample barcodes that differentiate a sample from one or more samples.

13. The system of claim 2, wherein the adapters are on both ends of the cfDNA molecule.

14. The system of claim 2, wherein the non-unique molecular barcode sequences comprise between 2 to 100,000 different barcodes.

15. The system of claim 14, wherein the non-unique molecular barcode sequences comprise between 10 to 100 different identifiers.

16. The system of claim 2, wherein the adapters comprising the non-unique molecular barcode sequences are randomly attached to the cfDNA molecules.

17. The system of claim 1, further comprising:

an electronic display in communication with the computer over a network, wherein the electronic display comprises a user interface for displaying results upon implementing (i)-(v).

18. The system of claim 17, wherein the user interface is a graphical user interface (GUI) or web-based user interface.

19. The system of claim 17, wherein the electronic display is in a personal computer.

20. The system of claim 17, wherein the electronic display is in an internet enabled computer.

21. The system of claim 20, wherein the internet enabled computer is located at a location remote from the computer.

22. The system of claim 1, wherein the method further comprises:

(vi) counting families mapping to each genetic locus of a plurality of genetic loci.

23. A system comprising:

a communication interface that receives, over a communication network, sequencing reads comprising cfDNA molecule sequences and non-unique molecular barcode sequences generated by a nucleic acid sequencer; and

a computer in communication with the nucleic acid sequencer over a communication network, wherein the computer comprises one or more computer processors and a computer readable medium comprising non-transitory machine-executable code that, upon execution by the one or more computer processors, reduces noise and distortion in the signal by;

(i) receiving, over the communication network, the sequencing reads generated by the nucleic acid sequencer;

(ii) mapping the sequence reads to one or more reference sequences from a human genome;

(iii) grouping the sequence reads into families, each of the families comprising sequence reads comprising identical barcodes and having the same start and stop positions, wherein each of the families comprises sequencing reads corresponding to a same original cell free deoxyribonucleic acid (cfDNA) molecule from the cfDNA molecules;

(iv) at each genetic locus of a plurality of genetic loci in the one or more reference sequences;

(1) collapsing sequence reads in each family to yield a base call for each family at the genetic locus; and

measuring a frequency of one or more bases called at the locus from among the families; and

(2) counting families mapping to each genetic locus of a plurality of genetic loci.

24. The system of claim 23, wherein the nucleic acid sequencer sequences a sequencing library generated from cfDNA molecules derived from a subject, wherein the sequencing library comprises the cfDNA molecules and adapters comprising the non-unique molecular barcode sequences.

25. The system of claim 24, wherein adapters comprising the non-unique molecular barcode sequences are located at both ends of the cfDNA molecules.

26. The system of claim 24, wherein the adapters comprising the non-unique molecular barcode sequences are randomly attached to the cfDNA molecules.

27. The system of claim 24, wherein the non-unique molecular barcode sequences comprise between 5 to 100 different identifiers.

28. The system of claim 23, wherein the nucleic acid sequencer performs next generation sequencing to generate the sequencing reads.

29. The system of claim 23, wherein the computer readable medium comprises a memory, a hard drive or a computer server.

30. The system of claim 23, wherein the communication network comprises a telecommunication network, an internet, an extranet, or an intranet.