Method for detecting a genetic variant

• US 10,640,819 B2
• Filed: 07/17/2015
• Issued: 05/05/2020
• Est. Priority Date: 07/18/2014
• Status: Active Grant

• Current Assignee: Cancer Research Technology Limited (Cancer Research UK)
• Original Assignee: Cancer Research Technology Limited (Cancer Research UK)
• Inventors: Rosenfeld, Nitzan, Forshew, Tim, Marass, Francesco, Murtaza, Muhammed
• Primary Examiner(s): Woitach, Joseph
• Application Number:
  US15/325,046
• Publication Number:
  US 20170204455A1
• Time in Patent Office: 1,754 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2527/146 Concentration of target or template
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection

Abstract

The present invention provides a method for detecting a genetic variant in a region of interest in a DNA sample comprising (i) determining, for a given sequencing platform, sequencing process and sequencing depth, the distribution of the number of reads supporting a genetic variant or plurality of genetic variants expected to be observed in the sequencing results of amplification reactions due to amplification and sequencing error (read count distribution); (ii) based on the read count distribution determined in step (i), establishing a threshold frequency at or above which the genetic variant must be observed in sequencing results of amplification reactions to assign a positive determination for the presence of the genetic variant in a given amplification reaction; (iii) partitioning the DNA sample into a plurality of replicate amplification reactions, so that the mean number of amplifiable template molecules of the region of interest in a replicate amplification reaction is fewer than the reciprocal of the threshold frequency determined in step (ii); (iv) performing the amplification reactions of step (iii) and sequencing the products of amplification reactions, (v) based on step (ii) and the results of step (iv), determining the presence/absence of the genetic variant in each replicate amplification reaction; and (vi) integrating the results of (v) to determine the presence/absence of the genetic variant in the region of interest in the DNA sample.

Claims

1. A method for detecting a genetic variant in a region of interest in a DNA sample comprising

(i) determining, for a given sequencing platform, sequencing process and sequencing depth, the distribution of the number of reads supporting a genetic variant or plurality of genetic variants expected to be observed in the sequencing results of amplification reactions due to amplification and sequencing error (read count distribution);

(ii) based on the read count distribution determined in step (i), establishing a threshold frequency at or above which each genetic variant must be observed in sequencing results of amplification reactions to assign a positive determination for the presence of the genetic variant in a given amplification reaction;

(iii) partitioning the DNA sample into a plurality of replicate amplification reactions, so that the mean number of amplifiable template molecules of the region of interest in a replicate amplification reaction is fewer than the reciprocal of the threshold frequency determined in step (ii);

(iv) performing the amplification reactions of step (iii) and sequencing the products of amplification reactions,

(v) based on step (ii) and the results of step (iv), determining the presence/absence of the genetic variant in each replicate amplification reaction; and

(vi) integrating the results of (v) to determine the presence/absence of the genetic variant in the region of interest in the DNA sample.

2. The method according to claim 1, wherein for the genetic variant to be determined as being present in the region of interest in the DNA sample in step (vi), a positive determination for the presence of the genetic variant must be made in more than one replicate amplification reaction in step (v).

3. The method according to claim 2, wherein for the genetic variant to be determined as being present in the region of interest in the DNA sample in step (vi), a positive determination for the presence of the genetic variant must be made in at least 3 replicate amplification reactions in step (v).

4. The method according to claim 1, wherein the threshold frequency of step (ii) is the frequency at which the cumulative distribution function value for the probability of observing the genetic variant at that frequency is 0.99 or greater.

5. The method according to claim 1, wherein threshold frequency of step (ii) is determined using a binomial, over-dispersed binomial, Beta, Normal, Exponential or Gamma probability distribution model.

6. The method according to claim 1, wherein the threshold frequency of step (ii) is the frequency where the z-score is 20.

7. The method according to claim 1, wherein establishing a threshold frequency at or above which the genetic variant must be observed in sequencing results of amplification reactions to assign a positive determination for the presence of the genetic variant in a given amplification reaction comprises

(a) based on the read count distribution determined for a plurality of genetic variants in step (i), establishing a plurality of threshold frequencies at or above which the genetic variants should be observed in sequencing results of amplification reactions to assign a positive determination for the presence of the genetic variant in a given amplification reaction, and

(b) based on step (a), establishing an overall threshold frequency at or above which a genetic variant must be observed in sequencing results of a given amplification reaction to assign a positive determination for the presence of the genetic variant in that amplification reaction, which is the threshold frequency at which at least 90% of the plurality of threshold frequencies determined in step (a) are less than this value.

8. The method according to claim 1, wherein the mean number of amplifiable template molecules to be present in a replicate amplification reaction is the number at which when the genetic variant is present in a single amplifiable template molecule of a replicate amplification reaction, the probability that a positive determination will be made for that replicate is 0.9 or greater.

9. The method according to claim 1, wherein step (i) comprises sequencing a DNA sample multiple times to determine the read count distribution for a genetic variant or plurality of genetic variants.

10. The method according to claim 1, wherein the read count distribution at which a genetic variant or plurality of genetic variants is expected to be observed in the sequencing results of amplification reactions due to amplification and sequencing error is determined in step (i) based on sequencer and/or polymerase error rates, optionally taking sequence context into account.

11. The method according to claim 1, wherein following partitioning of the DNA sample in step (iii), each replicate amplification reaction has more than a single amplifiable template molecule for the region of interest per replicate amplification reaction.

12. The method according to claim 11, wherein the mean number of amplifiable template molecules per replicate amplification reaction is more than 1 and less than 1000.

13. The method according to claim 1, wherein partitioning the DNA sample into a plurality of replicate amplification reactions comprises diluting the DNA sample and aliquoting into replicate amplification reactions.

14. The method according to claim 1, wherein replicate amplification reactions are performed in parallel, and sequencing of the products of amplification reactions is performed in parallel.

15. The method according to claim 1, wherein amplification reactions are performed using one or more primers flanking the region of interest and which integrate sample and/or amplification reaction replicate specific identifier sequences into the products of amplification and/or wherein the primers integrate sequence adapters into the products of amplification.

16. The method according to claim 1, wherein a plurality of regions of interest are analysed in parallel.

17. The method according to claim 1, wherein the method further comprises:

(vii) determining the frequency of the genetic variant in the DNA sample.

18. The method according to claim 1, wherein the read count distribution is defined as a normal distribution characterised by parameters which are the mean frequency and variance of the frequency at which a genetic variant or plurality of genetic variants is observed or expected to be observed in the sequencing results of amplification reactions due to amplification and sequencing error.

20. A method for diagnosis or prognosis or monitoring a disease, for selecting a patient for a therapy or identifying genetic variation predictive of susceptibility, or for evaluating response or resistance to a therapy, said method comprising carrying out the method of claim 1 on a DNA sample obtained from a subject who has or is suspected of having a disease or who is undergoing therapy to treat a disease.

19. A method for detecting and/or quantifying a genetic variant in a sample of cell-free DNA obtained from a cancer patient, the method comprising:

(i) determining, for a given sequencing platform, sequencing process and sequencing depth, the distribution of the number of reads supporting a genetic variant or plurality of such genetic variants expected to be observed in the sequencing results of amplification reactions due to amplification and sequencing error (read count distribution);

(ii) based on the read count distribution determined in step (i), establishing a threshold frequency at or above which the genetic variant must be observed in sequencing results of amplification reactions to assign a positive determination for the presence of the genetic variant in a given amplification reaction;

(iii) partitioning the DNA sample obtained from a subject into a plurality of replicate amplification reactions, so that the mean number of amplifiable template molecules of the region of interest in a replicate amplification reaction is fewer than the reciprocal of the threshold frequency determined in step (ii);

(iv) performing the amplification reactions of step (iii) and sequencing the products of amplification reactions,

(v) based on step (ii) and the results of step (iv), determining the presence/absence of a genetic variant in each replicate amplification reaction,

(vi) integrating the results of (v) to detect the presence of the genetic variant in the region of interest in the DNA sample, thereby detecting and/or quantifying the genetic variant in the sample of cell-free DNA from the cancer patient.