Nucleic acids and methods for detecting chromosomal abnormalities

• US 10,465,245 B2
• Filed: 07/29/2016
• Issued: 11/05/2019
• Est. Priority Date: 07/29/2015
• Status: Active Grant

• Current Assignee: Biora Therapeutics Incorporated
• Original Assignee: Progenity, Inc. (Athyrium Capital Management LP)
• Inventors: Mann, Tobias, Wang, Heng, Kim, Jung H., Sekedat, Matthew
• Primary Examiner(s): Brusca, John S
• Application Number:
  US15/224,463
• Publication Number:
  US 20170183731A1
• Time in Patent Office: 1,194 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• 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 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• 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
• CPC Class Codes: G16H 50/20 for computer-aided diagnosis, e.g. based on medical expert systems

Abstract

Methods and nucleic acid molecules for detecting chromosomal abnormalities such as aneuploidy. Methods for selecting nucleic acid molecules for use in the methods of the disclosure.

Claims

1. A method of characterizing fetal DNA in a maternal blood sample, comprising:

a) obtaining a nucleic acid sample isolated from a maternal blood sample;

b) capturing a plurality of target sequences of interest in the nucleic acid sample obtained in step a) by using one or more populations of molecular inversion probes (MIPs) to produce a plurality of replicons,

wherein each of the MIPs in the population of MIPs comprises in sequence the following components;

first targeting polynucleotide arm—

first unique molecular tag—

polynucleotide linker second unique molecular tag—

second targeting polynucleotide arm;

wherein the pair of first and second targeting polynucleotide arms in each of the MIPs are substantially complementary to first and second regions in the nucleic acid that, respectively, flank each sequence in the plurality of target sequences of interest;

wherein the first and second unique targeting molecular tags in each of the MIPs in combination are distinct in each of the MIPs;

c) sequencing a plurality of MIPs amplicons that are amplified from the replicons obtained in step b);

d) determining the number of capture events of each of a first population of amplicons of the plurality of amplicons provided in step c) based on the number of the unique molecular tags of each MIP that amplified a replicon, wherein the first population of amplicons is determined by the sequence of the target sequence of interest;

e) determining the number of capture events of each of a second population of amplicons of the plurality of amplicons provided in step c) based on the number of the unique molecular tags of each MIP that amplified a replicon, wherein the second population of amplicons is determined by the sequence of the target sequence of interest;

f) determining, for each target sequence of interest from which the first population of amplicons was produced, a site capture metric based at least in part on the number of capture events determined in step d);

g) identifying a first subset of the site capture metrics determined in step f) that satisfy at least one criterion;

h) determining, for each target sequence of interest from which the second population of amplicons was produced, a site capture metric based at least in part on the number of capture events determined in step e);

i) identifying a second subset of the site capture metrics determined in step h) that satisfy the at least one criterion;

j) normalizing a first measure determined from the first subset of site capture metrics identified in step g) by a second measure determined from the second subset of site capture metrics identified in step i) to obtain a test ratio;

k) detecting a difference between the test ratio and a plurality of reference ratios that are computed based on reference nucleic acid samples isolated from reference subjects known to exhibit euploidy or aneuploidy to characterize the fetal DNA in the maternal blood sample.

2. The method of claim 1, wherein at least one of the following is satisfied:

(1) the length of the first targeting polynucleotide arm is between 14 and 30 base pairs,

(2) the length of the second targeting polynucleotide arm is between 14 and 30 base pairs,

(3) each of the targeting polynucleotide arms has a melting temperature between 45°

C. and 80°

C., or

(4) each of the targeting polynucleotide arms has a GC content between 30% and 80%, or between 30% and 70%.

3. The method of claim 1, wherein the polynucleotide linker is not substantially complementary to any genomic region of the subject, and at least one of the following is satisfied:

(1) the polynucleotide linker has a length of between 20 and 1,000 base pairs,

(2) the polynucleotide linker has a melting temperature of between 45°

C. and 80°

C.,

(3) the polynucleotide linker has a GC content between 30% and 80%, or between 30% and 70%, or

(4) the polynucleotide linker comprises at least one of a forward amplification primer and a reverse amplification primer.

4. The method of claim 1, wherein each of the MIPs replicons provided in step b) is produced by:

i) the first and second targeting polynucleotide arms, respectively, hybridizing to the first and second regions in the nucleic acid sample, respectively, wherein the first and second regions flank a target sequence of interest; and

ii) after the hybridization, using a ligation/extension mixture to extend and ligate the gap region between the two targeting polynucleotide arms to form single-stranded circular nucleic acid molecules.

5. The method of claim 1, wherein the first plurality of target sequences of interest is on a single chromosome, and

wherein the second plurality of target sequences of interest is on multiple chromosomes.

6. The method of claim 1, further comprising computing a variability coefficient for a plurality of site capture metrics for a particular site, wherein each site capture metric in the plurality of site capture metrics is evaluated from a nucleic acid sample from a different subject, and wherein the at least one criterion used at steps g) and h) includes a requirement that the variability coefficient for the particular site is below a threshold value.

7. The method of claim 1, wherein the first measure determined at step j) is a sum of the first subset of site capture metric and corresponds to a chromosome of interest, and the second measure determined at step j) is a sum of the second subset of site capture metric and corresponds to chromosomes other than the chromosome of interest.

8. The method of claim 1, wherein the determining at step 1) comprises performing a statistical test to evaluate whether the test ratio obtained at step j) is statistically different from the plurality of reference ratios.

9. The method of claim 1, wherein the test ratio and the reference ratios are chromosomal fractions.

10. A method of characterizing fetal DNA in a maternal blood sample comprising:

a) obtaining a genomic DNA sample from a maternal blood sample;

b) adding the genomic DNA sample into each well of a multi-well plate, wherein each well of the multi-well plate comprises a probe mixture, wherein the probe mixture comprises a population of molecular inversion probes (MIPs) and a buffer;

wherein each MIP in the population of MIPs comprises in sequence the following components;

first targeting polynucleotide arm—

first unique molecular tag—

polynucleotide linker—

second unique molecular tag—

second targeting polynucleotide arm;

wherein the pair of first and second targeting polynucleotide arms in each of the MIPs are substantially complementary to first and second regions in the nucleic acid that, respectively, flank each sequence in a plurality of target sequences of interest;

wherein the first and second unique targeting molecular tags in each of the MIPs in combination are distinct in each of the MIPs;

c) incubating the genomic DNA sample with the probe mixture for the MIPs to capture the plurality of target sequences of interest;

d) adding an extension/ligation mixture to the sample of c) for the MIPs and the plurality of target sequences of interest to form a plurality of MIPs amplicons, wherein the extension/ligation mixture comprises a polymerase, a plurality of dNTPs, a ligase, and buffer;

e) adding an exonuclease mixture to the targeting and control MIPs amplicons to remove excess probes or excess genomic DNA;

f) adding an indexing PCR mixture to the sample of e) to add a pair of indexing primers, a unique sample barcode and a pair of sequencing adaptors to the plurality of amplicons;

g) using a massively parallel sequencing method to determine the number of sequencing reads of a first population of barcoded amplicons provided in step f) based on the number of the unique targeting molecular tags, wherein the first population of barcoded amplicons is identified by the sequence of the target sequence of interest;

h) using a massively parallel sequencing method to determine the number of sequencing reads of a second population of barcoded amplicons provided in step f) based on the number of the unique targeting molecular tags, wherein the second population of barcoded amplicons is identified by the sequence of the target sequence of interest;

i) computing a site capture metric based at least in part on the number of first sequencing reads determined in step g) and a plurality of control probe capture metrics based at least in part on the numbers of second sequencing reads determined in step h);

j) identifying a subset of site capture metrics of the population of the MIPs amplicons that have control probe capture metrics satisfying at least one criterion;

k) normalizing the site capture metric by a factor computed from the subset of control probe capture metrics satisfying the at least one criterion, to obtain a test normalized site capture metric;

l) detecting a difference between the test normalized site capture metric and a plurality of reference normalized site capture metrics that are computed based on reference genomic DNA samples obtained from reference subjects exhibiting known genotypes using the same target and control sites, target population, subset of control populations in steps b)-h) to characterize fetal DNA in the maternal blood sample.

11. The method of claim 10, wherein at least one of the following is satisfied:

(1) the length of the first targeting polynucleotide arm is between 14 and 30 base pairs,

(2) the length of the second targeting polynucleotide arm is between 14 and 30 base pairs,

(3) each of the targeting polynucleotide arms has a melting temperature between 45°

C. and 80°

C., or

(4) each of the targeting polynucleotide arms has a GC content between 30% and 80%.

12. The method of claim 10, wherein the polynucleotide linker is not substantially complementary to any genomic region of the subject, and at least one of the following is satisfied:

(1) the polynucleotide linker has a length of between 20 and 1,000 base pairs,

(2) the polynucleotide linker has a melting temperature of between 45°

C. and 80°

C.,

(3) the polynucleotide linker has a GC content between 30% and 80%,

(4) the polynucleotide linker comprises at least one of a forward amplification primer and a reverse amplification primer.

13. The method of claim 12, wherein the sequence of the forward amplification primer comprises the nucleotide sequence of 5′

-CTTCAGCTTCCCGATTACGG-3′

(SEQ ID NO;

     1); and

/or

wherein the sequence of the reverse amplification primer comprises the nucleotide sequence of 5′

-GCACGATCCGACGGTAGTGT-3′

(SEQ ID NO;

     2).

14. The method of claim 12, wherein the polynucleotide linker comprises the nucleotide sequence of 5′

-CTTCAGCTTCCCGATTACGGGCACGATCCGACGGTAGTGT-3′

(SEQ ID NO;

     3).

15. The method of claim 10, wherein the first targeting polynucleotide arm comprises the nucleotide sequence of 5′

-CACTGCACTCCAGCCTGG-3′

(SEQ ID NO;

     4).

16. The method of claim 10, wherein the second targeting polynucleotide arm comprises the nucleotide sequence of 5′

-GAGGCTGAGGCAGGAGAA-3′

(SEQ ID NO;

     5).

17. The method of claim 10, wherein the MIP comprises the nucleotide sequence of 5′

-CACTGCACTCCAGCCTGG(N_1-6)CTTCAGCTTCCCGATTACGGGCACGATCCGACGGTAGTGT(N_7-12)GAGGCTGAGGCAGGAGAA-3′

(SEQ ID NO;

     6), wherein (N_1-6) represents the first unique molecular tag and (N_7-12) represents the second unique molecular tag.

18. The method of claim 10, wherein the population of MIPs has a concentration between 10 fM and 100 nM.

19. The method of claim 10, wherein the size of the MIP replicon is between 80-90 base pairs.

20. The method of claim 10, wherein the sequencing step has a read depth of between 6-8 million reads.