Compositions and methods for identifying nucleic acid molecules

• US 10,533,219 B2
• Filed: 09/26/2017
• Issued: 01/14/2020
• Est. Priority Date: 12/07/2016
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Zimmermann, Bernhard, Swenerton, Ryan, Rabinowitz, Matthew, Sigurjonsson, Styrmir, Gemelos, George, Ganguly, Apratim, Sethi, Himanshu
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US15/716,058
• Publication Number:
  US 20180155775A1
• Time in Patent Office: 840 Days
• Field of Search: None
• 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 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present disclosure provides methods and compositions for sequencing nucleic acid molecules and identifying individual sample nucleic acid molecules using Molecular Index Tags (MITs). Furthermore, reaction mixtures, kits, and adapter libraries are provided.

Claims

1. A method of determining the number of copies of one or more chromosomes or chromosome segments of interest from a target individual in a sample of blood, or a fraction thereof, from the target individual or from the mother of the target individual, the method comprising:

forming a population of tagged nucleic acid molecules by reacting a population of sample nucleic acid molecules with a set of nucleic acid molecular index tags (MITs), wherein the number of different MITs in the set of MITs is between 10 and 1,000, wherein a ratio of the total number of sample nucleic acid molecules in the population of sample nucleic acid molecules to the diversity of MITs in the set of MITs is greater than 1,000;

1, wherein at least some of the sample nucleic acid molecules comprise one or more target loci of a plurality of target loci on the chromosome or chromosome segment of interest, and wherein the sample is 1.0 ml or less of blood or a fraction of blood derived from 1.0 ml or less of blood;

amplifying the population of tagged nucleic acid molecules to create a library of tagged nucleic acid molecules;

determining the sequences of the attached MITs and at least a portion of the sample nucleic acid segments of the tagged nucleic acid molecules in the library of tagged nucleic acid molecules, to determine the identity of a sample nucleic acid molecule that gave rise to a tagged nucleic acid molecule;

measuring a quantity of DNA for each target locus by counting the number of sample nucleic acid molecules that comprise each target locus using the determined identities; and

determining, on a computer, the number of copies of the one or more chromosomes or chromosome segments of interest using the quantity of DNA at each target locus in the sample nucleic acid molecules.

2. The method of claim 1, wherein the number of target loci and the volume of the sample provide an effective amount of total target loci to achieve a desired sensitivity and a desired specificity for determining the number of copies.

3. The method of claim 2, wherein the plurality of target loci comprises 1,000 target loci and wherein the sample is plasma.

4. The method of claim 2, wherein the total number of MIT molecules in the reaction mixture is greater than the total number of sample nucleic acid molecules in the reaction mixture, wherein the population of tagged nucleic acid molecules is formed using a ligation reaction, wherein the population of tagged nucleic acid molecules is enriched using hybrid capture before the amplifying, and wherein the number of total target loci in the sample is at least 4 times greater than the number of total target loci required to meet the desired specificity and the desired sensitivity.

5. The method of claim 2, wherein the number of target loci and the volume of the sample provide at least 500,000 total target loci in the sample, wherein the set of MITs comprises at least 32 MITs, wherein the sample is from the mother and comprises at least 3% fetal nucleic acids compared to maternal nucleic acids, and wherein the desired specificity is 99% and the desired sensitivity is 99%.

6. The method of claim 5, wherein the sample is 0.5 ml or less of blood or the sample is a fraction of blood derived from 0.5 ml or less of blood.