Compositions and methods for identifying nucleic acid molecules

• US 10,577,650 B2
• Filed: 09/26/2017
• Issued: 03/03/2020
• 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:
  US15/716,331
• Publication Number:
  US 20180155776A1
• Time in Patent Office: 889 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 reaction mixture comprising:

a population of sample nucleic acid molecules between 10 and 1,000 nucleotides in length;

a set of between 10 and 1000 different Molecular Index Tags (MITs) between 4 and 8 nucleotides in length; and

a ligase,

wherein the MITs are separate nucleic acid molecules from the sample nucleic acid molecules,

wherein a ratio of the total number of sample nucleic acid molecules in the reaction mixture to the diversity of the MITs in the set of MITs in the reaction mixture is at least 10,000;

1,

wherein the sequence of each of the MITs in the set of MITs differs from all other MIT sequences in the set by at least 2 nucleotides; and

wherein the reaction mixture comprises at least two copies of every MIT.

2. The reaction mixture of claim 1, wherein each MIT constitutes at least a portion of a double-stranded nucleic acid adapter molecule.

3. The reaction mixture of claim 1, wherein each MIT constitutes a portion of a Y-adapter nucleic acid molecule of a set of Y-adapter nucleic acid molecules, where each Y-adapter of the set comprises a base-paired, double-stranded polynucleotide segment and at least one non-base-paired single-stranded polynucleotide segment, wherein the sequence of each of the Y-adapter nucleic acid molecules in the set, other than the MIT sequence, is identical, and wherein the MIT is a double-stranded sequence that is part of the base-paired, double-stranded polynucleotide segment.

4. The reaction mixture of claim 3, wherein the double-stranded polynucleotide segment is between 5 and 25 nucleotides in length, not including the MIT, and the single-stranded polynucleotide segment is between 5 and 25 nucleotides in length.

5. The reaction mixture of claim 3, wherein the reaction mixture comprises circulating cell-free human DNA, wherein the number of combinations of any 2 MITs in the set of MITs exceeds the total number of sample nucleic acid molecules in the reaction mixture that span a target locus in the human genome, and wherein the total number of MIT molecules in the reaction mixture is at least two times greater than the total number of sample nucleic acid molecules in the reaction mixture.

6. The reaction mixture of claim 1, wherein the population of sample nucleic acid molecules comprises at least 500,000,000 sample nucleic acid molecules.