Methods, systems, and compositions for counting nucleic acid molecules

Abstract

Compositions and methods, systems, and kits for detecting and quantifying variations in numbers of molecules, particularly variations in gene dosage, e.g., due to gene duplication, or to variations from the normal euploid complement of chromosomes, e.g., trisomy of one or more chromosomes that are normally found in diploid pairs, without digital sequencing.

Description

[0001]The present application is a continuation of U.S. patent application Ser. No. 16 / 373,568, filed Apr. 2, 2019, which claims priority to U.S. Provisional Application Serial Nos. 62 / 651,676, filed Apr. 2, 2018, and 62 / 660,699, filed Apr. 20, 2018, each of which is incorporated herein by reference.SEQUENCE LISTING[0002]The text of the computer readable sequence listing filed herewith, titled “36313-304_SEQUENCE_LISTING_ST25”, created Jan. 14, 2021, having a file size of 3,000 bytes, is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION[0003]The present invention relates to compositions and methods for determining numbers of copies of individual molecules, such as nucleic acid molecules, without digital sequencing. The technologies find use, for example, in analysis of variations in copy numbers of specific nucleic acids sequences that may arise, e.g., from variations in chromosome number, gene copy number, expression level, etc. The technologies find particula...

AI Technical Summary

Benefits of technology

The patent describes a method for capturing target sequences on a nucleic acid sample and further manipulating the captured sequences for downstream analysis. This is achieved through the use of molecular inversion probes (MIPs) which are introduced to the nucleic acid fragments to improve their capture. After capture, the target sequences are subjected to enzymatic gap-filling and ligation steps to create a copy of the target sequence integrated into a circle-like structure. Nucleic acid analogs can also be incorporated for further processing. The efficiency of capture can be improved by lengthening the hybridization and gap-filling incubation periods. The technical effects include improved efficiency in capturing target sequences and the ability for downstream analysis and manipulation of the captured sequences.

Problems solved by technology

Variations in gene dosage arise due to errors in DNA replication and can occur in germ line cells, leading to congenital defects and even embryonic demise, or in somatic cells, often resulting in cancer.

Structural chromosome abnormalities affecting parts of chromosomes arise due to chromosome breakage, and result in deletions, inversions, translocations or duplications of large blocks of genetic material.

A large variety of congenital defects, growth deficiencies, and intellectual disabilities are found in children with chromosomal aneuploidies, and these present life-long challenges to families and societies.

There are a variety of prenatal tests that can indicate increased risk for fetal aneuploidy, including invasive diagnostic tests such as amniocentesis or chorionic villus sampling, which are the current gold standard but are associated with a non-negligible risk of fetal loss.

Current methods for quantifying variations in numbers of molecules, for example performing aneuploidy screening, that rely on next generation sequencing (NGS) are often time-consuming, expensive, and require extensive bioinformatics analysis.

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