US 2022/0177874 A1 · METHODS FOR LIBRARY PREPARATION TO ENRICH INFORMATIVE DNA FRAGMENTS USING ENZYMATIC DIGESTION

Abstract

The present disclosure provides methods and compositions for preparation of a nucleic acid library. In some embodiments, the nucleic acids comprise cell-free DNA, including cfDNA that is in need of analysis, such as by sequencing. The methods may comprise restriction enzyme digestion, adapter ligation, and subsequent amplification, and may provide improved approaches for reducing the number adapter dimers produced during the process. In an aspect, a method for preparing a library of nucleic acids may comprise: digesting DNA molecules with restriction enzymes to produce DNA fragments; ligating adapters to the DNA fragments by incubating with ligase to produce a mixture of adapter-ligated DNA fragments and adapter dimers; amplifying the adapter-ligated DNA fragments to produce amplified adapter-ligated DNA fragments; and reducing the quantity of the adapter dimers by differentiating between the junction between an adapter and a DNA fragment, and the junction between an adapter and another adapter.

Claims90

Dependent claims

A method for preparing a library of nucleic acids, comprising:

(a) digesting a plurality of DNA molecules with a first one or more restriction enzymes to produce DNA fragments;
(b) ligating adapters to the DNA fragments by incubating with ligase to produce a mixture of adapter-ligated DNA fragments and adapter dimers;
(c) amplifying the adapter-ligated DNA fragments to produce amplified adapter-ligated DNA fragments;
and
(d) reducing the quantity of the adapter dimers either after or during (b) and/or after (c), wherein the reducing comprises differentiating between the junction between an adapter and a DNA fragment, and the junction between an adapter and another adapter.

2 more elements

A method for preparing a library of nucleic acids, comprising:

(a) digesting a plurality of DNA molecules with a first one or more restriction enzymes to produce DNA fragments;
(b) ligating adapters to the DNA fragments by incubating with ligase to produce a mixture of adapter-ligated DNA fragments and adapter dimers;
and
(c) amplifying the adapter-ligated DNA fragments to produce amplified adapter-ligated DNA fragments,
subject to one or more of the following:
(1) performing (c) using a primer or primers that bind a junction between the end of the DNA fragment and the adapter, but does not bind a junction between the end of one adapter and the end of another adapter;
(2) digesting the mixture of adapter-ligated DNA fragments and adapter dimers with a second one or more restriction enzymes that digest the junction between the end of one adapter and the end of another adapter, but do not digest the junction between the end of the DNA fragment and the adapter;
(3) performing (a) and (b) in the same reaction mixture, and further comprising digesting the mixture with a second one or more restriction enzymes that digest the junction between the end of one adapter and the end of another adapter, but do not digest the junction between the end of the DNA fragment and the adapter;
(4) the adapter is an adapter dimer by design, and further comprising digesting the mixture of adapter-ligated DNA fragments and adapter dimers with a second one or more restriction enzymes that digest the junction between the end of one adapter and the end of another adapter, but do not digest the junction between the end of the DNA fragment and the adapter;
and/or
(5) (c) produces amplified adapter dimers that are digested with a third one or more restriction enzymes that digest the junction between the end of one adapter and the end of another adapter.

8 more elements

Dependencies are inferred from the claim text.

Methods for library preparation to enrich informative dna fragments using enzymatic digestion

Life of this patent

Methods for library preparation to enrich informative dna fragments using enzymatic digestion

Life of this patent

Abstract

Claims90