In Situ Library Preparation
AssigneeFactorial Diagnostics, Inc.
InventorsKatie Leigh Zobeck, Santiago Federico Sanchez, John Daniel Wells
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Abstract
Aspects of the present disclosure relate generally to methods, compositions, and kits for preparing a ligation-based or amplicon-based library in situ, e.g., for sequencing.
Claims38
A method for preparing a ligation-based library in situ for sequencing, the method comprising:
- (a) providing a sample comprising a prokaryotic or eukaryotic cell population;
- (b) performing, in each prokaryotic or eukaryotic cell of the prokaryotic or eukaryotic cell population, an enzymatic fragmentation reaction to form DNA fragments within the prokaryotic or eukaryotic cell population;
- (c) ligating, in each prokaryotic or eukaryotic cell, the DNA fragments to adapter sequences in situ to create a ligated library comprising ligated DNA fragments;
- (d) lysing each of the target prokaryotic or eukaryotic cells to collect the ligated DNA fragments;
- and
- (e) purifying the ligated DNA fragments.
The method of 11, wherein each PCR reaction comprises 5-15 PCR cycles in the thermocycler.
The method of 11, wherein each PCR reaction comprises 12-15 PCR cycles in the thermocycler.
The method of any one of 1-22, wherein the method comprises performing one or more wash steps on the prokaryotic or eukaryotic cells.
The method of any one of 1-23, wherein the method comprises performing one or more wash steps on the DNA fragments.
A kit for amplicon-based library preparation in situ, the kit comprising:
- a cell preservation agent capable of preserving a prokaryotic or eukaryotic cell population, the cell preservation agent selected from:
- a fixative, a permeabilizer, or a fixative and a permeabilizer;
- a primer pool set capable of amplifying a target sequence region of DNA within one or more prokaryotic or eukaryotic cells of the cell population;
- a polymerase chain reaction (PCR) Enzyme Master Mix comprising one or more of:
- an enzyme, a buffer, or an enzyme and a buffer;
- a cell lysis buffer;
- in an amount sufficient to prepare an amplicon-based library in situ for sequencing;
- and
- instructions for carrying out the amplicon-based library preparation in situ, the instructions providing the following steps:
- amplifying the target sequence region of DNA in the cell population to produce a first set of amplicon products for each cell;
- lysing each of the cells to isolate DNA fragments having the target sequence region within the first set of amplicon products;
- and
- purifying the DNA fragments.
A kit for ligation-based library preparation in situ, the kit comprising:
- a cell preservation agent capable of preserving a prokaryotic or eukaryotic cell population, the cell preservation agent selected from:
- a fixative, a permeabilizer, or a fixative and a permeabilizer;
- a fragmentation enzyme and buffer for performing an enzymatic fragmentation reaction to form DNA fragments within the cell population;
- an End repair and A tail (ERA) master mix and buffer for performing an end-repair and A-tailing reaction on the one or more DNA fragments;
- a ligation enzyme and buffer;
- adapter sequences, wherein the ligation enzyme and buffer, and adapter sequences are capable of ligating, in each cell, the DNA fragments to the adapter sequences in situ to create a ligated library comprising ligated DNA fragments;
- amplification primers for amplifying the ligated DNA fragments to form amplicon products;
- a polymerase chain reaction (PCR) enzyme master mix comprising one or more of:
- an enzyme, a buffer, or an enzyme and a buffer;
- a cell lysis buffer;
- in an amount sufficient to prepare a ligation-based library in situ for sequencing;
- and
- instructions for carrying out the ligation-based library preparation in situ, the instructions providing the following steps:
- performing, in each prokaryotic or eukaryotic cell of the cell population, an enzymatic fragmentation reaction to form DNA fragments within the cell population;
- ligating, in each cell, the DNA fragments to adapter sequences in situ to create the ligated library comprising ligated DNA fragments;
- lysing each of the cells to collect the ligated DNA fragments;
- and
- purifying the ligated DNA fragments.
Dependencies are inferred from the claim text.