Single cell workflow for whole genome amplification
AssigneeMission Bio, Inc.
InventorsDalia Dhingra, David Ruff
Life of this patent
Estimating the term…
Abstract
Disclosed herein is a single-cell analysis workflow involving whole genome amplification for developing single-cell whole genome DNA libraries. The single-cell analysis workflow involves encapsulating and lysing cells in individual droplets and releasing genomic DNA from chromatin within the droplet. Transposases access the released genomic DNA and insert adaptor sequences into the cleaved nucleic acid fragments, thereby generating tagmented genomic DNA fragments that span the whole genome. Tagmented genomic DNA undergo nucleic acid amplification and sequencing for generating single-cell whole genome DNA libraries.
Claims114
A method for performing whole genome sequencing, the method comprising:
- providing a cell and reagents within a first droplet, the reagents comprising a lysing reagent and a protease;
- lysing the cell using the lysing reagent within the first droplet;
- releasing genomic DNA using the protease within the first droplet by exposing the first droplet to a temperature between 30° C. and 60° C.;
- tagmenting, in either the first droplet or a second droplet, the released genomic DNA by:
- using a transposase at a temperature between 35° C. and 55° C., cleaving the released genomic and incorporating adaptor sequences into the released genomic DNA;
- and
- filling in one or more gaps in the released genomic DNA created by incorporating the adaptor sequences at a temperature between 40° C. and 100° C.;
- and
- amplifying, in the second droplet, the tagmented genomic DNA to generate whole genome amplicons.
A method for performing whole genome sequencing, the method comprising:
- encapsulating a cell and reagents within a first droplet, the reagents comprising a lysing reagent and a protease;
- lysing the cell using the lysing reagent within the first droplet;
- releasing genomic DNA using the protease within the first droplet;
- encapsulating the released genomic DNA and a reaction mixture in a second droplet, the reaction mixture comprising a transposase and a DNA polymerase;
- tagmenting the released genomic DNA in the second droplet by:
- using the transposase, cleaving the released genomic and incorporating adaptor sequences into the released genomic DNA;
- using the DNA polymerase, filling in one or more gaps in the released genomic DNA created by incorporating the adaptor sequences;
- and
- amplifying, in the second droplet, the tagmented genomic DNA to generate whole genome amplicons.
A method for performing whole genome sequencing, the method comprising:
- encapsulating a cell and reagents within a first droplet, the reagents comprising a lysing reagent, a protease, and either a reverse transcriptase or DNA polymerase;
- lysing the cell using the lysing reagent within the first droplet;
- releasing genomic DNA using the protease within the first droplet;
- tagmenting the released genomic DNA in the first droplet by:
- using the transposase, cleaving the released genomic and incorporating adaptor sequences into the released genomic DNA;
- using either the reverse transcriptase or DNA polymerase, filling in one or more gaps in the released genomic DNA created by incorporating the adaptor sequences;
- encapsulating the tagmented genomic DNA and a reaction mixture in a second droplet;
- and
- amplifying, in the second droplet, the tagmented genomic DNA using the reaction mixture to generate whole genome amplicons.
A system for performing whole genome sequencing, the system comprising:
- a device configured to perform steps comprising:
- providing a cell and reagents within a first droplet, the reagents comprising a lysing reagent and a protease;
- lysing the cell using the lysing reagent within the first droplet;
- releasing genomic DNA using the protease within the first droplet by exposing the first droplet to a temperature between 30° C. and 60° C.;
- tagmenting, in either the first droplet or a second droplet, the released genomic DNA by:
- using a transposase at a temperature between 35° C. and 55° C., cleaving the released genomic and incorporating adaptor sequences into the released genomic DNA;
- and
- filling in one or more gaps in the released genomic DNA created by incorporating the adaptor sequences at a temperature between 40° C. and 100° C.;
- and
- amplifying, in the second droplet, the tagmented genomic DNA to generate whole genome amplicons.
A system for performing whole genome sequencing, the system comprising:
- a device configured to perform steps comprising:
- encapsulating a cell and reagents within a first droplet, the reagents comprising a lysing reagent and a protease;
- lysing the cell using the lysing reagent within the first droplet;
- releasing genomic DNA using the protease within the first droplet;
- encapsulating the released genomic DNA and a reaction mixture in a second droplet, the reaction mixture comprising a transposase and a DNA polymerase;
- tagmenting the released genomic DNA in the second droplet by:
- using the transposase, cleaving the released genomic and incorporating adaptor sequences into the released genomic DNA;
- using the DNA polymerase, filling in one or more gaps in the released genomic DNA created by incorporating the adaptor sequences;
- amplifying, in the second droplet, the tagmented genomic DNA to generate whole genome amplicons.
A system for performing whole genome sequencing, the system comprising:
- a device configured to perform steps comprising:
- encapsulating a cell and reagents within a first droplet, the reagents comprising a lysing reagent, a protease, and either a reverse transcriptase or DNA polymerase;
- lysing the cell using the lysing reagent within the first droplet;
- releasing genomic DNA using the protease within the first droplet;
- tagmenting the released genomic DNA in the first droplet by:
- using the transposase, cleaving the released genomic and incorporating adaptor sequences into the released genomic DNA;
- using either the reverse transcriptase or DNA polymerase, filling in one or more gaps in the released genomic DNA created by incorporating the adaptor sequences;
- encapsulating the tagmented genomic DNA and a reaction mixture in a second droplet;
- and
- amplifying, in the second droplet, the tagmented genomic DNA using the reaction mixture to generate whole genome amplicons.
A whole genome sequencing library comprising a plurality of sequence reads derived from genomic DNA across each chromosome of a single human cell, wherein at least 20% of the plurality of sequence reads of the whole genome sequencing library are mapped.
A whole genome sequencing library comprising a plurality of sequence reads derived from genomic DNA across each chromosome of a single human cell, wherein at least 10% of sequence reads of the whole genome sequencing library have a correct structure.
A whole genome sequencing library comprising a plurality of sequence reads derived from genomic DNA across each chromosome of a single human cell, wherein less than 40% of the plurality of sequence reads are duplicated.
Dependencies are inferred from the claim text.