Single-Cell Open Chromatin Mapping With Transposome Barcoding
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Solution Overview
Problem
Current technologies lack effective methods for labeling and identifying open chromatin regions of single cell genomes in a massively parallel manner.
Innovation Solution
A method involving transposomes to generate dsDNA fragments with 5′ overhangs, followed by barcoding using oligonucleotide barcodes with target-binding regions, and ligating these barcodes to dsDNA fragments, with optional gap filling by DNA polymerases lacking exonuclease activity, to label and identify open chromatin regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If transposomes are used to generate dsDNA fragments with 5' overhangs for barcoding, then chromatin accessibility can be determined from single cell genomes, but the process complexity increases due to multiple enzymatic steps and reagents
Solution Approach 1:
The method segments the chromatin labeling process into distinct functional steps: transposome-mediated fragmentation and barcoding, followed by separate amplification and sequencing steps. Each step uses specialized reagents (transposomes with specific adaptors, oligonucleotide barcodes with target-binding regions) to achieve precise chromatin accessibility mapping while managing complexity through modularization
Solution Approach 2:
The patent introduces intermediary molecules including transposome complexes (comprising transposase and adaptors), oligonucleotide barcodes with target-binding regions, and coupling oligonucleotides. These intermediaries facilitate the labeling and identification of open chromatin regions by mediating between the transposome and the chromatin structure, enabling precise measurement without direct manipulation of the chromatin itself
2Quantity of substance
If oligonucleotide barcodes with target-binding regions are used to label DNA fragments, then single cell genome labeling is achieved, but amplification bias may affect quantification accuracy
Solution Approach 1:
The method incorporates feedback mechanisms through the use of coupling oligonucleotides that bind to the 5' overhangs of dsDNA fragments, and through gap-filling steps using DNA polymerases. These feedback steps ensure complete and accurate labeling of all chromatin regions, allowing for precise quantification by compensating for potential amplification biases through comprehensive initial labeling
Solution Approach 2:
The patent employs parameter changes in the form of using DNA polymerases with specific exonuclease activity profiles (lacking 5' to 3' and 3' to 5' exonuclease activity) for gap filling. This specific enzymatic parameter selection ensures accurate gap filling without degradation of the labeled fragments, maintaining quantification accuracy while achieving complete labeling of nucleic acid targets
3Manufacturing precision
If DNA polymerases lacking exonuclease activity are used for gap filling, then barcoding is completed accurately, but the step time increases
Solution Approach 1:
The method uses partial action by employing DNA polymerases that lack exonuclease activity specifically for gap filling steps. This partial enzymatic capability (only polymerization, no degradation) is sufficient to complete the barcoding accurately without the need for more complex enzymatic systems, achieving the required precision while minimizing the time required for the gap filling step
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables accurate determination of chromatin accessibility and methylome information from single cell genomes, correcting for amplification bias and providing precise quantification of nucleic acid targets.
Implementation Method 1
contacting double-stranded deoxyribonucleic acid (dsDNA) with a transposome to generate a plurality of dsDNA fragments each comprising a first 5' overhang and a second 5' overhang
Implementation Method 2
the 5' end of each oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a first target-binding region capable of hybridizing to the first 5' overhang of at least one of the plurality of dsDNA fragments
Implementation Method 3
ligating the second strand of said dsDNA fragment to said hybridized oligonucleotide barcode
Implementation Method 4
filling a gap between the second strand and said hybridized oligonucleotide barcode with a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for labeling DNA (e.g., open chromatin-associated gDNA). The method can comprise contacting double-stranded DNA (dsDNA), such as gDNA, with a transposome to generate a plurality of dsDNA fragments each comprising a first 5′ overhang and a second 5′ overhang. The transposome can comprise a transposase, a first adaptor having a first 5′ overhang, and a second adaptor having a second 5′ overhang. The first 5′ overhang can comprise a complement of a target-binding region of a bead oligonucleotide. The first 5′ overhang can comprise a coupling sequence. The second 5′ overhang can comprise a universal sequence. There are provided, in some embodiments, coupling oligonucleotides comprising a 5′ complement of the coupling sequence and a 3′ complement of a target-binding region of a bead oligonucleotide.