Barcode Adapters for High-Throughput Single-Cell DNA Methylation Sequencing
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Solution Overview
Problem
Current methods for DNA methylation sequencing, such as scBS and scRRBS, face challenges including high costs, low efficiency, poor data quality, and limited throughput, making it difficult to analyze large numbers of single cells effectively.
Innovation Solution
The development of multi-scRRBS (msRRBS) method using barcode adapters and APOBEC enzyme for converting non-methylated cytosine, allowing for high-throughput CpG methylation analysis by pooling and tagging multiple single cells with specific barcodes, reducing DNA damage and operational complexity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional scBS or scRRBS methods are used for single-cell DNA methylation sequencing, then methylation analysis can be performed, but the throughput is low and it is difficult to analyze large numbers of single cells effectively
Solution Approach 1:
The patent segments the library construction process by introducing barcode adapters that can be ligated to multiple single-cell DNA fragments simultaneously. Different barcode adapters with unique identifiers allow parallel processing of numerous single cells, transforming a sequential process into a parallel one, thereby dramatically increasing throughput without sacrificing analysis quality
Solution Approach 2:
The patent applies preliminary action by pre-designing and preparing a set of barcode adapters before the actual sequencing experiment. These adapters are ready-to-use modules that can be directly ligated to DNA fragments, eliminating the need for time-consuming in-situ adapter preparation during the experiment, thus reducing overall processing time while maintaining high throughput
2Reliability
If traditional scBS or scRRBS methods are used, then methylation sequencing is possible, but the experimental cost is high
Solution Approach 1:
The patent uses barcode adapters as standardized, reusable templates that can be copied and applied to numerous single-cell samples. Instead of performing separate library construction for each cell, the same adapter design is copied across hundreds or thousands of samples, reducing reagent consumption and operational costs while maintaining consistent data quality across all samples
Solution Approach 2:
The barcode adapters serve multiple functions simultaneously: they enable sample identification, facilitate parallel library construction, and provide sequencing compatibility. This multi-functionality eliminates the need for separate reagents and procedures for each function, reducing overall experimental cost while ensuring reliable methylation sequencing data
3Productivity
If traditional scBS or scRRBS methods are used, then methylation analysis can be performed, but the library construction process is complex and inefficient
Solution Approach 1:
The patent segments the complex library construction process into distinct, modular steps: DNA fragmentation, barcode adapter ligation, pooling, and sequencing. Each step uses standardized reagents and protocols, reducing operational complexity. The barcode adapter itself is segmented into functional domains (barcode region, adapter sequence, primer binding sites), allowing for easy customization and reducing design complexity
Solution Approach 2:
The barcode adapter acts as an intermediary molecule that bridges the gap between single-cell DNA fragments and the sequencing platform. It simplifies the interface between sample preparation and sequencing by providing a universal connection point, thereby reducing the complexity of coordinating multiple specialized reagents and procedures
4Reliability
If traditional scBS or scRRBS methods are used, then methylation sequencing is possible, but DNA damage occurs and data consistency is poor
Solution Approach 1:
The patent performs preliminary pooling of DNA fragments after barcode adapter ligation but before subsequent enzymatic treatments or amplifications. This preliminary pooling equalizes the treatment conditions across all samples, reducing variability and improving data consistency. It also allows for optimization of downstream conditions that benefit all samples simultaneously, minimizing DNA damage
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
msRRBS enables efficient, cost-effective, and consistent analysis of CpG methylation in multiple single cells, improving data quality and reducing experimental costs by simplifying library construction and sequencing processes.
Implementation Method 1
APOBEC enzyme for converting non-methylated cytosine
Implementation Method 2
ligating a DNA fragment of each of the samples to a barcode adapter with a different barcode
Implementation Method 3
pooling DNA fragments of the plurality of samples that are ligated with a barcode adapter to obtain a DNA fragment pool
Implementation Method 4
followed by a first round of PCR amplification
Data Source
AI summary
Disclosed is a set of adhesive adapters containing sample barcodes for specifically tagging different samples. Further disclosed is a method for simultaneously detecting CpG methylation in a high number of samples, which is multi-sample reduced-representation bisulfite sequencing (msRRBS); and an alternative method thereof, which is multi-sample reduced-representation APOBEC sequencing (msRRAS). The adapters are used to specifically tag the plurality of samples, including all DNA fragments of the plurality of samples; then the plurality of samples are pooled to allow a single-tube reaction of the plurality of samples; and then the subsequent conversion, sequencing library construction and sequencing, distribution and decoding of readings of each sample, and downstream analysis are conducted. The library construction technology of the present application has advantages such as high efficiency, low cost, and stable and convenient operations.