Partitioned Amplicon Sequencing for Accurate cfDNA Variant Calling
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Solution Overview
Problem
Existing sequencing methods for detecting cancer from cell-free DNA in bodily fluids face challenges due to low nucleic acid concentrations and diversity, leading to amplification biases and difficulties in distinguishing genuine genetic variations from sequencing errors.
Innovation Solution
A method involving linking sample indexes to nucleic acid molecules, partitioning them into aliquots, amplifying, sequencing, and grouping reads by sample and partition indexes to determine start and stop points, allowing for accurate alignment and variant calling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If nucleic acid molecules in body fluids are directly sequenced, then the detection process is simplified, but the low concentration and diversity of nucleic acids lead to amplification biases and sequencing errors
Solution Approach 1:
The patent segments the sequencing process by dividing nucleic acid molecules into different groups based on their molecular barcode assignments. Each group is processed separately through amplification and sequencing, allowing for more precise tracking and reduction of amplification biases while maintaining overall process simplicity
Solution Approach 2:
Molecular barcodes serve as intermediaries that link original nucleic acid molecules to their amplification products. These barcodes enable precise tracking of each original molecule through the amplification and sequencing process, allowing accurate distinction between genuine genetic variations and sequencing errors even in low-concentration samples
2Measurement precision
If molecular barcodes are used to group amplicons, then genuine genetic variations can be distinguished from sequencing errors, but the process complexity increases
Solution Approach 1:
The molecular barcodes serve multiple functions: they identify original nucleic acid molecules, track amplification products, enable grouping of reads, and facilitate variant calling. This multi-functionality reduces the need for separate systems for each task, thereby managing complexity while improving precision
Solution Approach 2:
Molecular barcodes are assigned to nucleic acid molecules before amplification occurs. This preliminary assignment creates a tracking system that simplifies subsequent data analysis and variant calling, as the grouping information is already established before the amplification process begins
3Measurement precision
If nucleic acid molecules are partitioned into aliquots before amplification, then amplification biases are reduced, but the processing time and steps increase
Solution Approach 1:
The patent partitions nucleic acid molecules into multiple aliquots based on molecular barcode assignments before amplification. This segmentation ensures that each amplification reaction starts with a diverse representation of original molecules, reducing amplification biases and improving uniformity across samples
Solution Approach 2:
After separate amplification of partitioned aliquots, the patent merges the results by grouping sequencing reads according to their molecular barcodes. This combining step consolidates the benefits of partitioning while recovering processing efficiency, as the barcode-based grouping automates the reintegration of data from multiple aliquots
Data Source
AI summary
Sequencing methods for sequencing populations of nucleic acid molecules in which sequencing reads of amplicons are grouped into families according to the nucleic acid molecule of origin by partitioning, sample indexes and information from the sequencing reads, such as start and end points. The methods described herein provide many advantages over other sequencing analysis methods, including the identification of sequencing reads deriving from the same nucleic acid in the original sample while minimizing the number of aliquots that are processed.