Sequential Sequencing for NGS Phasing Information
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Solution Overview
Problem
Current short read next-generation sequencing (NGS) methods face limitations in determining phasing information, particularly in diploid organisms, where it is challenging to identify which loci are co-located on the same chromosome, which is crucial for diagnostic and clinical applications such as haplotyping and distinguishing between functional and non-functional genes.
Innovation Solution
The method employs sequential paired sequencing reads from the same immobilized nucleic acid template, using pools of oligonucleotides as priming sites to target specific regions, allowing for the compilation and alignment of data to differentiate between closely related nucleic acid sequences and determine their co-location on the same template.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If short read sequencing is used to query regions of variable DNA sequence, then sequencing throughput and parallelization are improved, but the ability to determine phasing information (which loci are co-located on the same chromosome) is lost
Solution Approach 1:
The method segments the sequencing process into multiple sequential rounds, where each round targets specific regions of interest. By dividing the genome into manageable segments and sequencing them in successive rounds with different primer pools, the system maintains high parallelization while preserving phasing information through the sequential accumulation of reads from the same template molecules.
Solution Approach 2:
The method performs preliminary actions by immobilizing nucleic acid templates and designing specific primer pools that target conserved regions before the actual sequencing of variable regions. This preliminary setup ensures that subsequent sequencing rounds can efficiently target specific loci while maintaining the ability to trace reads back to their original template, thus preserving phasing information.
2Measurement precision
If methods like Sanger sequencing and subcloning are used to obtain phasing information, then phasing accuracy is improved, but labor intensity and cost increase
Solution Approach 1:
The method replaces manual, labor-intensive techniques like Sanger sequencing and subcloning with an automated next-generation sequencing platform. By using immobilized templates and pooled primers in a high-throughput sequencing instrument, the system achieves comparable or superior phasing accuracy while dramatically reducing manual labor and operational costs.
Solution Approach 2:
The sequencing platform is designed to perform multiple functions: it can sequence both conserved and variable regions, handle multiple primer pools, and generate phasing information alongside standard sequencing data. This multi-functionality allows a single platform to replace multiple specialized techniques, reducing overall complexity and cost.
3Quantity of substance
If short read sequencing is used with conserved sequence blocks interspersed between variable regions, then sequencing coverage is improved, but the ability to perform phasing analysis is reduced
Solution Approach 1:
The method dynamically adjusts the sequencing strategy by using different primer pools in successive rounds. First round primers target conserved regions to establish coverage, while subsequent round primers target variable regions for phasing analysis. This dynamic adaptation allows the system to optimize for both coverage and phasing capability depending on the sequencing round.
Solution Approach 2:
The conserved sequence regions serve as intermediaries that connect variable regions of interest. By designing primers that target conserved regions flanking variable regions, the system uses these conserved blocks as mediators to capture and sequence the variable regions in between, thereby maintaining both high coverage and phasing information.
Data Source
AI summary
The present invention provides improved methods, compositions and kits for short read next generation sequencing (NGS). The methods, compositions and kits of the present invention enable phasing of two or more nucleic acid sequences in a sample, i.e. determining whether the nucleic acid sequences (typically comprising regions of sequence variation) are located on the same chromosome and/or the same chromosomal fragment. Phasing information is obtained by performing multiple, successive sequencing reactions from the same immobilized nucleic acid template. The methods, compositions and kits provided herein are useful, for example, for haplotyping, SNP phasing, or for determining downstream exons in RNA-seq.