Mutational Load Estimation via PCR Amplicon Sequencing
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Solution Overview
Problem
Current methods for measuring tumor mutational load are inefficient and costly, particularly for liquid biopsies, as they require extensive sequencing and are not readily implementable in high-throughput formats due to the challenge of amplifying long DNA fragments from fragmented samples.
Innovation Solution
A PCR-based method using thermostable polymerase and primers that hybridize to repeated sequences in the genome, generating numerous amplicons of significant length (up to several megabases) for sequencing, allowing for the estimation of sequence variations and mutational load without the need for extensive enrichment or hybridization steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If whole-exome sequencing or sequencing panels are used to measure mutational load, then measurement precision is improved, but device complexity and cost increase due to requiring enrichment steps
Solution Approach 1:
The invention extracts only the necessary information (mutational load) directly from the sequenced regions without requiring prior enrichment steps. By sequencing random genomic regions and analyzing mutations directly, the method eliminates the complex hybridization-based enrichment processes while maintaining measurement capability.
Solution Approach 2:
The method uses PCR amplification to generate multiple copies of sequenced regions, enabling sufficient DNA quantity for sequencing without requiring enrichment. The amplification process creates adequate material from the limited input DNA while maintaining the random sampling approach.
2Measurement precision
If hybridization-based enrichment methods are used, then measurement precision is improved, but loss of time increases due to multi-step procedures
Solution Approach 1:
The invention removes the time-consuming hybridization enrichment steps from the workflow. By directly sequencing random genomic regions followed by PCR amplification, the method achieves mutational load measurement in fewer steps, significantly reducing processing time while maintaining precision.
3Productivity
If PCR is used to amplify DNA, then productivity is improved, but measurement precision deteriorates because PCR can only amplify a few tens of kb which is insufficient for mutation load estimation
Solution Approach 1:
The invention segments the genome into multiple random regions that are individually amplified by PCR and then sequenced. By analyzing mutations across many segmented regions collectively, the method achieves sufficient coverage for accurate mutational load estimation while maintaining the efficiency benefits of PCR amplification.
Solution Approach 2:
The method merges data from multiple PCR-amplified segments to achieve comprehensive mutational load measurement. By combining results from numerous small amplified regions, the approach recovers the precision needed for mutation load estimation while retaining PCR's productivity advantages.
4Measurement precision
If extensive sequencing is performed to detect rare mutations in fragmented DNA, then measurement precision is improved, but cost increases
Solution Approach 1:
The invention performs preliminary PCR amplification of random genomic regions before sequencing, generating sufficient DNA quantity from limited input. This preliminary action enables detection of rare mutations with adequate sequencing depth while reducing the need for extensive and costly sequencing of the entire genome.
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
This method enables the efficient and cost-effective estimation of mutational load by generating extensive sequence data from limited DNA samples, facilitating high-throughput analysis and improving the predictive capability for immunotherapy response.
Implementation Method 1
combining the sample of DNA with a thermostable polymerase, dNTPs and a set of primers to produce a reaction mix; thermocycling the reaction mix to produce a reaction product comprising at least 50 amplicons
Implementation Method 2
the 3′ end of each primer specifically hybridizes to a sequence that is repeated multiple times in the genome of the subject
Implementation Method 3
sequencing the amplicons or amplification products thereof to produce sequence reads; analyzing the sequence reads to estimate the number of sequence variations
Data Source
AI summary
Provided herein is method for, among other things, estimating the number of sequence variations in a sample of DNA. In some embodiments, the method can be used to estimate the mutational load of a sample. In some embodiments, the method makes use of a set of primers that have 3′ ends that specifically hybridizes to a sequence that is repeated multiple times in the genome. Thermocycling a reaction mix containing the primers may produce a reaction product comprising at least 50 amplicons having a total length of at least 100 kb. This product can be sequenced to provide an estimate of the number of sequence variations in the sample, and thus the mutational load of the sample.