Indexing Deep DNA Sequencing for Rare Variant Detection
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Solution Overview
Problem
Current deep sequencing approaches face challenges in accurately and sensitively quantifying human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV) minority populations, making it difficult to identify drug-resistant quasispecies within infected patients.
Innovation Solution
An assay is developed that uses high-throughput paired-end sequencing technology and a random sequencing tag strategy to produce single-stranded complements of target sequences, followed by PCR amplification and paired-end sequencing, allowing for the detection of mutant polynucleotides, including drug-resistant variants, within a large population of polynucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current deep sequencing approaches are used, then sequencing coverage is achieved, but accuracy and sensitivity in quantifying minority populations deteriorates
Solution Approach 1:
The patent divides the sequencing process into multiple distinct stages: initial PCR amplification with unique tags, nested PCR enrichment, and paired-end sequencing. Each stage processes specific portions of the polynucleotide population, allowing systematic identification of minority variants while maintaining accuracy through staged analysis rather than single-step processing
Solution Approach 2:
The patent performs preliminary tagging of polynucleotides with unique sequences during initial PCR amplification, before the actual sequencing occurs. This preliminary action allows for subsequent tracking and quantification of individual polynucleotide molecules, enabling accurate measurement of minority populations that would otherwise be indistinguishable in bulk sequencing
2Measurement precision
If deep sequencing is performed to identify rare sequences, then detection sensitivity improves, but technical artifacts and biases increase
Solution Approach 1:
The patent incorporates feedback mechanisms through the use of unique tags that allow tracking of individual polynucleotide molecules through multiple amplification cycles. By monitoring the distribution and frequency of tagged sequences, the system can identify and correct for amplification biases and technical artifacts, providing feedback that improves the accuracy of rare variant detection
Solution Approach 2:
The patent uses nested PCR amplification to create multiple copies of tagged polynucleotide sequences, enabling sufficient material for deep sequencing while maintaining the unique identification tags. This copying process, when properly controlled, allows rare sequences to be amplified to detectable levels without introducing excessive technical artifacts, as the tags preserve the original molecular identity through the amplification process
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
The assay achieves high sensitivity and accuracy in detecting minority populations and drug-resistant mutations, correcting technical artifacts and biases, thereby improving the detection of low-abundance viral variants and guiding antiviral therapy.
Implementation Method 1
PCR amplifying the single stranded complements produced in step a) or isolated in step b) using a first primer set comprising an outer PCR primer and a first 5' target primer to produce multiple double stranded copies
Data Source
AI summary
The invention pertains to an assay that is capable of detecting a mutant polynucleotide in a plurality of polynucleotides. In one embodiment, the assay of the invention is capable of detecting one copy of a mutant polynucleotide in about 50,000 to about 100,000 copies of polynucleotides. The assay of the invention can be used to identify a mutant viral quasispecies or a mutant mRNA encoding an oncogenic protein from a tumor sample. The assay of the invention involves producing the single stranded complements of each of a plurality of polynucleotides containing the target sequence, wherein each of the single stranded complements contain a unique tag sequence and amplifying the single stranded complements by PCR using several sets of primers designed to introduce the sequences appropriate for a paired-end sequencing analysis of the amplified polynucleotides. The invention also pertains to kits for carrying out the assays of the invention.