Nested-ClickSeq Sequencing for Low-Abundance Provirus/Transgene Junctions
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Solution Overview
Problem
Existing methods for sequencing low-abundance provirus/transgene integration sites are inefficient and require complex, technically demanding procedures, often necessitating host DNA fragmentation and multiple amplification steps, which are not feasible for routine use.
Innovation Solution
A nested-ClickSeq method using template-specific primers in both reverse transcription and PCR steps, combined with click-ligation of functionalized adaptors, allows for targeted amplification of provirus/transgene junctions, enhancing specificity and sensitivity without the need for additional ligation or PCR steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If non-targeted random next-generation sequencing is used, then virus isolates can be sequenced, but sensitivity is insufficient for low viral genome copy numbers
Solution Approach 1:
The method segments the sequencing process into targeted steps: first using a virus-specific primer to capture only viral sequences from the complex host-virus mixture, then using a second primer to amplify the captured viral cDNA. This segmentation allows sensitive detection of low-abundance viral genomes by isolating them from the overwhelming host background before amplification and sequencing.
2Measurement precision
If targeted PCR amplification is used to increase sensitivity, then low-abundance targets can be detected, but the method becomes technically complex requiring host DNA fragmentation and multiple amplification steps
Solution Approach 1:
The method extracts only the viral sequences of interest from the complex host DNA background through a first PCR step using a virus-specific primer. This extraction eliminates the need for host DNA fragmentation and subsequent complex enrichment steps, as the viral cDNA is directly captured and amplified in a single targeted reaction, simplifying the overall procedure while maintaining high sensitivity.
Solution Approach 2:
The first PCR step performs preliminary capture and amplification of viral cDNA before the second amplification step. This preliminary action enriches the viral templates to sufficient levels for downstream sequencing, eliminating the need for complex host DNA processing and multiple sequential amplification steps that would otherwise be required.
3Measurement precision
If conventional amplicon approaches are used for provirus sequencing, then integration sites can be identified, but the method requires knowledge of flanking host genome sequences which are not known a priori
Solution Approach 1:
Instead of designing primers that anneal to known flanking host sequences (conventional approach), the method inverts the strategy by using a virus-specific primer to capture the provirus along with its unknown flanking host sequences. The second primer then amplifies the captured material, allowing identification of previously unknown integration sites without requiring prior knowledge of the host genome context.
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 method provides a straightforward, sensitive, and specific protocol for sequencing provirus/transgene integration sites, suitable for routine use, even with low-abundance targets, maintaining compatibility with existing sequencing platforms.
Implementation Method 1
performing a reverse transcription reaction with a target nucleic acid, the one or more first primers and one of more terminating nucleotides selected from modified-deoxyGTP, modified-deoxyTTP, modified-deoxyUTP, modified-deoxyCTP and modified-deoxyATP, dNTPs and a reverse transcriptase to form azido-terminated cDNAs
Implementation Method 2
one or more first primers comprising one or more first sequences complementary to a target of interest
Implementation Method 3
chemically ligating a functionalized 5′ first adaptor to the terminated cDNAs
Implementation Method 4
amplifying the chemically-ligated terminated cDNA into an amplification product using one or more second primers
Data Source
AI summary
Provided herein are composition and methods for characterizing target nucleic acid sequences comprising: contacting a target nucleic with first primers comprising first sequences complementary to a target of interest; performing a reverse transcription reaction with a nucleic acid, the first primers and one of more terminating nucleotides, dNTPs and a reverse transcriptase to form azido-terminated cDNAs of various lengths; chemically ligating a functionalized 5′ first adaptor to the terminated cDNAs; and amplifying the chemically-ligated terminated cDNA into an amplification product using second primers comprising second sequences complementary to the target of interest, wherein the one or more second primers comprises a sequence nested between the one or more first primers and a 3′ end of the azido-terminated cDNA molecule, wherein the cDNA comprises sequences from the target of interest.