Multiplexed Whole-Genome Library Depletion Using Removable Blockers
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Solution Overview
Problem
Whole genome sequencing libraries contain high-copy sequences such as repetitive regions and microbial DNAs that are uninformative and costly to deplete individually, leading to increased sequencing and computational costs.
Innovation Solution
A method involving the use of removable blocker oligonucleotides to anneal to adaptor sequences in a multiplexed reaction, followed by sequence capture and depletion of non-target nucleic acids, and subsequent removal of free blocker oligonucleotides to create a depleted sequencing library.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If high-copy sequences are depleted individually for each sample, then depletion effectiveness is improved, but time consumption and cost increase substantially
Solution Approach 1:
The patent combines multiple individual depletion reactions into a single multiplexed reaction by pooling samples with unique adaptor labels. This allows simultaneous depletion of high-copy sequences across multiple samples, maintaining depletion effectiveness while substantially reducing time consumption and resource requirements.
Solution Approach 2:
The patent employs universal blocker molecules that can bind to multiple adaptor sequences with different labels. This universal blocker design enables a single reaction mixture to effectively deplete high-copy sequences from multiple samples simultaneously, achieving both effectiveness and efficiency.
2Reliability
If high-copy sequences are depleted individually for each sample, then depletion effectiveness is improved, but sequencing and computational costs increase
Solution Approach 1:
By merging multiple depletion reactions into one multiplexed reaction, the patent reduces the total number of sequencing reactions required. This consolidation maintains depletion effectiveness while reducing sequencing costs through fewer library preparations and lower computational resources needed for data processing.
Solution Approach 2:
The universal blocker molecules work across multiple samples with different adaptor labels, enabling a single depletion reaction to serve multiple purposes. This multi-functionality reduces the overall quantity of sequencing and computational resources required compared to individual sample processing.
3Productivity
If multiple samples are pooled in a single reaction, then resource efficiency is improved, but adaptor self-hybridization occurs
Solution Approach 1:
The patent introduces blocker molecules as intermediary substances that bind to adaptor sequences and prevent adaptor self-hybridization. These blockers act as mediators between the pooled samples and the adaptor sequences, allowing multiplexed reactions to proceed efficiently without harmful self-hybridization events.
Solution Approach 2:
The patent applies preliminary anti-action by adding blocker molecules before the adaptor sequences can self-hybridize. The blockers pre-occupy the adaptor binding sites, preventing the harmful self-hybridization reaction from occurring in the first place, thus enabling resource-efficient multiplexed processing.
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
Substantially reduces sequencing and computational costs while maintaining minimal impact on information content by effectively depleting high-copy sequences.
Implementation Method 1
adding removable blocker oligonucleotides to the composition, wherein the removable blocker oligonucleotides comprise: (i) first removable blocker oligonucleotides that can anneal to the first adaptor sequence; (ii) second removable blocker oligonucleotides that can anneal to the second adaptor sequence
Implementation Method 2
removing non-target nucleic acids from the composition by sequence capture to bait oligonucleotides thereby depleting non-target nucleic acids from the composition
Data Source
AI summary
A depleted sequencing library can be prepared by providing a composition comprising a heterogeneous mixture of linear nucleic acids having a first terminus and a second terminus. A first subset of target nucleic acids and a second subset of non-target nucleic acids can include a first adaptor region at the first terminus and a second adaptor region at the second terminus. A third subset of the target nucleic acids and a fourth subset of the non-target nucleic acids include the second adaptor region at the first terminus and at the second terminus. Removable blocker oligonucleotides can be added to the composition, non-target nucleic acids can be removed from the composition by sequence capture to bait oligonucleotides, and the composition can be treated to reduce a quantity of free blocker oligonucleotides that are not annealed to an adaptor sequence or to a sequence substantially complementary to an adaptor sequence.