Indexed Nucleic Acid Libraries With 3' End Blocking Against Index Hopping
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Solution Overview
Problem
Index hopping occurs during sequencing of pooled polynucleotides from different libraries, leading to incorrect assignment of library origin and confounding sequencing results due to unligated adaptor molecules and incomplete products.
Innovation Solution
Blocking the 3' ends of polynucleotides and degrading unincorporated adapters using exonucleases to prevent index hopping, specifically employing 5' and 3' exonucleases to target unligated adaptors and incomplete products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If unligated adaptors are present during cluster amplification, then library polynucleotides can be amplified, but index hopping occurs leading to incorrect library origin assignment
Solution Approach 1:
The patent removes unligated adaptors from the reaction mixture through purification steps (e.g., bead-based purification, gel electrophoresis) before cluster amplification. This extraction of the harmful component (unligated adaptors) prevents index hopping while preserving the library polynucleotides for amplification, thus resolving the contradiction between productivity and reliability
Solution Approach 2:
The patent applies preliminary anti-action by blocking the 3' ends of adaptors with modified nucleotides (e.g., dideoxynucleotides, phosphorothioate bonds) before the amplification step. This preliminary modification prevents unligated adaptors from serving as primers during cluster amplification, thereby preventing index hopping before it can occur while allowing library polynucleotides to be amplified normally
2Reliability
If 3' ends of polynucleotides are blocked, then unincorporated adapters cannot serve as primers, but library polynucleotide amplification may be inhibited
Solution Approach 1:
The patent applies local quality by selectively blocking 3' ends only in specific contexts - namely, unligated adaptors and incomplete products - while leaving library polynucleotides with intact 3' ends for amplification. This is achieved through controlled 3' end blocking reactions that occur preferentially on adaptor molecules, thus preventing index hopping without inhibiting library amplification
Solution Approach 2:
The patent performs preliminary 3' end blocking of adaptors before cluster amplification. By blocking the 3' ends of unligated adaptors in advance, the patent prevents them from serving as primers during amplification. The library polynucleotides, which have not been blocked, remain capable of amplification, thus resolving the contradiction between reliability and productivity
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
Mitigates index hopping by preventing unincorporated adapters from serving as primers during cluster amplification, resulting in a nearly 100-fold decrease in index hopping events.
Implementation Method 1
degrading unincorporated adapters using exonucleases to prevent index hopping, specifically employing 5' and 3' exonucleases to target unligated adaptors and incomplete products
Data Source
AI summary
The present invention is concerned with compositions and methods for improving the rate of correct sample identification in indexed nucleic acid library preparations for multiplex next generation sequencing by blocking the 3′ ends of pooled indexed polynucleotides from multiple samples prior to amplification and sequencing, by exonuclease treatment and optionally blocking the 3′ ends of pooled indexed polynucleotides from multiple samples prior to amplification and sequencing, by exonuclease treatment after protective adapters are ligated to target polynucleotides to degrade unincorporated adapters prior to amplification and sequencing, and/or by modifying or blocking 5′ and 3′ ends of pooled indexed polynucleotides from multiple samples, with an optional exonuclease treatment, prior to amplification and sequencing.