Indexed Nucleic Acid Library Adapter Blocking to Prevent 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 unincorporated adapters with index tags hybridizing to other libraries' adapters and serving as primers during cluster amplification.
Innovation Solution
Blocking the 3′ ends of polynucleotides, including unincorporated adapters, and optionally protecting 5′ and 3′ ends with exonucleases to prevent them from serving as primers or being degraded, thereby reducing index hopping.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If unincorporated adapters with index tags are present during library preparation, then library-specific identification is enabled, but index hopping occurs causing incorrect library origin assignment
Solution Approach 1:
The patent removes unincorporated adapters from the reaction mixture through purification steps (e.g., bead-based cleanup, column purification) before cluster amplification. This extraction of the harmful component (free adapters) prevents them from hybridizing to target sequences and causing index hopping, thereby resolving the contradiction between maintaining library identification capability and ensuring sequencing accuracy
Solution Approach 2:
The patent applies preliminary actions during library preparation to prevent index hopping before it occurs. This includes using adapters with modified 3' ends that cannot serve as primers, performing purification to remove excess adapters, and optimizing ligation conditions to minimize unincorporated adapters. These preventive measures address the root cause while preserving the index tagging function
2Adaptability or versatility
If adapters are ligated to polynucleotides for indexing, then library origin can be identified, but unincorporated adapters hybridize to other libraries causing index hopping
Solution Approach 1:
The patent introduces purification intermediaries (e.g., magnetic beads, spin columns) between the ligation step and cluster amplification to remove unincorporated adapters. This intermediary step acts as a filter that separates the desired ligated products from the harmful free adapters, allowing multiplexed sequencing to proceed without index hopping contamination
Solution Approach 2:
The patent modifies adapter design parameters, specifically altering the 3' end structure of adapters to prevent them from serving as primers during cluster amplification. This parameter change (modifying the chemical structure at the 3' end) maintains the adapter's ability to provide index identification while eliminating its ability to cause index hopping through inappropriate priming
3Productivity
If cluster amplification is performed on pooled libraries, then sequencing throughput is increased, but index hopping increases due to adapter hybridization between libraries
Solution Approach 1:
The patent performs preliminary purification of each library to remove unincorporated adapters before pooling and performing cluster amplification. This preliminary action ensures that when libraries are pooled and amplified together, there are no free adapters available to hybridize across libraries, thus maintaining both high throughput and accurate library assignment
Solution Approach 2:
The patent converts the potential harm of having excess adapters into a benefit by using controlled purification methods. The purification process, initially designed to simply clean up samples, is leveraged to specifically remove the source of index hopping while enriching for properly ligated products. This transforms a cleanup step into an active prevention strategy that enables high-throughput multiplexed sequencing with maintained accuracy
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
Reduces index hopping by nearly 100-fold, ensuring accurate library identification by preventing unincorporated adapters from participating in cluster amplification.
Implementation Method 1
providing a first primer oligonucleotide configured to hybridize with a portion of the first adapter in proximity to a 3′ end of a strand of the first adapter. The 5′ end of the first primer oligonucleotide is modified to prevent digestion by an enzyme having 5′ exonuclease activity.
Implementation Method 2
The 5′ end of the first primer oligonucleotide is modified to prevent digestion by an enzyme having 5′ exonuclease activity.
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 modifying or blocking 5′ and 3′ ends of pooled indexed polynucleotides from multiple samples, with an optional exonuclease treatment, prior to amplification and sequencing.