3' End Blocking for Index Hopping in Nucleic Acid Libraries
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Solution Overview
Problem
Index hopping occurs during sequencing of polynucleotides from multiple indexed libraries, where index tag sequences from one library are inadvertently added to polynucleotides from another library, leading to incorrect assignment of library origin, due to unincorporated adapters serving as primers during cluster amplification.
Innovation Solution
Blocking the 3' ends of polynucleotides and unincorporated adapters with dideoxynucleotides prevents them from serving as primers, thereby mitigating index hopping by ensuring accurate library origin identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If unincorporated adapters are present during cluster amplification, then library polynucleotides can be amplified efficiently, but index hopping occurs where index tag sequences are incorrectly assigned to polynucleotides from different libraries
Solution Approach 1:
The patent applies preliminary action by blocking the 3' ends of unincorporated adapters before the cluster amplification step. This preventive measure ensures that while adapters can still hybridize and initiate amplification of library polynucleotides, they cannot serve as primers for unwanted amplification that would cause index hopping. The blocking is performed in advance using enzymes like TdT to add dideoxynucleotides or other blocking groups to the 3' ends of free adapters, eliminating the harmful effect before it occurs during amplification.
2Reliability
If 3' ends of polynucleotides are blocked with dideoxynucleotides, then unincorporated adapters cannot serve as primers during cluster amplification, but the blocking process adds complexity to library preparation
Solution Approach 1:
The patent applies parameter changes by modifying the chemical state of the 3' ends of polynucleotides and adapters. Specifically, dideoxynucleotides are incorporated at the 3' ends, which changes the chemical parameter from a hydroxyl group to a dideoxy group that cannot form phosphodiester bonds. This parameter change effectively blocks the 3' ends without requiring complex structural modifications or additional components, achieving high reliability in index tag assignment while adding minimal complexity to the library preparation procedure.
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 3' blocking method significantly reduces index hopping, improving the accuracy of sequencing results by preventing incorrect index tag associations, as demonstrated in both Illumina TruSeq Nano and PCR-Free library preparations.
Implementation Method 1
Blocking the 3' ends of polynucleotides and unincorporated adapters with dideoxynucleotides prevents them from serving as primers
Implementation Method 2
The 3' ends of the first and second pluralities of polynucleotides and the first and second oligonucleotide adapters that are not attached to target polynucleotides may be blocked by incorporating a dideoxynucleotide onto the 3' ends of the oligonucleotides and polynucleotides
Implementation Method 3
The universal extension primer sequence may, among other things, hybridize to a first oligonucleotide coupled to a solid surface
Implementation Method 4
Hybridization of the sequencing primer may occur at a location on the coupled polynucleotide strand
Implementation Method 5
a polymerase may add nucleotides to extend the sequence using the hybridized library polynucleotide as a template
Implementation Method 6
Sequencing may occur through multiple rounds of addition of nucleotides to the sequencing primer using the coupled polynucleotide as a template
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.