NGS Library Negative Selection via Adapter-Mediated Restriction
Solution Overview
Problem
Current methods for removing non-desired DNA sequences from next-generation sequencing (NGS) libraries, such as ribosomal RNA, are inefficient and can distort the sample population, particularly when using oligo dT priming which is not applicable to prokaryotic organisms and misses non-polyadenylated regulatory transcripts.
Innovation Solution
A method involving sequence-specific oligonucleotide primers and restriction endonucleases to selectively remove non-desired nucleic acid sequences from NGS libraries after library generation, using adaptors with recognition sequences for restriction enzymes, allowing for unbiased template populations and enrichment of desired sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If oligo dT priming is used to remove non-desired DNA sequences, then ribosomal RNA can be depleted, but the method distorts the sample population and cannot be used for prokaryotic organisms
Solution Approach 1:
The patent changes the priming parameters from oligo dT (polyadenylate-specific) to random hexamers, fundamentally altering the selection mechanism to be organism-independent while maintaining depletion effectiveness through subsequent adapter-based selection
Solution Approach 2:
The patent introduces adapter molecules as intermediaries that carry restriction enzyme recognition sequences. These adapters mediate the selection process by enabling restriction enzyme-based removal of non-desired sequences after random priming, thus achieving both broad applicability and effective depletion
2Productivity
If oligo dT priming is used to enrich for polyadenylated transcripts, then mRNA can be selected, but non-polyadenylated regulatory transcripts are lost
Solution Approach 1:
Instead of selecting for polyadenylated transcripts using oligo dT priming, the patent inverts the approach by using random priming to capture all transcripts and then using adapter-based restriction enzyme selection to remove non-desired sequences, thereby preserving non-polyadenylated regulatory transcripts
Solution Approach 2:
The patent extracts non-desired sequences through adapter-mediated restriction enzyme digestion rather than excluding them during the priming stage. This extraction approach allows comprehensive representation of the transcriptome while removing only the unwanted portions
3Adaptability or versatility
If random priming is used to create unbiased template population, then all sequences are represented, but non-desired sequences remain in the library
Solution Approach 1:
The patent performs preliminary random priming to create an unbiased template population where all sequences are represented. Subsequently, adapters are ligated and restriction enzymes are applied to remove non-desired sequences, achieving both unbiased representation and selective depletion
4Manufacturing precision
If sequence-specific primers are used to target non-desired sequences, then depletion specificity is improved, but primer design complexity increases
Solution Approach 1:
The patent creates universal adapters that can be ligated to various DNA fragments and serve multiple functions: they provide restriction enzyme recognition sites for non-desired sequence removal and serve as handles for PCR amplification of desired sequences. This multi-functionality simplifies the overall process compared to designing multiple sequence-specific primers
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
Effectively depletes undesired sequences, such as ribosomal RNA, while retaining desired sequences in an unbiased manner, improving the quality of NGS libraries by reducing distortion and enabling more accurate sequencing results.
Implementation Method 1
annealing a sequence-specific oligonucleotide primer or a set of sequence-specific oligonucleotide primers to the single-stranded DNA templates
Implementation Method 2
extending the sequence-specific primers with a DNA polymerase beyond the adaptor-DNA template junction, thereby creating double-stranded DNA fragments
Implementation Method 3
treating the population of DNA fragments (both single-stranded and double-stranded) with a restriction endonuclease specific for double-stranded DNA, thereby cleaving only double-stranded DNA fragments at the adaptor restriction endonuclease site
Data Source
AI summary
The present invention provides methods, compositions and kits for the generation of next generation sequencing (NGS) libraries in which non-desired nucleic acid sequences have been depleted or substantially reduced. The methods, compositions and kits provided herein are useful, for example, for the production of libraries from total RNA with reduced ribosomal RNA and for the reduction of common mRNA species in expression profiling from mixed samples where the mRNAs of interest are present at low levels. The methods of the invention can be employed for the elimination of non-desired nucleic acid sequences in a sequence-specific manner, and consequently, for the enrichment of nucleic acid sequences of interest in a nucleic acid library.