Nucleic Acid Sequencing With Unique Molecular Tags
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Solution Overview
Problem
Current sequencing technologies face challenges in efficiently sequencing long nucleic acid sequences, particularly the 16S rRNA gene, which limits taxonomic resolution and is prone to recombination errors during amplification, making it difficult to accurately identify and distinguish between original and recombinant sequences.
Innovation Solution
A method involving the use of unique molecular tags at both ends of target nucleic acid molecules, followed by amplification, fragmentation, and sequencing, allows for the reconstruction of consensus sequences and identification of recombinant products, enabling accurate sequencing of long sequences and improving taxonomic resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If amplification steps are performed to analyze nucleic acid samples, then the quantity of nucleic acid molecules increases, but recombination events occur during amplification leading to sequencing errors
Solution Approach 1:
The patent applies preliminary action by performing molecular tagging of individual nucleic acid molecules before amplification. Unique molecular tags are introduced to each template molecule in advance, allowing later identification and tracking of original molecules versus recombination products generated during subsequent amplification steps.
Solution Approach 2:
The patent implements feedback by using the unique molecular tags to detect and identify recombination events that occur during amplification. The tags provide information feedback that enables computational methods to distinguish between original template sequences and recombination artifacts, allowing for correction or exclusion of erroneous sequences.
2Productivity
If sequencing focuses on short regions of 16S gene, then sequencing speed and volume increase, but taxonomic resolution decreases
Solution Approach 1:
The patent applies segmentation by dividing the long 16S rRNA gene into multiple shorter readable fragments through amplification and sequencing of individual tagged molecules. Each fragment carries unique molecular tags that allow computational reassembly, enabling high-throughput sequencing of short regions while maintaining the ability to reconstruct the complete long sequence for accurate taxonomic classification.
Data Source
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AI summary
The present invention relates to methods for generating sequences of template nucleic acid molecules, methods for determining sequences of at least two template nucleic acid molecules, computer programs adapted to perform the methods and computer readable media storing the computer programs. In particular the present invention relates to methods for generating sequences of at least one individual target template nucleic acid molecule comprising: a) providing at least one sample of nucleic acid molecules comprising at least two target template nucleic acid molecules; b) introducing a first molecular tag into one end of each of the at least two target template nucleic acid molecules and a second molecular tag into the other end of each of the at least two target template nucleic acid molecules to provide at least two tagged template nucleic acid molecules wherein each of the at least two tagged template nucleic acid molecules is tagged with a unique first molecular tag and a unique second molecular tag; c) amplifying the at least two tagged template nucleic acid molecules to provide multiple copies of the at least two tagged template nucleic acid molecules comprising the first molecular tag and the second molecular tag; d) sequencing regions of the at least two tagged template nucleic acid molecules comprising the first molecular tag and the second molecular tag; and e) reconstructing a consensus sequence for at least one of the at least two target template nucleic acid molecules.