Sequencing Adapter Barcodes for Parallel Real-Time DNA Analysis
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Solution Overview
Problem
Current Illumina sequencing technologies face challenges in achieving parallel real-time analysis of multiple DNA samples due to high turnaround times and limitations in sequence diversity, especially in the initial sequencing cycles, which hinder their application in time-critical clinical and diagnostic scenarios.
Innovation Solution
The method involves connecting first and second adapter oligonucleotides to the DNA fragments, with the barcoding sequence positioned downstream of the read 1 sequencing primer site, enabling real-time sequence analysis by ensuring high sequence diversity and allowing immediate sample identification during the sequencing process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If standard Illumina sequencing protocol is used, then sequencing quality is maintained, but turnaround time is at least 24-48 hours
Solution Approach 1:
The barcoding sequence is positioned downstream of the read 1 sequencing primer site, enabling it to be read during the initial sequencing cycles. This preliminary positioning allows sample identification to occur concurrently with the sequencing process itself, eliminating the sequential bottleneck where barcoding would normally occur after sequencing completion.
Solution Approach 2:
The adapter oligonucleotide structure enables continuous useful action by allowing the barcoding sequence to be read throughout the sequencing process rather than requiring a separate post-processing step. The sequencing machine continuously reads both the DNA fragment and the barcode in parallel, maximizing productivity without sacrificing quality.
2Measurement precision
If barcoding sequence is positioned upstream of read 1 primer site, then sample identification is enabled, but sequence diversity in initial cycles is reduced
Solution Approach 1:
Instead of placing the barcoding sequence upstream of the read 1 primer site (conventional approach), the invention inverts the arrangement by positioning the barcoding sequence downstream of the read 1 primer site. This inversion ensures that the barcoding sequence is read after the initial high-diversity region, maintaining sequence diversity while still enabling accurate sample identification.
3Loss of time
If sequential sequencing and data analysis is used, then analysis accuracy is maintained, but total turnaround time increases
Solution Approach 1:
Sample identification through barcoding occurs during the sequencing process itself rather than after data generation. This preliminary identification action enables parallel execution of sequencing and analysis operations, reducing total turnaround time without compromising analytical accuracy since the barcode is read in the same sequencing cycle as the DNA fragment.
Solution Approach 2:
The invention merges the barcoding function with the sequencing function into a single integrated process. The adapter oligonucleotide structure combines both the sequencing primer site and the barcoding sequence, allowing both functions to be performed simultaneously in the same sequencing cycle, thereby eliminating the sequential time loss.
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
This approach enables parallel real-time sequencing of multiple DNA samples by distinguishing clusters based on barcoding sequences within the first sequencing cycles, reducing turnaround time and improving sequencing efficiency for clinical and diagnostic applications.
Implementation Method 1
connecting one kind of first and second adapter oligonucleotides to the 5′ and 3′ ends of a DNA strand of the DNA fragments
Implementation Method 2
Fluorescent molecules linked to the nucleotides allow the identification of the DNA sequence for each of the analyzed stretches of DNA
Data Source
AI summary
A method for real-time sequence analysis of DNA fragments includes providing a DNA fragments sample and connecting first and second adapter oligonucleotides to 5′ and 3′ ends of a DNA strand of the DNA fragments, respectively. A first adapter oligonucleotide includes from 5′ to 3′ a) a first flow cell binding sequence, b) a read 1 sequencing primer site, c) optionally a random sequence, and d) a sample-specific barcoding sequence. A second adapter oligonucleotide includes from 5′ to 3′ d) a sequence complementary to the sample-specific barcoding sequence, c) optionally a sequence complementary to the random sequence, b) a read 2 sequencing primer site that might be complementary to the read 1 sequencing primer site, and a) a second flow cell binding sequence. First and second adapter oligonucleotides of one kind have complementary barcoding sequences. A sequencing by synthesis process is used for sequencing the connected adapter oligonucleotides.