Sample Indexing Barcodes for Multiplexed Polynucleotide Sequencing
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Solution Overview
Problem
Existing methods for sequencing multiple polynucleotide templates from different sources lose individual sample information when pooling samples, as they require separate analysis of each DNA sample and lack efficient methods to identify the source of each nucleic acid sequence after sequencing.
Innovation Solution
A method for indexing samples by attaching unique nucleic acid tags to each sample, allowing simultaneous sequencing and identification of the source, using universal adaptors and sample-specific primers to differentiate between samples, enabling multiple reads on a single array.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If samples are pooled for sequencing to reduce cost and time, then productivity improves, but individual sample information is lost
Solution Approach 1:
The patent divides the sequencing process into segments by assigning unique molecular identifiers (UMIs) and sample-specific barcodes to individual molecules and samples respectively. This segmentation allows pooled sequencing to maintain both high throughput and individual sample identification capability.
Solution Approach 2:
The patent introduces barcodes as intermediary elements that link sample identity to sequenced reads. These barcodes act as mediators that enable traceability of pooled samples back to their individual sources without requiring separate sequencing reactions.
2Measurement precision
If separate analysis of individual DNA samples is performed to retain sample information, then measurement precision improves, but productivity deteriorates
Solution Approach 1:
The patent merges multiple individual sample analyses into a single pooled sequencing reaction by incorporating barcodes. This combining approach maintains the precision of individual sample identification while achieving the productivity benefits of pooled processing.
3Device complexity
If universal adaptors are used for multiple samples, then device complexity is reduced, but sample differentiation becomes difficult
Solution Approach 1:
The patent applies local quality by maintaining universal adaptor sequences for consistent processing while incorporating sample-specific barcode regions. This allows the same basic adaptor structure to serve multiple samples while locally differentiated barcodes enable sample identification.
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
Enables simultaneous sequencing and identification of nucleic acid sequences from multiple sources with retained individual sample information, allowing for high-throughput analysis of complex genomes and multiplexing smaller genomes, with the potential to sequence millions of reads in a single experiment.
Implementation Method 1
The polynucleotide molecules are denatured to provide single-stranded templates
Implementation Method 2
Sequencing primers are hybridized to complementary sequences
Data Source
AI summary
The invention relates to methods for indexing samples during the sequencing of polynucleotide templates, resulting in the attachment of tags specific to the source of each nucleic acid sample such that after a sequencing run, both the source and sequence of each polynucleotide can be determined. Thus, the present invention pertains to analysis of complex genomes (e.g., human genomes), as well as multiplexing less complex genomes, such as those of bacteria, viruses, mitochondria, and the like.