Random Priming V(D)J Sequencing for Accurate Full-Length Copy Counting
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Solution Overview
Problem
Existing methods for full-length expression profiling of V(D)J-containing transcripts face challenges in accurately determining the copy number and sequencing of nucleic acid targets, particularly in single-cell analyses, due to amplification bias and stochastic processes in PCR, which affect gene expression measurements.
Innovation Solution
A method involving oligonucleotide barcodes with molecular labels is used to label nucleic acid targets, generating barcoded molecules, extending their ends, and amplifying them using universal primers, followed by random priming and sequencing to determine the copy number based on distinct molecular label sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used to amplify nucleic acid targets, then the quantity of target molecules is increased, but amplification bias and stochastic processes affect measurement precision
Solution Approach 1:
The patent divides the amplification process into multiple independent PCR reactions, each targeting specific regions of the nucleic acid molecule. By segmenting the amplification, the method reduces amplification bias because each region is amplified independently, allowing accurate counting of original molecules based on the distribution of amplification products across multiple reactions rather than relying on a single biased amplification process.
Solution Approach 2:
The patent performs barcoding of nucleic acid molecules before amplification, assigning unique molecular identifiers to each original molecule. This preliminary action allows subsequent PCR amplification to proceed without introducing bias in the counting process, as the barcodes are already attached to the original molecules and can be used to track and count them accurately through the amplification steps.
2Loss of information
If full-length sequencing is performed on V(D)J transcripts, then sequencing coverage is improved, but the complexity of the sequencing process increases
Solution Approach 1:
The patent segments the full-length sequencing task by first amplifying specific regions of the nucleic acid molecules using multiple target-specific primers. This creates multiple shorter amplicons that can be sequenced more efficiently and with lower complexity than the full-length molecule would require. The segmentation allows full-length information to be reconstructed from multiple shorter sequences without requiring a single complex full-length sequencing run.
Solution Approach 2:
The patent performs barcoding and amplification of specific regions before sequencing. By preparing multiple barcoded amplicons in advance, the method simplifies the sequencing process because each amplicon can be sequenced independently using standard protocols, and the barcodes provide information about the original molecules without requiring complex sequencing methodologies.
3Loss of information
If multiple target-specific primers are used for amplification, then coverage of different regions is improved, but the number of amplification reactions increases
Solution Approach 1:
The patent uses multiple target-specific primers to segment the nucleic acid molecule into different regions, each amplified in separate PCR reactions. This segmentation improves region coverage by ensuring that different parts of the molecule are amplified and sequenced independently, providing comprehensive coverage without requiring a single complex reaction that would be less efficient.
Solution Approach 2:
The patent employs universal barcoding mechanisms that work across all amplification reactions. The same barcoding and molecular labeling approach is used in each PCR reaction, allowing the method to scale efficiently. The universal barcodes enable consistent tracking and counting across multiple amplification reactions, reducing the overall complexity despite the increased number of reactions.
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 allows for accurate determination of nucleic acid targets' copy numbers and full-length sequencing, correcting for amplification bias and improving the precision of gene expression analysis in single-cell studies.
Implementation Method 1
contacting copies of a nucleic acid target with a plurality of oligonucleotide barcodes, wherein each oligonucleotide barcode comprises a first universal sequence, a molecular label (e.g., a first molecular label), and a target-binding region capable of hybridizing to the nucleic acid target
Implementation Method 2
extending 3'-ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules
Implementation Method 3
amplifying the plurality of extended barcoded nucleic acid molecules using a target-specific primer capable of hybridizing to a sequence of the nucleic acid target and a primer comprising the first universal sequence
Implementation Method 4
hybridizing random primers to the first plurality of barcoded amplicons and extending the random primers to generate a plurality of extension products
Data Source
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AI summary
Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3' end and are subsequently barcoded on the 5' end following a template switching reaction and intermolecular and/or intramolecular hybridization and extension. 5'- and/or 3'-barcoded nucleic acid targets can serve as templates for amplification reactions and/or random priming and extension reactions to generate a sequencing library. The method can comprise generating a full-length sequence of the nucleic acid target by aligning a plurality of sequencing reads. Immune repertoire profiling methods are also provided in some embodiments.