Sequential Hybridization Barcoding for High-Multiplex Transcript Profiling
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Solution Overview
Problem
Existing methods for profiling transcripts in cells, such as single cell RNA-seq and qPCR, are labor-intensive, costly, and prone to artifacts, while in situ sequencing is inefficient, and spectral mRNA barcoding is limited in barcode generation and data complexity.
Innovation Solution
A sequential barcoding scheme using detectably labeled oligonucleotides that target nucleic acids with different detectable moieties in multiple rounds of hybridization and imaging, followed by optional removal steps to reduce noise and bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single cell RNA-seq or qPCR is used for transcript profiling, then gene expression can be measured, but the process becomes cost prohibitive, labor intensive and prone to artifacts
Solution Approach 1:
The patent segments the transcript profiling process into in situ hybridization steps with sequential barcoding, avoiding the need for cell isolation and multi-step processing required by RNA-seq and qPCR. This maintains measurement precision while reducing process complexity
Solution Approach 2:
The patent performs preliminary barcoding of transcripts in situ before any processing steps, assigning unique molecular identifiers to individual transcripts within their native cellular context. This preliminary action eliminates the need for subsequent cell isolation and processing steps
2Measurement precision
If in situ sequencing technologies are used to convert mRNA into DNA template, then transcript detection is achieved, but the conversion efficiency is low (1% for RT and 10% for PLA) introducing significant noise and bias
Solution Approach 1:
The patent extracts the conversion step entirely by using direct in situ hybridization with detectably labeled oligonucleotides that bind to mRNA without requiring reverse transcription or enzymatic conversion. This eliminates the efficiency losses and noise introduction associated with RT and PLA steps
Solution Approach 2:
The patent uses detectably labeled oligonucleotides as intermediaries that directly hybridize to target transcripts, replacing the inefficient enzymatic conversion process. These oligonucleotide intermediaries provide direct detection without the noise and bias introduced by enzymatic reactions
3Adaptability or versatility
If spectral mRNA barcoding technologies using smFISH are used, then multiple targets can be detected, but the number of barcodes is limited and data analysis complexity increases
Solution Approach 1:
The patent adds a temporal dimension to barcoding by using sequential hybridization steps with different detectable labels across multiple imaging cycles. This creates expanded barcode capacity beyond the limitations of simultaneous multi-color detection, enabling detection of many more targets without proportionally increasing data analysis complexity
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 efficient multiplexing of targets with reduced noise and bias, allowing for accurate detection and quantification of transcripts and DNA loci in cells.
Implementation Method 1
contacting a cell comprising a plurality of nucleic acids with a first plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid
Data Source
AI summary
The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. In some embodiments, through sequential barcoding, the present invention provides methods for high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci.