DNA Microscopy In Situ Barcoding for Spatial Transcriptomics
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Solution Overview
Problem
Current DNA microscopy methods require dissociation of cells for analysis, which is problematic for structurally fragile cell types like neurons, and existing RNA sequencing technologies face challenges with sequence-dependent bias and inaccuracy, especially at low copy numbers.
Innovation Solution
The method involves randomized barcoding of nucleic acids before and during in situ amplification, generating unique molecular identifiers (UMIs) and unique event identifiers (UEIs) to enable sequencing of DNA or RNA in non-dissociated cells or tissue at single-cell or subcellular resolution, using commercial DNA sequencers for high coverage and single-base resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cells are dissociated for analysis, then sequencing accuracy is improved, but structural integrity of fragile cell types is lost
Solution Approach 1:
The method performs preliminary barcoding of nucleic acids with unique molecular identifiers (UMIs) before cell dissociation. This preliminary action tags each nucleic acid molecule with a unique identifier that persists through subsequent processing steps, allowing accurate reconstruction of original molecular information even when cells are dissociated for sequencing analysis.
Solution Approach 2:
The method creates copies of nucleic acid molecules through in situ amplification while maintaining spatial information. Amplified copies retain the original barcodes and can be detected through the cell membrane without requiring physical extraction, effectively creating information copies that enable sequencing while preserving structural context.
2Productivity
If bulk lysis is performed for RNA sequencing, then throughput is improved, but spatial information and pairing information is lost
Solution Approach 1:
The method performs preliminary barcoding with UMIs and spatial encoding before bulk processing. This allows the subsequent bulk lysis and high-throughput sequencing to proceed efficiently while the pre-established barcodes preserve information about original spatial locations and molecular pairings.
Solution Approach 2:
The barcode sequences serve as intermediaries that carry spatial and pairing information through the bulk processing pipeline. These molecular tags act as information carriers that bridge the gap between in situ spatial organization and bulk sequencing throughput, allowing both high productivity and information retention.
3Quantity of substance
If exponential PCR amplification is used, then signal amplification is improved, but sequence-dependent bias and inaccuracy increase
Solution Approach 1:
The method uses self-service amplification through in situ synthesis that occurs within the fixed cell structure. The amplification process is driven by the template molecules themselves and their associated barcodes, reducing external interference and sequence-dependent biases that plague conventional PCR methods.
Solution Approach 2:
The method replaces conventional enzymatic PCR amplification with in situ synthesis approaches that occur within the fixed cell matrix. This substitution reduces mechanical and enzymatic biases inherent in traditional PCR while maintaining signal amplification, thereby improving measurement precision.
4Manufacturing precision
If sectioning is performed for tissue imaging, then resolution is improved, but volumetric information is lost
Solution Approach 1:
The method creates information copies through barcode tagging of all nucleic acid molecules throughout the entire tissue volume before any sectioning. This allows multiple sections to be analyzed while reconstructing the complete three-dimensional spatial distribution of molecules through computational integration of the barcode data from different planes.
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 volumetric imaging without sectioning, high read lengths, and low error rates, enabling the analysis of single template molecules with high accuracy and precision, even in complex tissues like tumor or neural tissue.
Implementation Method 1
mRNA transcripts are reverse-transcribed into cDNA and fixed to the cellular matrix
Implementation Method 2
mRNA transcripts are reverse-transcribed into cDNA and fixed to the cellular matrix
Implementation Method 3
the cDNA subsequently circularized so that polonies comprising long repeats of cDNA sequence may be formed by rolling circle amplification
Implementation Method 4
fluorescent primers using fluorescence microscopy
Implementation Method 5
fluorescent primers hybridize to target sequences
Data Source
AI summary
The present invention relates to DNA microscopy methods to record the cellular co-localization and/or spatial distributions of arbitrary nucleic acid sequences, or other biomolecules tagged with nucleic sequences. The method involves sequence-components which may identify the targeted sequences-of-interest themselves and/or spatial beacons relative to which their distances are measured.