Synthetic 3D Matrix Indexing for Spatially Resolved Sequencing
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Solution Overview
Problem
Fluorescent in situ sequencing (FISSEQ) faces limitations in read length, read speed, spatial resolution, and sensitivity due to diffraction limits and inefficiencies in library construction, which hinder accurate and high-throughput nucleic acid sequencing.
Innovation Solution
A method for constructing a synthetic three-dimensional (3D) matrix with spatial molecular indices (SMIs) that attach to nucleic acid molecules, allowing for 3D spatial positioning and sequencing, followed by amplification and release for in situ and in vitro sequencing to determine both spatial position and sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent in situ sequencing (FISSEQ) is used to detect nucleic acid molecules, then spatial positioning information can be obtained, but read length and sequencing accuracy are limited due to diffraction limits and library construction inefficiencies
Solution Approach 1:
The patent segments the sequencing process into distinct in situ and in vitro phases. In situ, spatial positioning is captured through imaging of fluorescently labeled nucleic acids. In vitro, the same molecules are subjected to high-throughput sequencing to obtain full read lengths. This segmentation allows each method to operate in its optimal performance regime without the trade-offs that plague a single integrated approach.
Solution Approach 2:
The patent introduces spatial molecular indices (SMIs) as intermediary elements that bridge the in situ and in vitro sequencing processes. These SMIs are attached to nucleic acid molecules during in situ imaging, serving as molecular barcodes that link the spatial position captured by fluorescence microscopy with the sequence information obtained from subsequent in vitro sequencing. This intermediary mechanism enables accurate reconstruction of spatial information from pooled sequencing data.
2Productivity
If traditional FISSEQ library construction is used, then in situ sequencing can be performed, but sensitivity and throughput are reduced due to construction inefficiencies
Solution Approach 1:
The patent performs preliminary actions by attaching spatial molecular indices to nucleic acid molecules before the main sequencing operation. This pre-attachment of SMIs during in situ imaging ensures that spatial information is captured and preserved before molecules are extracted and pooled for high-throughput sequencing. This preliminary action prevents information loss that would otherwise occur during library construction and enables more sensitive detection.
Solution Approach 2:
The patent creates a copy of spatial information in the form of molecular barcodes (SMIs) that are attached to each nucleic acid molecule. These barcode copies serve as permanent records of spatial position that can be read during in vitro sequencing. This copying mechanism allows the spatial information to be preserved and accurately reconstructed even though the original molecules are extracted from their in situ environment for pooled sequencing.
3Measurement precision
If in situ sequencing is performed directly on fixed samples, then spatial position can be determined, but sequencing depth and accuracy are limited
Solution Approach 1:
The patent introduces dynamic flexibility into the sequencing workflow by allowing samples to transition between in situ and in vitro environments. After initial in situ imaging to capture spatial positions, the same samples can be dynamically transferred to in vitro conditions for enhanced sequencing. This dynamic approach enables optimization of sequencing depth and accuracy without being constrained by the limitations of purely in situ methods.
Solution Approach 2:
The patent creates a universal platform that combines both in situ and in vitro sequencing capabilities. The spatial molecular index attachment protocol serves multiple functions: it captures spatial information for imaging, provides molecular barcodes for sequencing, and enables sample tracking throughout the multi-stage process. This multi-functional approach simplifies the overall workflow compared to separate dedicated protocols while achieving superior results.
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
Enhances sequencing accuracy, sensitivity, and spatial resolution by enabling high-throughput, efficient detection of nucleic acids with improved spatial fidelity, overcoming limitations of traditional FISSEQ methods.
Implementation Method 1
Fluorescent in situ sequencing (FISSEQ) may be used to detect one or more fluorescent signals emanating from each sequencing template
Implementation Method 2
ribonucleic acid (RNA) may be converted into complementary deoxyribonucleic acid (cDNA) with the addition of a known accessory sequence domain, such as by using a reverse transcription primer
Implementation Method 3
The cDNA molecules may be subsequently circularized, such as by a splint ligation or ssDNA circularization ligation (e.g., by CircLigase) reaction
Implementation Method 4
The cDNA molecules may be subsequently circularized, such as by a splint ligation or ssDNA circularization ligation (e.g., by CircLigase) reaction, and locally amplified, such as by rolling circle amplification (RCA), in situ within the 3D matrix
Data Source
AI summary
The present disclosure provides methods and systems for generating indices in a synthetic three-dimensional (3D) matrix within a biological sample (e.g., a cell or a tissue sample). The present disclosure also provides methods and systems for using indices in the synthetic 3D matrix to index target molecules and for sequencing the target molecules in vitro.