Single-Cell RNA Processing with rRNA Depletion and Early Barcoding
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Solution Overview
Problem
Current methods for obtaining full-length transcript information from biological samples are limited, and ribosomal RNA (rRNA) abundance can exhaust resources and introduce bias in RNA analysis.
Innovation Solution
A method for depleting rRNA from RNA samples and barcoding non-poly-adenylated RNA molecules within partitions, using enzymatic and chemical fragmentation processes, followed by nucleic acid reactions to generate barcoded nucleic acid molecules for further analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If rRNA is not depleted from RNA samples, then total RNA can be processed, but rRNA abundance exhausts resources and introduces bias in analysis
Solution Approach 1:
The patent applies rRNA depletion by extracting and removing ribosomal RNA molecules from the total RNA sample before further processing. This is achieved through specific biochemical methods that selectively target and eliminate rRNA, allowing the remaining non-rRNA species (mRNA, lncRNA, miRNA, etc.) to be analyzed without resource exhaustion or bias from abundant rRNA sequences
2Adaptability or versatility
If current methods are used to obtain full-length transcript information, then transcript data can be obtained, but the methods are limited and do not adequately capture underexplored RNA species
Solution Approach 1:
The patent applies preliminary barcoding to RNA molecules before fragmentation and processing steps. By attaching unique molecular identifiers (barcodes) to RNA molecules in advance, the method enables tracking and identification of original transcript sources throughout subsequent processing, allowing comprehensive capture of diverse RNA species including underexplored types without requiring complex post-processing differentiation
3Reliability
If RNA samples are processed without partitioning, then processing is simpler, but accurate control of respective environments cannot be achieved
Solution Approach 1:
The patent applies partitioning to divide the RNA sample into multiple discrete droplets or wells, with each partition containing individual RNA molecules or small groups. This segmentation enables independent environmental control in each partition, allowing precise manipulation of reaction conditions (temperature, pH, enzyme activity) for different RNA species while maintaining overall process reliability
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 streamlined sample processing to obtain full-length transcript information while avoiding rRNA bias, allowing for the analysis of underexplored RNA species like microRNA and long non-coding RNA.
Implementation Method 1
depleting ribosomal RNA (rRNA) molecules from the RNA molecules, in the cell, to yield a plurality of remaining RNA molecules
Implementation Method 2
fragmenting, in the cell, the plurality of remaining RNA molecules to yield a plurality of RNA fragments
Implementation Method 3
using enzymatic and chemical fragmentation processes
Implementation Method 4
using the non-poly-adenylated RNA fragment and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded nucleic acid molecule
Implementation Method 5
the nucleic acid reaction is a nucleic acid extension reaction... the enzyme is a thermostable group II intron reverse transcriptase (TGIRT)
Data Source
AI summary
Provided herein are methods, compositions, and systems for processing nucleic acids from individual cells or cell populations. Cells are co-partitioned and processed, allowing for analysis of a variety of types of RNA from cells while minimizing ribosomal RNA species. The present invention enables high-throughput measurement of full length RNA from single cells with a streamlined workflow.