mRNA Sequencing Across Poly(A) Tails With Barcode-Guided Priming
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Solution Overview
Problem
Direct mRNA sequencing is challenging due to the presence of poly(A) tails, which are uninformative and require expensive labeled nucleotides for sequencing, leading to increased costs and time consumption.
Innovation Solution
The method involves hybridizing polynucleotides derived from mRNA with a primer, using non-terminating nucleotides to sequence through a homopolymer region, thereby omitting the poly(A) tail, and employing labeled nucleotides to determine the sequence of the target region of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If labeled nucleotides are used to sequence the poly(A) tail region, then complete sequencing coverage is achieved, but sequencing costs and time consumption increase significantly
Solution Approach 1:
The patent extracts and removes the poly(A) tail region from the sequencing process by using a primer that hybridizes to a region downstream of the poly(A) tail. This allows the sequencing reaction to start immediately at the informative region without wasting time and resources sequencing the uninformative poly(A) tail, thereby reducing sequencing time and costs while maintaining complete coverage of the target region.
Solution Approach 2:
The patent performs preliminary action by designing a primer with a pre-defined hybridization region that is located downstream of the poly(A) tail. This preliminary positioning of the primer ensures that the sequencing reaction begins directly at the informative region, avoiding the need to sequence the poly(A) tail beforehand and thus eliminating time and resource waste.
2Loss of information
If labeled nucleotides are used for complete mRNA sequencing, then full sequence information is obtained, but sequencing costs increase
Solution Approach 1:
The patent applies local quality by using labeled nucleotides only in the region where they are needed - the informative target region downstream of the poly(A) tail. The primer is designed to hybridize to a specific location that excludes the poly(A) tail, so labeled nucleotides are consumed only for sequencing the biologically relevant portion of the mRNA, reducing costs while maintaining complete information for the target region.
3Reliability
If sequencing includes the poly(A) tail region, then complete mRNA structure is captured, but sequencing efficiency decreases
Solution Approach 1:
The patent extracts the poly(A) tail from the sequencing process by positioning the primer hybridization site downstream of the tail. This extraction removes the uninformative region from the sequencing reaction, improving sequencing efficiency by allowing the reaction to focus only on the informative region while still capturing the complete structure of the target mRNA sequence through appropriate primer design.
Solution Approach 2:
The patent uses partial action by sequencing only the necessary portion of the mRNA molecule - the informative region downstream of the poly(A) tail - rather than sequencing the entire mRNA including the uninformative tail. This partial sequencing approach maintains productivity by reducing the sequencing burden while still achieving complete and reliable information for the biologically relevant region.
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 reduces sequencing costs and time by minimizing the use of labeled nucleotides and efficiently sequencing the informative regions of mRNA while avoiding the poly(A) tail, thus enhancing the sequencing process.
Implementation Method 1
hybridizing a plurality of polynucleotides derived from mRNA with a primer to form a plurality of hybridized templates
Implementation Method 2
determining the sequence of the barcode region using labeled nucleotides that lack a complement base of the same base present in the homopolymer region
Implementation Method 3
extending the primer within the homopolymer region using nucleotides complementary to the base present in the homopolymer region
Data Source
AI summary
Described herein are methods for determining a sequence of a region of interest from an mRNA molecule. Sequenced polynucleotides can include a barcode region, a homopolymer region (e.g., a poly-A region), and a target region associated with the mRNA molecule. According to some methods, the barcode region omits the same base present in the homopolymer region. According to some methods, extension of the primer used for sequencing is stalled within the homopolymer region. According to some methods, sequencing flow cycles and the different barcode regions of the polynucleotides configured are such that the primer is extended to the end of the barcode region across the plurality of polynucleotides before being extended into the homopolymer region. According to some methods, two primers or a cleavable primer is used to separately sequence the barcode region and the target region.