SPRI Bead Size Selection for Long-Read Nucleic Acid Libraries
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Solution Overview
Problem
Existing methods for size selection in nucleic acid sequencing, particularly for long-read sequencing, face challenges in efficiently recovering high yields of target fragments greater than 10 kb, are not tolerant to a wide range of input amounts, and add complexity to laboratory workflows, making them unsuitable for automation.
Innovation Solution
A method using paramagnetic Solid Phase Reversible Immobilization (SPRI) beads in a binding buffer comprising 10% PEG6000, 1900 mM NaCl, and 10 mM TrisHCl pH8, with a ratio of beads to sample between 0.97× and 0.91×, for selecting nucleic acid fragments greater than 5 kb in length.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If gel size selection is used to remove short fragments, then sequencing yield is improved, but input DNA requirements and workflow complexity increase
Solution Approach 1:
The invention changes the chemical parameters of the binding buffer by specifying exact concentrations of PEG6000 (10%), NaCl (1900 mM), and TrisHCl (10 mM pH 8), which fundamentally alters the size selection mechanism to work with SPRI beads rather than gel electrophoresis, thereby simplifying the workflow while maintaining high sequencing yield
Solution Approach 2:
The invention replaces the mechanical gel electrophoresis system with a chemical-based SPRI bead system that uses paramagnetic separation and controlled binding conditions, eliminating the need for gel casting, electrophoresis runs, and manual fragment excision while achieving equivalent or superior size selection
2Device complexity
If commercial bead-based size selection methods are used, then workflow is simplified, but size selection effectiveness for fragments >10 kb is limited
Solution Approach 1:
The invention achieves high precision size selection for fragments >10 kb by changing the binding buffer parameters to include 10% PEG6000, 1900 mM NaCl, and 10 mM TrisHCl pH 8, which creates optimal conditions for SPRI beads to selectively bind only long fragments, overcoming the limitations of commercial methods that typically target smaller fragments
Solution Approach 2:
The invention introduces dynamic control over the bead-to-sample ratio (0.8× to 1.2×) and binding buffer composition, allowing optimization of size selection precision for different fragment size ranges, particularly enabling effective selection of fragments greater than 10 kb that were previously difficult to isolate
3Reliability
If strict size selection is applied to enrich long fragments, then long-read sequencing quality is improved, but recovery yield of target fragments decreases
Solution Approach 1:
The invention optimizes the binding buffer parameters (10% PEG6000, 1900 mM NaCl, 10 mM TrisHCl pH 8) and bead-to-sample ratio (0.8× to 1.2×) to create a selective binding condition where only long fragments (>10 kb) bind to the beads, while short fragments remain in solution and are washed away, thereby achieving both high sequencing quality and high recovery yield of target fragments in a single step
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 method effectively removes shorter fragments and retains longer fragments, achieving high recovery rates of up to 100% of selected nucleic acid fragments, suitable for generating nucleic acid libraries for long-read sequencing, particularly for PacBio CCS, with improved sequencing yield and reduced workflow complexity.
Implementation Method 1
selecting for nucleic acid fragments greater than 5 kb in length from a fragmented nucleic acid sample using paramagnetic Solid Phase Reversible Immobilization (SPRI) beads
Implementation Method 2
using paramagnetic Solid Phase Reversible Immobilization (SPRI) beads
Implementation Method 3
The binding buffer comprises specified concentrations of PEG6000, NaCl and TrisHCl at pH8
Data Source
AI summary
The invention relates to a method of preparing a nucleic acid library for sequencing comprising selecting for nucleic acid fragments greater than 5 kb in length from a fragmented nucleic acid sample using paramagnetic Solid Phase Reversible Immobilization (SPRI) beads in a binding buffer. The binding buffer comprises specified concentrations of PEG6000, NaCl and TrisHCl at pH8, and the ratio of SPRI beads in binding buffer to sample is between 0.97× and 0.91×. Also provided are methods of sequencing the genome of an organism and generating novel genome assemblies.