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Published on: February 7, 2025
459
Sequencing Strategy to Ensure Accurate Plasmid Assembly
Sarah I Hernandez1, Casey-Tyler Berezin1, Katie M Miller1
1Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, Colorado, 80523, United States of America.
Biorxiv : the Preprint Server for Biology
|April 8, 2024
Summary
Accurate plasmid sequencing is crucial for research and production. A hybrid approach using both short and long reads provides the most reliable de novo assembly, identifying mutations effectively.
Area of Science:
- Molecular Biology
- Genomics
- Bioinformatics
Background:
- Plasmid sequence verification is an underestimated bottleneck in research and clinical production.
- Current sequencing technologies and assembly pipelines impact final plasmid assembly accuracy.
Purpose of the Study:
- To evaluate short-read, long-read, and hybrid de novo assembly pipelines for plasmid sequencing.
- To identify the most accurate and consistent method for plasmid assembly and mutation detection.
Main Methods:
- Comparative analysis of de novo assembly pipelines (short-read, long-read, hybrid).
- Testing across a 24-plasmid library with three replicates.
- Evaluation of assembly accuracy, consistency, and mutation identification.
Main Results:
- Short-read assemblies struggled with GC-rich regions.
- Long-read assemblies exhibited insertions and deletions in repetitive regions.
- Hybrid assemblies demonstrated superior accuracy, consistency, and mutation detection.
Conclusions:
- Hybrid sequencing approaches offer the most robust de novo plasmid assembly.
- Challenges remain in resolving highly GC-rich and repetitive regions.
- Prioritizing easily sequenced genetic elements in construct design is recommended.
