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Related Experiment Video

Updated: Jun 8, 2025

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

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.

ACS Synthetic Biology

|November 7, 2024

Summary

This summary is machine-generated.

Accurate plasmid sequencing is crucial for research and production. A hybrid approach using both short and long reads provides the most reliable de novo plasmid assembly, identifying mutations effectively.

Keywords:

NGS assembly nanopore reproducibility whole-plasmid sequencing workflows

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Last Updated: Jun 8, 2025

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

Use of In Vivo Assembly for High-efficiency Plasmid Construction

Use of In Vivo Assembly for High-efficiency Plasmid Construction

Published on: February 7, 2025

Improving Student Outcomes with an Adaptable Molecular Cloning Course-Based Undergraduate Research Experience

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Published on: November 15, 2024

Area of Science:

Molecular Biology
Genomics
Bioinformatics

Background:

Plasmid sequence verification is an underestimated bottleneck in research and clinical production.
Existing sequencing and assembly methods present challenges, particularly for de novo assembly.

Purpose of the Study:

To evaluate short-read, long-read, and hybrid de novo assembly pipelines for plasmid sequencing.
To compare the accuracy and consistency of different sequencing strategies.

Main Methods:

Evaluation of three de novo assembly pipelines: short-read, long-read, and hybrid (short + long reads).
Testing across three replicates of a 24-plasmid library.

Main Results:

Short-read assemblies struggled with GC-rich regions.
Long-read assemblies exhibited insertions and deletions, especially in repetitive regions.
Hybrid assemblies demonstrated the highest accuracy, consistency, and mutation identification.

Conclusions:

Hybrid sequencing approaches offer superior plasmid de novo assembly.
Challenges remain in resolving highly GC-rich and repetitive regions.
Prioritizing easily sequenced genetic parts in construct design is recommended.