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Related Concept Videos

Genome Annotation and Assembly03:36

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The genome refers to all of the genetic material in an organism. It can range from a few million base pairs in microbial cells to several billion base pairs in many eukaryotic organisms. Genome assembly refers to the process of taking the DNA sequencing data and putting it all back together in a correct order to create a close representation of the original genome. This is followed by the identification of functional elements on the newly assembled genome, a process called genome annotation.
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The genome of most prokaryotic organisms consists of double-stranded DNA organized into one circular chromosome in a region of cytoplasm called the nucleoid. The chromosome is tightly wound, or supercoiled, for efficient storage. Prokaryotes also contain other circular pieces of DNA called plasmids. These plasmids are smaller than the chromosome and often carry genes that confer adaptive functions, such as antibiotic resistance.
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Plasmids are extrachromosomal DNA molecules found in bacteria, archaea, and some eukaryotic microbes like yeast. These small, circular DNA structures typically contain fewer than 30 genes, although some may exist linearly. Plasmids vary in their number within a cell, known as copy number. Single-copy plasmids are present in one copy per cell and multi-copy plasmids are present in multiple copies, reaching over 100 copies per cell.Plasmids usually replicate independently of the chromosomal DNA...
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Updated: Jul 29, 2025

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies
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Long read genome assemblers struggle with small plasmids.

Jared Johnson1, Marty Soehnlen1, Heather M Blankenship1

  • 1Michigan Department of Health and Human Services, Bureau of Laboratories, Lansing, MI, 48906, USA.

Microbial Genomics
|May 24, 2023
PubMed
Summary
This summary is machine-generated.

Long-read genome assemblers often miss bacterial plasmids, especially smaller ones. Unicycler, a hybrid assembler, successfully recovered all plasmid sequences, making it ideal for comprehensive bacterial genome analysis.

Keywords:
Plasmidshybrid genome assemblylong-read sequencingwhole-genome sequencing

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Area of Science:

  • Genomics
  • Bioinformatics
  • Microbiology

Background:

  • Whole-genome sequencing is crucial for bacterial studies, often assuming complete genome capture.
  • Long-read assemblers (Flye, Raven, Miniasm, Canu) may miss plasmid sequences, particularly those of smaller sizes.
  • Plasmid recovery is essential for understanding bacterial genetics and evolution.

Purpose of the Study:

  • To investigate the relationship between bacterial plasmid size and recovery rates using different genome assemblers.
  • To compare the performance of long-read-only assemblers against a hybrid assembler for plasmid recovery.
  • To identify optimal assembly strategies for maximizing plasmid sequence capture.

Main Methods:

  • Evaluated four long-read-only assemblers (Flye, Raven, Miniasm, Canu) and one hybrid assembler (Unicycler).
  • Assessed recovery of 33 bacterial plasmids (1,919–194,062 bp) from 14 isolates using Oxford Nanopore long reads.
  • Compared assembler performance using both long-read and Illumina short reads for Unicycler.

Main Results:

  • Long-read-only assemblers (Canu, Flye, Miniasm, Raven) frequently missed plasmid sequences.
  • Unicycler achieved 100% plasmid recovery across all tested plasmids.
  • Plasmid loss by long-read assemblers was primarily associated with plasmids <10 kb.

Conclusions:

  • Long-read-only assemblers are unreliable for complete bacterial plasmid recovery.
  • The hybrid assembler Unicycler is recommended for maximizing plasmid sequence capture in bacterial genome assembly.
  • Plasmid size is a critical factor influencing recovery success with long-read sequencing data.