Shotgun Library Construction & Sequencing

The target DNA is randomly segmented into small fragments using the mechanical shearing method. Then the fragments are connected with the vector to construct the shotgun library.

Shotgun library construction has the following applications:

  1. Small genome sequencing such as virus, phage, plasmid, etc.;
  2. BAC/Fosmid clone sequencing;
  3. Gene screening;
  4. Thorough sequencing with fragment lengths > 9 k and without reference sequence.


Figure 2. Workflow of shotgun library construction and sequencing

Bioinformatics Contents:

The main results of library qualification include storage capacity, insert segment size, homologous alignment, and randomness analysis;
The results of assembly include average coverage, low quality range, special construction, etc.

Sample Requirements:

  • ŸSmall genome sequencing
    Fresh tissue samples:

    Plant samples should be etiolated seedlings, and animal samples should be fresh tissues.
    DNA samples:

    Concentration should be ≥ 50 ng/μL,total DNA ≥ 20 μg, and OD260/280 = 1.8 to 2.0.
    Virus, phage, and wild plasmid samples should be purified DNA samples.
  • Ÿ BAC/Fosmid clone sequencing
    Transformed strains

    Spread the plate overnight, shaking cultivated bacterial strains in a 96-well plate (single bacterial strains). Use streak or stab cultured strains.

Turnaround Time:

The standard turnaround time for using the workflow (above) is approximately 30 working days;
The standard turnaround time for library construction and sequencing using the workflow (above) is approximately 60 working days.

Completion Milestone and Results Submission:

ŸCompletion milestone:

  • Shotgun library construction projects:
    Depending on the insert fragment length, a shotgun library can be divided into the following categories: 2-3Kb, 4-6Kb, and 6-8Kb.
    The qualification ratio of the right fragment size is ≥ 80%.
    The capacity of the library depends on the genome size and coverage.
  • Library construction and sequencing projects:
    Phase II:  1scafford, 2end, < 5 gap

Results submission:

For library construction projects, all transformed bacterial liquid and the library appraisal report are included;
For library construction and sequencing projects, assembly results are included.