Clone Verification

Clone verification is the process of confirming that a cloned construct contains the expected insert sequence. By comparing sequencing results to the intended reference sequence, researchers can quickly identify mutations, insertions, deletions, or cloning errors before proceeding with downstream experiments.

Visualize and Complete Clone Verification in Just a Few Clicks

Step 1: Select Reference

Step 2: Select and Group Clones

Step 3: View Results

Step 4: Inspect Differences

CodonCode Aligner simplifies clone validation by quickly mapping your cloned sequences to the reference, checking for differences, and providing a full verification report in a single workflow. The results show clone accuracy and highlight potential problems clearly for each clone. The detailed report contains sequence coverage maps and discrepancy tables with links directly to the automatically created alignment for further visual inspection.

Compare Sequencing Results to Expected Constructs

Whether you are verifying a plasmid, cloned insert, mutagenized construct, or synthetic DNA fragment, CodonCode Aligner can help you compare observed sequencing results from Sanger or NGS sequencing to the expected reference. The clone verification wizard in Aligner makes the process easy and fast - no need to align or interpret the results yourself - let the wizard do the work for you.

In the result overview, you can see at one glance how accurate your clones are and which clones are usable.

The results also show specific problems, so you know immediately if a clone has a any discrepancies, a reversed insert, only partial insert coverage, or, for example, no insert at all.

Each clone verification result can be expanded to see details about coverage, discrepancies, and a visual overview of the alignment.

Save Time When Screening Multiple Clones

Projects often require screening multiple candidate clones to find the correct construct. CodonCode Aligner streamlines this process by helping researchers automatically group and align sequencing reads, compare clones to the expected sequence, and focus their attention on clones that contain meaningful differences.

Automatically group reads by name to sort them into the different clones. CodonCode Aligner can guess a name scheme for you, or you can have full control and define the different name parts yourself to group your sequences.

Detect Mutations and Cloning Errors

Unexpected sequence changes can affect gene expression, protein function, or experimental outcomes. CodonCode Aligner makes it simple to identify mutations and cloning errors early by providing easy-to-understand results and detailed sequence comparisons.

Each analyzed clone has a detailed analysis result with a visual overview of sequence coverage and a list of all discrepancies that has direct links to the differences in the alignment.

Review Chromatograms to Confirm Variants

When a sequence difference is detected, CodonCode Aligner allows researchers to inspect the original Sanger chromatograms to verify whether the change represents a genuine mutation or a sequencing artifact. This integrated direct link to the alignment and ability to review the traces helps to make sure you can be confident in your clone verification results.

Each discrepancy in the result list has a link to the alignment for the clone verification. From there simply double click on the position in the contig to see the traces that make up your clone.

Explore More Virtual Cloning Features in CodonCode Aligner

CodonCode Aligner supports a variety of molecular cloning techniques. Explore these additional pages to learn how Aligner helps you design, simulate, and verify different cloning workflows:

• Gibson Assembly
  Seamless DNA assembly without the need for restriction enzymes — ideal for multi-fragment cloning and scarless constructs.
• HiFi Assembly
  A high-fidelity version of Gibson-style cloning that joins DNA fragments with short overlaps while reducing errors and improving efficiency for complex assemblies.
• Restriction Cloning
  Simulate restriction enzyme digests, ligation, and insert orientation with support for hundreds of enzymes.
• TA Cloning
  Learn how to simulate TA cloning and verify insert orientation before going to the bench.
• Blunt-End PCR Cloning
  Plan and test blunt-end ligation of PCR products and vectors, with annotation and map preview features.
• Directional TOPO Cloning
  Learn how to simulate directional TOPO cloning and verify insert orientation before going to the bench.
• Cloning History
  See how the cloning history automatically documents how each construct created in CodonCode Aligner was made.
• Molecular Cloning
  Overview of virtual molecular cloning methods in CodonCode Aligner.
• Primer Design
  Design primers using Primer3. Adjust parameters like primer characteristics and reaction conditions to create PCR, cloning, and sequencing primers that specifically match your workflow.
• Virtual Gels and RFLP Analysis
  Use virtual gels to verify and plan any gels you use in the lab. You can look at restriction maps and virtual gels for single sequences, or even compare multiple sequences (for example your cloning product in both directions) using the RFLP analysis tool in Aligner.