Perform Cloning Validation

Introduction

In this tutorial, we will take Sanger sequencing reads from several cloning samples and learn how to use the Cloning Validation tool to rapidly map the sequences to their respective reference, check each for variants, validate sequences that meet our criteria, and output a summary table of each clone's validation status.

Sequence Naming Schema

To automate the grouping and mapping process you will need to have your reads and reference(s) in a consistent naming format. In the sample data you will find a pair of Sanger sequence reads for each clone and for each gene of interest.

Notice that each sequence name also contains the reference name. There must be a part of your read name that matches a part in your reference name for this automatic mapping assignment function. If grouping sequences into clones, there must also be a part of your read name that specifies the clone. The parts of your sequence names can be separated by standard delimeters (e.g. comma, underscore, period, or space). The Batch Rename tool can help to quickly name your sequences in this format.

Since our sequences already have the sequence names formatted, you can select all of the sequences reads and the reference sequences and start the cloning validation tool. Select all of the sample sequences (Sanger sequences and references) and start the tool through Cloning > Cloning Validation.

Automated Grouping of Sequences

Click on the Automatically group by... button to open the visual grouping feature.

In our samples, the clone identifier is in the second section of the name in each sequence, so select this section, and click OK.

We now have a pair of reads (forward and reverse) for each clone automatically grouped for us.

Next, click on the Automatically map by... button.

Select the section of your reference sequence names that corresponds to the naming of our clone sequences and click OK.

Sequences from each clone will now be further grouped to their corresponding reference sequence.

Checking the tick boxes within each group will set that group for mapping. To simply map all clones that have grouped, click the three dots on the upper right of the reference group, and click "Select All".

Once you've selected the clones you wish to map, click Continue.

Mapping Options

Here we can select the Sequencing technology used for our reads and our trimming preferences. Select Sanger and Re-trim sequences. You can also expand the advanced options to tweak the mapping parameters. We'll leave these as default for this tutorial. Next, click Continue to begin mapping your reads.

Results

In the results page, you can see the reads for each clone have been aligned to their respective reference sequence. Sequences where variants were found are highlighted. At the top of the results window you can see the Length, Average Quality, the number of Variants identified, and the Validation status for each sequence.

Let's first look at Reference B, since we have only one clone here. For now, collapse the clones and sequences for Reference A by clicking the down arrow at the left of the reference sequence. You can hover your mouse over the reference name to see the full name of the sequence.

In the first sequence of cloneB3 we have 2 variants. Scroll the sequence viewer to the right until you can see the two variant annotations.

We can see that these two annotations are near the tail end of the sequence where the sequence quality is poor. Furthermore, we can see in the reverse read that these bases are correct. We can therefore manually validate both sequences by clicking the check box on the upper right of each sequence.

You should now see that cloneB3 has been validated and because at least one sequence is valid, this automatically marks the reference sequence as valid.

Note: If you first mark any sequence within a clone as not valid and then mark additional sequences as valid within that clone this will not automatically validate the clone. However, you can still manually validate the clone by clicking the check box on the clone.

Automated Validation

Next, well look at Reference A. Collapse Reference B, and expand Reference A.

Here we have some reads with many variants. If we already know we cannot accept any variants within a particular region, we can filter these reads from our results. At the top of the results window, you can see the Filter results options. Click on Variants, select Without Variants, and click Apply. Next, click In entire construct, select Part of construct, choose Based on annotation type... > CDS and the annotation mCT3 VH, and click Apply.

This action has removed the reads where there were any variants within the mCT3 VH CDS annotation and these wont be validated. In the remaining reads, we are now zoomed into the chosen region. Since the remaining reads have no variants in the region of interest, we can automatically validate all of them.

To do so click the Actions tab and select Mark all shown reads as valid.

Next, we will reverse the filter so that we can mark the invalid sequences. Click on the Variants button again, but this time select With variants > Include Silent Mutations, and click Apply. Mark all of these sequences (in our case only one) as not valid, either through Actions or by clicking the X option on the sequence.

Notice that the cloneA3 validation status remains unmarked. Whenever the validation status for both sequences agrees (either both valid or both not valid) the status for the clone with be automatically applied. In this is case, we previously marked one of the reads as 'valid' and now the second read is marked 'not valid'. Therefore, we now need to manually choose whether to validate this clone. I'll chose to not validate the clone based on this sequence.

Now that we have validated our sequences, click Save to apply the validation status to the sequences.

Validation Results

The Results table now contains the assemblies of each clone, with their assembly reports. Each read has a new meta-data column added: Validated. Here we can see that the sequences for cloneA3 were not validated and the clone A3 assembly is also not validated. All other sequences and clone assemblies were validated. Each reference sequence also shows which clone was used to validate the reference within the Validating clone column.

Additional meta-data can be found in the document history. For example, if multiple clones were validated for a given reference, this data can be found within the document history.

Exporting Validation Results

You may wish to export your validation results for record keeping or sharing validation status of the clones. There are a couple ways to do this:

1. Export the document table You can select all of your sequence export any columns from the data table you wish in a table format (e.g. CSV/TSV). For example, select all of the ABI files and use Export > Export Documents, choose the format TSV/CSV, and select the document table columns you wish to export.

2. If you wish to export detailed information on each variant identified in your clones, you can export this table within the Cloning Validation tool. Simply click Actions > Export As and choose TSV or CSV.