Visually group your reads into clones and then map them to your reference(s). If you have a naming format for your reads and your reference(s) that meets the requirements below then this process can be automated. Please follow the steps below that are relevant to your workflow.
Naming format requirements to automate use of the Cloning Validation tool:
All your reads must have a consistent naming format.
This means they require the same number of parts with the same type of part in the same place for all reads. For example:
reference0-clone1_forwardprimer_A01.ab1
All your references must have a consistent naming format.
This means they require the same number of parts with the same type of part in the same place for all references. For example:
experiment1_reference0_location1_A1
If grouping into clones, there must be a part of your read name that specifies the clone.
For example, the following would be grouped into two clones: clone1 and clone2.
reference0-clone1_forwardprimer_A01.ab1
reference0-clone1_reverseprimer_B01.ab1
reference0-clone2_forwardprimer_C01.ab1
reference0-clone2_reverseprimer_D01.ab1
If you have identical clone names that map to different references, select the part of the name that specifies the reference as well as the clone. For example:
reference0-clone1_forwardprimer_A01.ab1
reference0-clone1_reverseprimer_B01.ab1
reference1-clone1_forwardprimer_C01.ab1
reference1-clone1_reverseprimer_D01.ab1
There must be a part of your read name that matches a part in your reference name.
To automatically map your reads to your reference(s), there must be a part of the name of both read and reference that match. For example:
1_reference0-clone1_forwardprimer_A01.ab1
reference0-clone1
How to use the Cloning Validation tool:
1. Select your data and open the tool
Select all your reads and reference(s) and click on Cloning Validation in the Cloning menu.
2. Reference Reads can be automatically detected. If not, drag the references to the right to assign as a reference. If you need to add additional reference sequences from your database. Use the "+" button at the top right of the reference panel.
3. (Optional) Group your reads into clones
Automatically
Click on the Group reads into clones button and select the parts of your read name(s) that define the clone. You will see colour coded highlighting as a preview for the grouping. If this looks correct, click OK to apply the grouping.
Manually
Select the reads from your clone, click on the Actions button and select Group Selected. Once you have grouped reads, you can drag additional reads into groups.
3. Map your reads to your reference(s)
Automatically
Click on the Automatically map by button and select the part of your reference name(s) that match the part of a name in your reads/clones, then click OK.
Manually
You can drag and drop selected reads/clones from the list of reads to the relevant reference. Alternatively, you can select your reads/clones and click on the Actions button or the [...] button within grouped reads.
4. Configure mapping options
When you are satisfied with the optional grouping of reads into clones and mapping to reference(s), click Continue. The default trimming and mapping settings should be appropriate for most circumstances but are configurable in this step. When you are satisfied with these settings, click Continue to perform batch cloning validation.
5. Validate your reads/clones
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. To view individual sequences, click the arrow to the left of the sequence name to expand the viewer. Use the 'Scroll to start of sequence" button to jump to where this sequence starts on the mapping.
Manually validate sequences by clicking the check box on the upper right of each sequence. If at least one clone sequence is valid, this also automatically marks the reference sequence as valid. Note that 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
If, for example, you already know you cannot accept any variants within a particular region, you can filter these reads from your 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 the appropriate range, and click Apply.
The remaining reads have no variants in the region of interest, so you can automatically validate all of them. To do so click the Actions tab and select Mark all shown reads as valid.
Results
Click Save to complete validation and apply the results to the documents. The document 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. These results can also be exported in table format by selecting the documents and choosing Export Documents > CSV/TSV format.
Tips
Formatting your data for automation
- To edit the names of your sequences in batch, select the sequences (before using the Cloning Validation tool) then click on the Edit menu from the main menu and select Batch Rename.
- If you want to remove parts of a name for automation purposes but want to keep the name for meta data purposes you can add a metadata field and move the data to that field or copy the name to description for safekeeping. Use Batch Rename to copy the contents of the name to another field.
Automating grouping and mapping with multiple naming formats
- If you have multiple naming formats simply perform the automatic grouping and mapping steps multiple times with the relevant read/reference template.
Using automation as well as manual grouping and mapping
- In addition to being able to use the automatic grouping and mapping, you can partially automate and partially manually assign your clones and reference mapping.
- You can manually group reads into clones after they are mapped to their reference but you can only perform this step automatically prior to mapping to their reference.
Adding more reads or reference sequences
- To add more reads, select + Add more reads, found at the bottom of the reads list
- To add more references, select + Add another reference, found at the end of the reference list.
Converting reads to references and references to reads
- Assign reads as reference sequences by selecting the read, clicking on the Actions button and then selecting Convert read to reference.
- Convert a reference to a read by clicking on the [...] button to the right of the reference name and selecting Convert reference to read.
Resetting grouping and mapping
- To reset all groupings, select all, click on the Actions button and select Ungroup all.
- To reset mapping to references click the Reset mappings button.