A quick guide on how to use WebRECall.
To get started with WebRECall, first enter your login name and password; this will bring you to the Start Page.
Upload Sequencer FilesEdit
Once you have loaded the Start Page, you will need to locate the files generated by your sequencer. This will vary greatly depending on your system configuration, so ask an administrator if you are unsure. Currently, .AB1 or .SCF files are accepted, however, they need to be zipped before being uploaded. This ZipFile will be the basis for your sample collection. Currently, samples cannot be added to a collection once it has been uploaded, though samples can be removed.
When collecting all of your sequencer files into the zipfile, please do not include the .qual or .poly files that are sometimes placed with the .ab1 files; These will not be used for the analysis. ZipFiles larger than 10Mb will not be accepted.
Choose Reference SequenceEdit
Once the ZipFile containing your sequencer files has been selected, choose the reference sequence with which to align your sequences. If no reference sequences are listed in the dropdown menu, you will need to ask a group SuperUser or an Administrator to upload one for you. (this can be done via the Settings page.)
Hit the 'Process Data' button at the bottom of the page, and your sequences will be uploaded to our server for aligning and analysis. The button will change into a progress baton with a snippet of descriptive text above it. This text will describe what stage of the analysis is currently being attempted. This process takes some time, so it may be a good opportunity to go get some coffee. If, for any reason you need to interrupt the process, click on the link to 'kill process'. This will interrupt the analysis, however, sequences which were successfully analyzed before the kill command was issued will still be retained.
Upon the completion of the analysis, your sequences will be displayed in the list on the right. By default, the date of the upload and the number of sequence files will be used to label the collection of sequences. If the various sequence files representing each primer associated with your sample sequence are not aligning properly, you may want to check the Settings page to ensure that the Naming syntax is correct. If your Syntax is off, WebRECall will be unable to properly determine which sequencer files belong together.
Once the sequences have been properly aligned and analyzed by WebRECall, you can click on a sample collection label to expand it, displaying all of the sample IDs in the collection. Each sample ID will be displayed in one of three colours. A label with a Red background will indicate that the sequencing has failed to pass one or more of the quality control checks that WebRECall imposes upon it and will likely need to be resequenced or at least patched. It is still possible to view the sequence alignment, however, this is usually because of large stretches of poor quality data; the number of nucleotide mixtures exceeded the 'mixture cutoff' or there was a long stretch of single coverage. The 'mixture cutoff' and the maximum acceptable length of single coverage can be set via the Settings page. A label with a Green background indicates that the sample has passed the basic quality control checks and is ready for export or review and a plain black label with a white background indicates that the sequence had been interrupted during processing, was never completed and needs to be re-uploaded for re-processing.
Sequences which failed quality control checks can be deleted from the collection via the 'Delete' Button, as can sequence IDs whose analyses were interrupted.
Double-clicking a sample ID label will open the chromatogram view in another window or tab. Also, clicking on the 'View' button beneath the collection list will serve the same purpose.
WebRECall is sensitive, and will accurately detect most of the base calls, however, dye blobs will occasionally cause erroneous readings in poor-quality areas, so it is recommended that you review all of your sequences by eye. Any changes you make to the sequence will be recorded on the server and remembered between sessions.
Exporting Your ResultsEdit
Having checked your sequences for errors, you are now ready to download the results. There are two options for doing this.
In the Settings Page, is is possible to register an email address which will be sent all results after immediate processing.
If you have edited any of the sequences by hand, you will be able to download the sequence directly to your computer by clicking on the 'Download' button. If you have selected a single Sample ID, that will be downloaded. Alternatively, it is possible to select a collection of sequences and download that as well, however it is not possible to select more than one individual sequence or collection for downloading at this time.