Importing Trace Data

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Before importing reads you should create a new folder for them, normally named after the plate you are working with (eg. "M062"). To create a new folder, select File > New Folder... from the menu.

Downloading Traces from LIMS

An example of downloading two sequencing plates (one for each direction).

The easiest way to import your traces into Geneious is to download them directly from the LIMS database (if you have previously attached your trace files to a sequencing plate). This is the best way to import traces because the read directions are set automatically and the FIMS data is attached at the same time. These steps will have to be performed manually if you import your traces directly from disk (see below).

To download your traces from the LIMS, select the sequencing plates you want to download in the Biocode plugin search, then select Biocode > Download Traces from LIMS. Normally you would choose one forward and reverse sequencing plate for a set of sequences. You may just select a single plate if it holds all your forward and reverse reads. Click OK and Geneious will ask you to choose a folder, and begin downloading your sequences.

Once complete, you will have all of the traces from the plates you entered and they will already have their read directions set and be annotated with the necessary data from the FIMS. Therefore you can skip the Annotate with FIMS Data step further down the pipeline.

Tip: If you know the names of your sequencing plates, you can download them directly without having to perform a search. Select the folder you want to download to in Geneious, then select Biocode > Download Traces from LIMS, and enter your plate names manually.

Importing Traces from Disk

Locate the ab1 or scf files on disk then click and drag them from the file manager on to the new folder in Geneious. Alternatively you can import from inside Geneious using File > Import > From File... in the menu.

Note: If the traces you plan to work with have already been attached to a cycle sequencing reaction in the LIMS then you can download the traces directly from the LIMS. If so, you should skip to the Download Traces from LIMS section. It is not necessary to set read direction as described below.

Setting Read Directions

Once you have imported the reads it is currently necessary to tell Geneious which reads are in the forward or reverse direction. This is so that the correct read is reversed during assembly and downstream steps are able to identify the direction of the reads.

Select all of either the forward or reverse reads from the ones you have imported and select Biocode > Set Read Direction... in the toolbar. Choose either Forward or Reverse for the read direction and click OK. It is only necessary to mark either the forward or reverse reads, Geneious will work out the rest by process of elimination.

After running this, an extra column will be added to the reads named "Is Forward Read" with a value of true or false.

Tip: If your forward and reverse reads are in different folders, it's easiest to import all of the reads from one folder then set the read direction for those then import the second folder.

Tip: You can use Edit > Batch Rename... to add _F or _R to the names of your reads if the names don't have any indication of direction (not required)

Search box.png

Tip: If you have imported both forward and reverse reads in to Geneious before setting read direction you can use Search or Filter in the top right corner of the Geneious window to locate a particular direction of read based on names.

Annotating with FIMS/LIMS data

You can either enter a forward and reverse plate, or you can use the annotated plate and well if you are updating sequences you have previously annotated.

To aid downstream analysis and submission it is extremely useful to annotate sequences with the associated data from the FIMS. This must be done pre-assembly (with the reads) because forward and reverse reads can come from different sequencing plates. Annotating is the first step in the assembly pipeline that utilizes the FIMS/LIMS database so you will need to connect to the Biocode service before proceeding. To do this, right-click on Biocode in the sources panel on the left-hand side and select login.

Select all of the reads which you imported and go to Biocode > Annotate with FIMS Data... in the toolbar.

You need to enter the forward and reverse sequencing plate names (from the LIMS) which correspond to your reads and identify which part of the sequence names identify the well location. If both forward and reverse reads are on a single plate then you can leave the reverse plate field blank or enter the same name twice.

Click OK and the operation will add many new columns to the table for each of the reads. These include things like Specimen ID, Taxonomy and Collector. The values should be identical for each forward and reverse pair of reads.

Often there will be reads which don't have entries in the FIMS due to sequencing results coming through from wells which were essentially empty. This operation will tell you about any of these and the extra columns will be left blank.

Annotating with FIMS data only

If you have plate data in your FIMS database, and you do not wish to enter reaction information for your data in the LIMS, choose Biocode > Annotate with FIMS data only..., and enter the name of your FIMS plate.

Please note that you will not get primer information for your sequences using this method, so you may have to annotate those yourself if you want to use the sequences to generate a genbank submission.

Tip: To get the empty well reads out of your way, you can easily select them all by sorting the table by one of the FIMS attributes (eg. Tissue ID) then selecting the ones with no value. You can then either delete them or create a new subfolder called "empties" and move them in to there.

Tip: If you want to change the names of your reads to reflect some aspect of the FIMS data, you can use Edit > Batch Rename... to copy your choice of fields in to the name column.