Aug 302016
 
EDNA label printer

The EDNA label printer in the office

Over the years, many different people have used the molecular laboratories at RBGE, to work on a multitude of projects on a multitude of plants and fungi. Some are staff members who stay for decades, others students who are only in the lab for a matter of months. Every time DNA is extracted and used in a molecular project, the amplified gene regions are processed and then the plastic tubes that they were in are sent for recycling – but the extracted DNA is kept in a DNA bank, in case it is needed for further research. Logistically, managing this DNA can be problematic. Scientists like to use their own numbering systems when they’re working (mine used to be one of the commonest – my initials followed by consecutive numbers, a system which worked perfectly until some of my extractions ended up in the same freezer as extractions by Dr Linda Fuselier), something quick and easy to scrawl onto the plastic tubes. This can link to collection information written in a lab-book, including who collected the plant, what date it was collected, and what country it came from. However, as people move on, and as the years pass, it becomes increasingly difficult to find any particular sample or set of samples, particularly when several sets of people share the same initials – and this is compounded by having to rummage through boxes of frozen DNA samples being kept at either -20° or -80°C. Few places at the Botanics are less pleasant than the dank room that contains our -80°C freezers!

 

Printed labels and EDNA tubes, Lab 32

Printed labels getting stuck onto EDNA tubes, Lab 32

The frustrations associated with rooting through inconsistently labelled DNA collections led Dr Michelle Hart and Alex Clarke, in 2006, to instigate a standardised format for DNA labelling, with samples of DNA identified as part of the RBGE DNA bank and assigned EDNA numbers, the format of which consists of the year the DNA was banked, followed by a multi-digit identification number. For example, the last EDNA number that we have issued is EDNA16-0045851, for DNA extracted from the moss Weissia controversia. Due to uncertainties about institutional databases, in its early years the DNA bank was curated through Excel spreadsheets; this was revamped and upgraded in 2011 to the database that we still use today. Information about the methods and date of DNA extraction, the material’s collector, and the place of collection are all stored and easily retrieved, critical information if the DNA is going to be used to provide data for future publications. The EDNA number stays on all downstream files that are created from the DNA – lab books, raw sequence files, and it is also included as the isolate number in GenBank submissions – meaning that all molecular data generated at RBGE is still valuable after people have moved on and lab books have been mislaid.

 

EDNA tube

A labelled EDNA tube ready for the DNA sample, Lab 32

As to what happens to the actual DNA extraction, long-term storage involves transferring the liquid into a small barcoded and labelled tube in a lockable and numbered 96-tube rack, which will be kept on a labelled shelf in a -80°C freezer. The system is not perfect, however – banking or recovering the DNA samples still involves a trip to our mildewy bank room…

 

Pipelling DNA samples into EDNA tubes, Lab 31

Pipelling DNA samples into labelled EDNA tubes, Lab 31

Aug 232016
 

When people extract DNA in the RBGE molecular lab, we insist that it’s given something we call an EDNA (Edinburgh DNA) number. This links to a database that is internal to RBGE.

evilednaThe EDNA number is used for all internal molecular lab processes – it’s written on the tube of DNA, used to refer to the sample in lab books, and part of the file name for all DNA sequences that are generated from that sample. Using this standard system across all projects means that we can keep track of what DNA we have, we can store it in a way that makes it relatively easy to retrieve, it can be used in other projects, and critical information like which specimen voucher is linked to a DNA extraction is not lost if people move on from RBGE.

Getting an EDNA number involves filling in a simple Excel spreadsheet with some basic collection information, and uploading it to a database. The Excel spreadsheet is accessible to RBGE lab users on an internal server (DNA, Molecular lab registration forms, EDNA (DNA), EDNA_submission_sheet_v01), and has two sets of fields, required and additional. If anything’s missing from the required fields, an EDNA number will not be issued, whereas the additional data is recommended but not essential… However, the more fully complete the data entry is, the faster it is to use it to generate GenBank submissions and publication voucher tables, justifying spending a little extra time on getting the forms completed.

Two points to remember when filling in the spreadsheet are not to use special characters, and not to make any of the entries too long, as there’s a maximum character number.

 

REQUIRED INFORMATION

Taxon name: this should not have authority information (Bellis perennis L.), just the genus and specific epithet (Bellis perennis).

Collector name: this cannot begin with an initial (J. Smith) as it will be rejected by the database; either use a full Christian name (John Smith), or put the surname first (Smith, J.).

Collector number: if there is none, s.n. is accepted.

Country code: two-letter standard codes; when filling in the spreadsheet, there is a tab with all the codes that you can look up (e.g. DE for Germany).

Material type: drop-down menu choices – fresh, frozen, herbarium, seed, silica gel dried.

Extraction type: drop-down menu choices include tissue maceration type, e.g. pestle, or mixer mill, and chemistry used, e.g. CTAB, Plant DNeasy minikits, Qiextractor.

 

ADDITIONAL INFORMATION

User DNA ID: this is the number that was given to the extraction in the lab; it’s extremely useful to have this for various troubleshooting in the lab – it can help match accessions to tubes, sort out issues with sample order, etc.

Extraction Date: entered in standard format year-month-date. Again, this can be useful for later troubleshooting, e.g. for separating batches of extractions by date, in case something went wrong on a particular day.

Herbarium barcode: this is ONLY for RBGE herbarium barcodes, not those from other institutes. If this is available, filling this in will propagate specimen data from the herbarium database. However, the required fields still need to be filled in.

Living Accession Number: this is ONLY for RBGE living accessions, not those from other institutes. If this is available, filling this in will propagate specimen data from the living collection database. However, the required fields still need to be filled in. The qualifier letter should not be filled in here.

Living Qualifier: this field is for any alphabetical character after the Living Accesion Number.

Silica Gel Box Number: this field is best left empty unless silica material came from a box numbered in the same format as “SGN12345”.

Sample note: free field, but there is a limit on how many characters are allowed, so should be kept short, and free from special characters. It may be useful to note e.g. if the extraction was from sporophyte versus gametophyte tissue, or flower versus leaf.

Location: free field, but there is a limit on how many characters are allowed, so should be kept short, and free from special characters.

Coordinates: free field, but there is a limit on how many characters are allowed, so should be kept short.

Decimal longitude:

Decimal latitude:

Collection Date Verbatum: this is for dates that cannot be turned into the correct date format, e.g. “Spring 1920”, “October 1976”.

Collection Date: entered in standard format year-month-date. This can be very useful in relation to DNA quality. If this is filled in, there is no point also filling in the Collection Date Verbatum field.

Note: free field, but there is a limit on how many characters are allowed, so should be kept short, and free from special characters.

 

Once the EDNA form is filled in, it can be uploaded to the EDNA database, which is available to users at RBGE who have a Username and Password.

Once logged on, the tab ‘Importer’ becomes highlighted; at the bottom of the Importer screen is a “Load” button.  The information in the excel sheet should be pasted into the ‘Load data’ window, and mapped to the fields. This will leave four fields that need to be filled in manually, three required fields: User (the lab user’s name, available from a drop-down list); Project (again, from a drop-down, e.g. MSc, barcoding, Leguminosae); Contact (a permanent staff member who will take long-term responsibility for the project, chosen from the drop-down list) – and one optional field, EDBANK Format (how the DNA will be stored long term – Plate, Strip or Tube; for most phylogenetics projects DNA will be stored in individual tubes, while for some population genetic project it will be stored in strips or plates – check with the molecular lab staff if unsure which format to chose).

After this information is filled in, the tab “Validate” becomes available. The entered data is screened for things like collector names that start with initials, accession numbers, dates, latitudes and longitudes that are in the wrong format, or other errors. If any are found, then these need corrected in the excel spreadsheet and the information all needs reloaded and re-entered. If there are no validation errors, the “Import to EDNA” button becomes available. At this point, the data will either successfully import, or other errors will be identified (e.g. non-standard characters, or too many characters). Unfortunately errors identified at this later point only stop EDNA numbers being generated for individual samples rather than for the whole batch, and it is not possible to cancel the issued EDNA numbers. This means that, for example, if entering a plate of 96 DNA extractions to EDNA, it’s quite possible for some samples in the middle of the plate to not be assigned a number. Obviously this becomes a sample labelling headache that is optimally sorted by redoing the entire batch to get consecutive EDNA numbers for all the samples, although this will lead to apparent duplicates of samples in the database. Molecular lab staff should be informed of redundant numbers, so that the duplicates are not also assigned places in the DNA bank.

When the numbers have been generated, they can be downloaded from the database by clicking on the “Tasks” tab, and the “As Spreadsheet” option – this will return all the information that has just been entered, along with the EDNA accession numbers for each sample.

 

See also:

The RBGE DNA bank