Workflow Geneious/Biocode LIMS

A handbook for the practical implementation of barcode sequencing with Geneious and the Biocode LIMS plugin.

Geneious is a platform for managing and analysing molecular sequences.

• Workflow Geneious/Biocode LIMS (this document)
• Creating the Extraction Plate
• Creating the PCR Plate
• Creating the Sequencing Plates
• Download the Raw Sequences into Geneious
• De Novo Assemble
• Quality Check and GBOL Gold Standard
• Generate Consensus Sequences
• Flag Entries in DiversityCollection
• Mark Sequences as passed

The entire data flow from the creation of the extraction/sequencing plates to sequencing, assembly, error analysis and the final barcode is mapped using the LIMS/FIMS plugin for Geneious. The plates are created in the Biocode LIMS plugin and annotated with the collection information („Field Information Data, FIMS“). The workflow from extraction to PCR and raw sequence data is reproduced in the LIMS via different copies of the first extraction plate, which are all linked together.

Finished sequencing plates are plates to which the raw sequences („traces“) from the sequencing lab are attached in ABI format. These are transferred to Geneious, where they are assembled and checked. Finished sequences are marked as passed and copied back to the LIMS. From there, a script takes them and copies the sequence information into DiversityCollection.

• Biocode LIMS Plugin
• Two logins are required to use the system: A personalised login for the Geneious Shared Database and a general login for the FIMS/LIMS system. Please refer to this documentation: Geneious for CaBOL - Installation and Database Login.
• A Primer database: there is one already setup in the shared database. Please add new primers there for all to use.
• The Binning profiles for vertebrates and invertebrates. Available from here: https://oc.zfmk.de/index.php/f/831677

Here are listed some basic agreements for the naming of plates, sequences and files, which have proven to always achieve the same results.

In a name, different components are always separated with an underscore.

Plates:

A plate name for e.g. an extraction plate is composed of the project name (here: CaBOL), the plate type and a consecutive number. All three parts are connected by underscores:

<Project name>_<Plate type>_<running number with 4 digits>

Example: CaBOL_Extr_0001.

The abbreviations for the different plate types are:

Extr

Extraction plate

PCR

PCR plate

SeqF

Sequencing plate forward

Seqr

Sequencing plate reverse

Indicators for failed sequences as used in DiversityCollection:

CON

obvious contamination like Homo sapiens or a completely different order

XCON

cross-contamination with another sample on the plate like neighboring wells

PCON

contamination with the DNA of a parasite e.g. Wolbachia

CHK

different species or order – determination must be verified by specialist

LUMP

two or more species share one barcode (same percentage) → cannot be distinguished by COI

BOLD

identification of an animal not precisely determined by matching with the database

The detailed installation and login process for the Biocode Plugin is described here: Geneious for CaBOL - Installation and Database Login.

The official Biocode Wiki: https://github.com/biocodellc/biocode-lims/wiki

When installed you find the Biocode icon in your Geneious and can log in: