Genomic Diversity

The service has all the required equipment to implement the following services:

1) Preparation, purification or quantification of DNA and RNA samples
 

(i) Preparation, purification or quantification of DNA samples. The KingFisher Flex nucleic acid purification technology is based on the use of magnetic spheres for sample extraction in a 96- well format. High quality processing minimises the risk of reagent carry-over and cross- contamination between samples. Standard processing includes DNA extraction of 94 samples plus 2 controls per run.

Quantification of DNA concentration of samples in 96-well plates (94 samples) is performed with an Infinite® M Nano+. These measurements can be performed by fluorescence detection (Qubit® homologue) or absorbance (Nanodrop® homologue) on the DNA samples, as required

2) Barcoding, Metabarcoding and Metagenomics

ii) Metazoa barcoding. Quantification, normalisation and PCR amplification of a 658 bp fragment of the 3' end of the COI gene (fragment considered the standard barcode in Metazoa).

Quantifications of DNA extracts are done by absorbance measurements on the Infinite® MNano+, which allows working with 96-well plates. Normalisation and PCR preparation will be carried out entirely on the Tecan Freedom EVO 150 workstation.
After positive amplification of the samples, Sanger sequencing will be carried out at the customer's expense and can be sent to the agreed sequencing service directly from our facilities.

(iii) Metabarcoding of Metazoa. An automated service is offered for the development of metabarcoding libraries for a 418 bp COI gene fragment (3' end of the gene) for high-throughput sequencing (Illumina® platform).

The service includes quantification and normalisation of the DNA concentration of the samples, PCR amplification of the 3' end (418 bp) of the COI gene, and indexing of the samples with the Nextera XT Index Kit v2, including the necessary sample clean-up steps at different steps of the protocol using magnetic spheres (AMpure XP). The service includes the check of each XT library generated by measuring the concentrations of fragment ranges in a Bioanalyzer 2100, as well as a final measurement of the dsDNA concentration by fluorescence in the Infinite® MNano+ reader, and the final normalisation and pooling for sequencing in the MiSeq platform using a 2x300 cycles kit.

We also offer the possibility of producing Nextera XT libraries of fragments other than the 3' end (418bp) of the COI gene. In these cases, the customer must supply the PCR products, carrying out the indexing, normalisation, pooling and sequencing process on the MiSeq platform at our facilities. Sequencing in this case can be carried out with 2x150 or 2x200 cycle kits. The PCR products received will be checked before the start of the process to confirm the quality of the samples.

iv) Sequencing of total genomic DNA. The service offered consists of the preparation of Illumina® TruSeq or TruSeq Nano libraries and their sequencing on the Illumina® MiSeq platform (2x300 cycles or 2x150 cycles). The service includes the sonication of the samples in a Covaris sonicator and the quantification of the samples by absorbance (homologous method to Qubit) before and after the library preparation process. If required, the service will include the re-concentration of the initial DNA, or of the final product after the preparation of the libraries, using a DNA Clean&Concentrator kit from Zymo Research.

Requirements

1. Preparation, purification or quantification of DNA samples: The samples to be extracted shall be delivered to the service in 1,5 ml vials (1 sample per tube), labelled with indelible alcohol- based ink.
2. Metazoan barcoding: In case DNA extractions have not been performed by this service, DNA extracts shall be sent in individual tubes or in a 96-well plate, leaving wells 95 and 96 as blank.
3. Metabarcoding of Metazoa: For the elaboration of Nextera XT libraries of the 3' end of the COI gene, samples will be delivered to the service in individualised vials labelled with indelible alcohol-based ink. For the production of Nextera XT libraries other than those mentioned above, the customer must supply the PCR products. The PCR products received will be checked before the start of the process to confirm the quality of the samples.
4. Total genomic DNA sequencing: DNA extracts shall be delivered to the facility in individual tubes or in a 96-well plate. DNA extracts received shall be checked before the start of the process to confirm the quality and concentration of the samples.

The Excel file provided by this service shall be filled in, where data on the samples are collected for their correct identification.

The user will be provided with the technician's e-mail address and contact telephone number for any queries they may have.

Quantification of the DNA concentration of samples is automated with an Infinite® MNano+ fluorescence multi-mode microplate reader. These measurements can be performed by fluorescence detection (Qubit® homologue) or absorbance (Nanodrop® homologue) on DNA samples, as required.

• KingFisher Flex.
• Robotic workstation (Feedom EVO 15workstation).
• Absorbance and Fluorescence Reader (Infinite® MNano+).
• Agilent Bioanalyzer 2100.