Got a second gene!

We could finally amplify a second gene from the DNA of the samples collected last year. This was a fragment of the mitochondrial gene Cytochrome Oxidase I (COI). This gene is the standard for the Barcode of Life project (at least for metazoans), but problems with the universality of the primers and the obtained sequence length made it less used in metabarcoding applications.

The agarose gel of the COI amplification

Starting from published primers and modifying them through database mining, we obtained highly degenerate primers that amplified successfully in all samples. We will now send this DNA for high throughput sequencing

We cannot wait to have the results and compare them with what we get with the 18S gene. It is reasonable to expect that a multi-locus approach will give us the best picture of the biodiversity present.

The comparison of molecules with morphology

One of the objectives of the project was to validate the obtained results from molecular data by using a traditional morphological approach. We welcome in the project Manuel Orobitg, a student in the Master in Oceanography and Marine Environment Management of the University of Barcelona. In his MSc Thesis Manuel will analyse morphologically the samples from the Maërl community in Islas Cíes and Cabrera that were collected at the same time than the samples for genetics and were preserved in formalin.

Manuel will identify morphospecies of the major groups, and will identify to species level the polychaetes with the help of Dr. Daniel Martin. He will also obtain sequences from the main species so that we will be able to identify them unequivocally in our genetic metabarcodes.

Aspect of a Maërl sample

Manuel in action sieving samples

We expect that this morphological dataset will confirm the usefulness and completeness of molecular techniques, but it might also uncover any bias, if present.

We filtered the samples with the same protocol used for the genetic samples, and Manuel is now doing the long and time-consuming process of sample sorting and group separation...best of lucks, Manuel!

Drowned in sequences!

Well, we did receive in February the sequences of 18S of the first round of samples from Islas Cíes and Cabrera... not bad to start with: over 14 million sequences!!

Now we are working in the analyses of this huge amount of data. We had to filter out bad quality reads, sequencing errors, and contaminants. Then we combined all sequences that were equal.

From this we went on and grouped similar sequences in Molecular Operational Taxonomic Units, our proxy for species, and our raw data for ecological analyses. These MOTUs were assigned a taxonomy using reference databases in GenBank

We had to fight a little bit with the different programs and pipelines, we wrote some code, and kept the processors of the CEAB computer cluster busy for some time. But we got finally over 4200 MOTUs!!! our preliminary analyses showed a clear distinction in biodiversity structure in both parks, and also between communities.

We are excited about the amount of MOTUs we obtained, and are looking forward to start the ecological/biodiversity analyses... will keep you updated!