Sample processing, library preparation, and sequencing

1. Optimization of sampling and DNA isolation protocols

I conducted a pilot sampling at Kilifi Creek to determine the best storage media, transport conditions, and extraction protocols. Following this, a total of 75 coral samples were collected. Verification gels yielded positive results, which meant that DNA isolation was successful (see the attached gel image); however, due to the holobiont nature of corals, further confirmation of coral DNA was necessary

 

Figure 1: Verification gel for genomic DNA (gDNA). The multiple bands in a single sample indicates gDNA from multiple organisms, confirming the fact that coral is a holobiont

2. Confirming the presence of coral-specific DNA in the holobiont

To confirm the presence of coral DNA, PCR was performed using published 12s and COI primers (Shinzato et al, 2021). I successfully validated that coral DNA was indeed present in my samples, as shown in the gel image below. This step was crucial for ensuring the accuracy of subsequent analyses.

 

Figure 2: Coral-specific verification gel. All samples tested were positive for coral DNA

3. Optimization of the 3RAD library preparation protocol

I went ahead to start library preparation, focusing on optimizing protocols for 3RAD libraries suitable for Illumina sequencing (Novaseq 6000). Briefly, this involved normalizing gDNA to approximately 20 ng/uL and digesting with 3 restriction enzymes (BamHI-HF, ClaI, and MSPI)—hence the name "3RAD." After digestion, I conducted adapter ligation, pooling (based on the unique adapter combinations), limited cycle PCR to add sequencing primers, and finally size selection. I selected a size between 300 and 450 bp. I successfully optimized the library preparation protocol as shown below:

 

Figure 3: A gel image showing sie-selected libraries, ready for sequencing. "A. Library" means "amplified library." This was amplified by PCR to make it visible in agarose

4. Pilot sequencing

To validate the methods, a pilot library consisting of five samples was sequenced, and the sequences were analyzed using BLAST in NCBI. The results confirmed that all sequences were positive for corals, indicating that the optimization methods were successful. The following barplot shows the hits obtained from BLAST.

 

Figure 4: A bar plot showing the distribution of hits from NCBI BLAST algorithm. All samples tested positive for corals (26 unique genera and 11 unique species). Up to this point, all my lab protocols are working!

5. Final library preparation and sequencing

I have prepared a library for all samples, which has already been shipped to Macrogen, Europe, for sequencing. I am currently waiting the sequence data to finalize the project