Validation of 515F and 806R primers for M. capitata V4 microbiome analyses

The workflow below provides step-by-step instructions for how the Bhattacharya Lab validated the 515F and 8069 (targeted the V4 region of 16S rDNA in bacteria) for microbiome analyses in Montipora capitata. These primers, while working for most other taxa, have proven troublesome in M. capitata, amplifying what seems to mostly be coral DNA. For the below samples, we performed a DNA extraction protocol that enriches for bacterial DNA, in hopes that this would alleviate the issues observed previously. For the validation, 3 Montipora capitata samples, 2 sediment samples, and 2 Galaxea fascicularis samples were tested. We find that while the primers do amplify the mitochondrial genome in M. capitata, enough 16S DNA was sequenced to perform the QIIME2 workflow for microbiome analysis. Qimme2 workflow was adapted from Dr. Emma Strand’s Open Lab Notebook Post, “Holobiont Integration 16S V4 QIIME2 Analysis Pipeline”.

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Protocol for coral colony genotyping using the 18S-28S rDNA marker

The protocol detailed below provides step-by-step instructions for how the Bhattacharya Lab genotyped the Galaxea spp. corals in our coral culturing facility and is based on the methodology outlined in Takabayashi et al. 1998. In short, the 18S-28S region was amplified from extracted DNA by PCR with forward and reverse primers A18S and ITS4, respectively. The resulting amplicons were sequenced at Azenta Life Sciences using the Sanger method. The resulting sequencies were quality-trimmed and aligned using MUSCLE. Pairwise distances between samples were used to inform relatedness.

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Development of controls for Pocillopora acuta triploidy detection via qPCR

The goal of this notebook post is to explain how I developed the standards we will use for triploidy detection for the Pacu Ploidy project. We are developing a qPCR test to determine the ploidy of P. acuta individuals by detecting copy number of the ITS amplicon after several rounds of qPCR. All DNA used for the method will be normalized by mass, so it should take more cycles to amplify the DNA from the triploid samples because the they have a larger genome size; fewer copies of the amplicon will be present in the same quantity of sample. This process is based off of the geneBlock design protocol (link) used by the Frank Laboratory at Kewalo Marine Lab

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2022 Hawaii Field Plan

This post describes the timeline and methodology for all work planned during the 2022 Hawaii field season (May 3 - 24, 2022). Four or five studies will be undertaken during this trip. A short description of each will be provided below. More information can be found on the fieldwork Project Planning Board and Gannt chart made by Dr. Tim Stephens.

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