Uncovering the cryptic ecological diversity of Caribbean sponges

Methods

Summary

Methods justification

Metagenomics can identify which microbial genes are present in a DNA sample and their predicted functions. This approach is particularly powerful for characterizing the ecological roles of non-culturable microbes, which comprise the majority of Ircinia’s microbiome (Hardoim and Costa 2014).

Overview and technical hypothesis

I will conduct metagenomics to test the hypothesis that incipient Ircinia species differ in relative enrichment levels of microbial genes associated with the hosts’ dissimilar ecological profiles.

Reproducibility

All DNA sequences will be made publicly available on GenBank as assembled microbial contigs. Additionally, I will publish all code necessary to replicate the analysis on Github.

1) DNA isolation

MoBio PowerSoil kit: this DNA extraction kit is optimized for obtaining genomic DNA in soil samples and performs well for the microbiomes in sponges.

2) Library preparation

I will use a non-PCR (polymerase chain reaction) DNA library preparation, which is required to make reliable inferences regarding which ecological processes are dominant in my sponges’ microbiomes (Jones et al. 2015). PCR is a method that amplifies segments of DNA and results in higher DNA concentrations, however, at the cost of introducing errors (alterations to the DNA sequence due to polymerase infidelity during DNA replication and amplification biases that change the relative abundances of different DNA sequences). Thus, I need to use a reagent kit that is free from PCR biases.

3) DNA sequencing

Data generation will consist of paired-end sequencing of sonication-sheared DNA fragments on an Illumina HiSeq4000.

4) Bioinformatics: microbiome metagenome assembly

Assembly of microbial contigs and functional annotation will be performed in the DOE-JGI MAP pipeline using the KEGG database (Huntemann et al. 2016).

5) Analysis: test of the hypothesis

I will perform a PERMANOVA on a relative abundance matrix of gene copies followed by a SIMPER post hoc test. The SIMPER analysis will show which genes are driving the among-species differences. Finally, pairwise PERMANOVAs will determine which species pairs host significantly dissimilar suites of microbial genes.

Challenges

The data collection and analyses planned for my project are low-risk procedures with well-established precedents in the literature. I am also proficient in the wetlab and computational techniques required to perform metagenomics, and thus few technical obstacles are likely to present in the execution of my project, provided that I perform quality control on my finished DNA library. This step is required to ensure the ligation of Illumina adapters onto the microbial DNA and will involve Sanger sequencing plasmids that contain ligated sequences from the finished DNA library. This confirms that the sample-specific barcodes and next-generation sequencing adapters are intact in the final constructs and are ready to be sequenced on the HiSeq4000. The costs of cloning and the Sanger sequencing runs are included in the ‘reagents’ section of the budget.

Pre Analysis Plan

Please see 'summary.'