Population genomic analysis

November 4, 2025

Today, I started using pyani-plus to determine which reference genome – the more complete KBHIv3 genome (97% completeness) or the ULFMv1 genome (85% completeness). Pyani will give us metrics of how closely related the two genomes are by nucleotide similarity. I am currently running a “simplified workflow” with just those two genomes (screen -r pyani-simple) and a more complex experimental design (screen -r pyani) that includes Acropora millepora (JSIDv2.1) as an outgroup, congeners M. efflorescens (SLJPv2) and M. cactus (SLJPv2), and conspecific from the Big Island, M. capitata WTHIv1.1. The simplified workflow is using the ANI algorithm that is considered the “gold standard” ANIb, which uses BLAST+. The complex design uses fastANI, which is faster and perhaps less accurate.

Traits of new, lineage-specific genes:

• Have higher tissue-specific expression in Green tea cultivar (Zhao & Ma 2021).
• In mangrove plants, new genes had lower overall expression and higher tissue-specific expression (Ma et al. 2021).
• Shorter (Lipman et al. ~2002~ found in a comparison between two prokaryotes, yeast, Drosophila, and humans), lower GC-content, lower codon usage bias, fewer exons (Domazet-Loso & Tautz 2003 orphan genes in Drosophila).
  • LSGs have shorter gene lengths, fewer exons, higher GC content and higher isoelectric point (Green tea Zhao & Ma 2021).
    • Paper linking gene length, GC content, and transcriptional plasticity: ~Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis - Nature Communications~.
• More orphan genes expressed in Drosophila embryos (Domazet-Loso & Tautz 2003).
• Orphans often show conditional expression, with ~10% only expressed under specific conditions (Seçkin et al. 2025).
• Many dynamically regulated in specific tissues, developmental stages, tumors, or in response to stressors like COVID-19 (Seçkin et al. 2025).
• Orphans often exhibit tissue- or stage-specific expression profiles supporting roles in lineage-specific traits and ecological adaptations (Seçkin et al. 2025).

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”.

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.

This protocol is based on the Zymo Quick-RNA™ Miniprep Plus Kit protocol and the Putnam Lab protocol using the Zymo Duet RNA/DNA Extraction Kit written by E. Strand.

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

The goal of running this procedure is to capture the V4 16S rDNA region of the microbial community in reef sediment for amplicon sequencing.

The Bhattacharya Lab houses an Eppendorf Mastercycler (catalog number 5333). The manual can be found online (here). The instructions here provide abridged instructions for running the machine. Please refer to the manual for more detailed instructions.

This protocol is based on the Zymo Quick-DNA™ Miniprep Plus Kit protocol and the Putnam Lab protocol using the Zymo Duet RNA/DNA Extraction Kit written by E. Strand.

Below based on Bhattacharya Lab Protocol written by Pinky Liau and modified by Erin Chille and Stephanie D’Elia.

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.

Below based on Putnam Lab Protocol written by M. Schedl.

The following protocol describes the process that I used to create a phylogeny of our P. acuta samples from a candidate amplicon region, using the profilin region as an example.

Alignment to a concatenated reference transcriptome compared to extraction from a de novo transcriptome assembly

A pipeline using DIAMOND Blast search, InterProScan, Blast2GO, UniProt, & KofamScan

Data uploaded and analyzed on the URI HPC bluewaves server

Overview:

Acropora hyacinthus eggs and sperm from FACE PUF Morea Project

Eggs and sperm from FACE PUF Morea Project from FACE PUF Morea Project

Eggs and sperm from FACE PUF Morea Project

Larvae from Biomineralization Project

Eggs and sperm from FACE PUF Morea Project

Larvae from Biomineralization Project

Larvae from Biomineralization Project

Larvae from Biomineralization Project

Larvae from Biomineralization Project

Materials:

• Zymo Duet DNA/RNA Extraction Kit HERE**
• Thermoixer
• Centrifuge capable of 16,000 rcf spin (Eppendorf)**

Larvae from Biomineralization Project

Larvae from Biomineralization Project

Larvae from Biomineralization Project

Larvae from Biomineralization Project

Larvae from Biomineralization Project

Larvae from Biomineralization Project

Larvae from Biomineralization Project

Posted orginally by Maggie Schedl

Larvae from Biomineralization Project