Whole genome sequencing century-old ethanol-preserved Philippine fishes

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Roberts_Roy_Thesis.pdf (2.12 MB)

Date

2023-08

Authors

Roberts, Roy Lonnie

ORCID

https://orcid.org/0000-0002-9056-7208

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https://hdl.handle.net/1969.6/97716

Abstract

The genomes of organisms stored in museums hold a wealth of information that is challenging to access. Recent success in sequencing desiccated museum insects involved using Whole Genome Amplification (WGA) and enzymatic repair of deoxyribose nucleic acid (DNA) damage. However, these techniques have not been tested on fishes preserved in ethanol for over a century. Here, I tested for the effects of WGA, repair, and the amount of template DNA on whole genome sequencing of historical (1908-09) and contemporary (2017-19) Philippine reef fishes. I attempted to construct 178 shotgun libraries (Illumina 2 x 150bp, 92 contemporary, 86 historical) using commercial kits and successfully produced 130 libraries (86 contemporary, 44 historical). Contrary to expectation, WGA had a negative effect on success of libraries, and repair had a small number of minor positive effects. A greater proportion of historical libraries treated with WGA failed than those that were not (37% +WGA, 65% -WGA, p=0.007). While the -WGA, historical libraries that succeeded produced less reads per unit effort than contemporary libraries (p=1.60E-07), this can be addressed by increasing the amount of DNA from historical specimens in the sequencing library. Increasing the template DNA mass was one of the best ways to improve library success, except when WGA was employed. However, only a limited amount of DNA can be obtained from historical samples using a standard commercial kit, and it would be useful to explore alternative protocols. Overall, I found the basic library preparation protocol without WGA or repair to be the best alternative for sequencing historical ethanol-preserved fish. This methodology is relatively cost-effective and can unlock the genomes of historical, ethanol-preserved fishes stored on the shelves of museums.

Description

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Fisheries and Mariculture

Keywords

bioinformatic processing, museomics, next generation sequencing, reef habitat conservation

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