**2. Aquatic animal models in biomedical research**

In recent years, aquatic animal models have been widely used in human disease research. These model systems have demonstrated the usefulness for improving our understanding of disease pathology at the molecular and cellular biology levels and have facilitated the development of new diagnostic and therapeutic methods. A few examples of diseases modeled by aquatic models are summarized in Table 1.

An example of the use of an aquatic model for human disease research is the *Xiphophorus* model. In the 1920s, it was found that F1 interspecies hybrids between *X. maculatus* (*X. maculatus*) and *X. hellerii*, when backcrossed to *X. hellerii,* result in melanoma development among 25% of the backcross progeny (Gordon-Kosswig cross [4–6]). The melanoma develops from naturally occurring macromelanophores that are found in *Xiphophorus*. In this cross, melanoma development is the result of interaction of a melanoma locus *Tu* and a tumor suppressor locus (*R/Diff*) that is capable of inhibiting *Tu*'s oncogenic effect in the parental *X. maculatus* fish. Since *Tu* and *Diff* are on different chromosomes, the segregation of *Tu* and *Diff* into backcross hybrids results in 25% of the animals with inherited *Tu* but do not inherit melanoma suppression by the *R/Diff* and thus exhibit melanomagenesis. The gene corre‐ sponding to *Tu* was discovered to be a mutant copy of the human epidermal growth factor receptor (EGFR) termed *Xmrk*, while a candidate gene for *R/Diff* is a *Xiphophorus* homologue of human *cdkn2a/b* (i.e., p15/16) [7–9]. It has been found that the mutational inactivation of human *cdkn2a* (p16) is associated with human melanoma (for a review, see [10]), and EGFRdriven downstream signaling by Ras-Raf-MAPK activation is a marker of human melanoma


**Table 1.** Aquatic models for human diseases

to clinical application. Aquatic animal models are widely used in genomics, development biology, toxicology, pathology, and cancer research (for a recent review, see [1]). The genomes of several aquatic models had been sequenced using NGS technology over the past few years [2, 3]. NGS technology has been trending toward reduced cost with greater sequencing length and accuracy. While this has facilitated the sequencing process, sequence assembly remains a significant challenge for bench-top scientists, and especially for species with complicated

In this chapter, we will focus on the application of NGS in aquatic genome and transcriptome assemblies. Although our focus will be on the genome sequencing of aquatic models, the associated techniques, problems, concerns, and solutions can also be applied to genome sequencing of other model systems. Using *Xiphophorus maculatus* (*X. maculatus*), *X. couchia‐ nus*, and *X. hellerii* genome sequencing as examples, we will discuss the technical details of NGS, data processing, and genome assembly using guided approaches. We will also discuss the problems encountered in genome sequencing of several feral fish models (ice fish, blind cave fish, etc.) and alternative approaches to sequence and assemble these genomes. Some problems remain and these are causing a bottleneck to broadening the representation of aquatic models with genome assemblies. These problems are summarized and methods to

In recent years, aquatic animal models have been widely used in human disease research. These model systems have demonstrated the usefulness for improving our understanding of disease pathology at the molecular and cellular biology levels and have facilitated the development of new diagnostic and therapeutic methods. A few examples of diseases modeled

An example of the use of an aquatic model for human disease research is the *Xiphophorus* model. In the 1920s, it was found that F1 interspecies hybrids between *X. maculatus* (*X. maculatus*) and *X. hellerii*, when backcrossed to *X. hellerii,* result in melanoma development among 25% of the backcross progeny (Gordon-Kosswig cross [4–6]). The melanoma develops from naturally occurring macromelanophores that are found in *Xiphophorus*. In this cross, melanoma development is the result of interaction of a melanoma locus *Tu* and a tumor suppressor locus (*R/Diff*) that is capable of inhibiting *Tu*'s oncogenic effect in the parental *X. maculatus* fish. Since *Tu* and *Diff* are on different chromosomes, the segregation of *Tu* and *Diff* into backcross hybrids results in 25% of the animals with inherited *Tu* but do not inherit melanoma suppression by the *R/Diff* and thus exhibit melanomagenesis. The gene corre‐ sponding to *Tu* was discovered to be a mutant copy of the human epidermal growth factor receptor (EGFR) termed *Xmrk*, while a candidate gene for *R/Diff* is a *Xiphophorus* homologue of human *cdkn2a/b* (i.e., p15/16) [7–9]. It has been found that the mutational inactivation of human *cdkn2a* (p16) is associated with human melanoma (for a review, see [10]), and EGFRdriven downstream signaling by Ras-Raf-MAPK activation is a marker of human melanoma

genomes.

address them in the next five years are proposed.

62 Next Generation Sequencing - Advances, Applications and Challenges

by aquatic models are summarized in Table 1.

**2. Aquatic animal models in biomedical research**

(for a review, see [11, 12]). This makes *Xiphophorus* a good model for genetic study of melano‐ ma, a cancer that shows increasing worldwide incidence but has forwarded very few experi‐ mentally tractable animal models [13–15]. In addition to this spontaneous melanoma model, different *Xiphophorus* interspecies hybrids have been shown to be melanoma inducible after exposure to DNA damaging agents such as UVB light. Some of these inducible melanoma models involve hybridization of *X. maculatus* and *X. couchianus* with a following backcross of the F1 hybrid to the *X. couchianus* parent. Both the heavy pigmented backcross progeny and F1 hybrids can develop melanoma after UVB or MNU exposure in their early life stage [16–20].

Genomes of aquatic disease models serve as bridges to link phenotypic changes to genetic responses and allow physiological and pathophysiological discoveries from animal models to be applied to human disease research. The sequencing of model system genomes offers researchers great resources for biomedical research. Genome sequences allow researchers to (a) find sequence variation among genomes and transcriptomes between different species and populations; (b) compare genetic response between different phenotypes, development stages, disease conditions, drug treatment, etc.; and (c) discover gene/gene and gene/environment interactions and use these findings to direct medical applications.

For *Xiphophorus,* genome sequencing, assembly, and annotation for 3 *Xiphophorus* species (*X. maculatus*, *X. couchianus*, and *X. hellerii*) were accomplished in 2014 ([3, 21] and unpublished data). In the post-*Xiphophorus* genome era, these genomes resources have strengthened the *Xiphophorus* melanoma models by establishing high similarity in gene expression patterns for *Xiphophorus* and human melanoma tumors. The genome assemblies for both parents of an interspecific disease model are now allowing regulatory dissection of melanoma relevant gene expression in hybrids and after tumor-inducing treatments [22]. The gene expression features that characterize metastatic melanoma progression in humans closely mimic those found in *Xiphophorus* melanoma tumors (unpublished data). For the purpose of screening potential antimelanoma compounds, a mutant *Xmrk* gene has been used to make a transgenic medaka (*Oryzias latipes*) fish model that develops melanoma very early after hatching [23, 24]. Whole transgenic melanoma medaka at 3–4 weeks post hatch are being utilized to characterize melanoma disease markers and for use in screening of small compounds for inhibitors of melanoma progression. In this way, several aquatic models systems represent a direct connection from "fish tank" discovery to "bedside" therapeutic application (for additional information on this topic, see https://dpcpsi.nih.gov/sites/default/files/orip/document/ zebrafish\_workshop\_final\_report\_orip\_website.pdf).
