Enhancement of de novo sequencing, assembly and annotation of the Mongolian gerbil genome with transcriptome sequencing and assembly from several different tissues

Background The Mongolian gerbil (Meriones unguiculatus) has historically been used as a model organism for the auditory and visual systems, stroke/ischemia, epilepsy and aging related research since 1935 when laboratory gerbils were separated from their wild counterparts. In this study we report genome sequencing, assembly, and annotation further supported by transcriptome sequencing and assembly from 27 different tissues samples. Results The genome was sequenced using Illumina HiSeq 2000 and after assembly resulted in a final genome size of 2.54 Gbp with contig and scaffold N50 values of 31.4 Kbp and 500.0 Kbp, respectively. Based on the k-mer estimated genome size of 2.48 Gbp, the assembly appears to be complete. The genome annotation was supported by transcriptome data that identified 31,769 (> 2000 bp) predicted protein-coding genes across 27 tissue samples. A BUSCO search of 3023 mammalian groups resulted in 86% of curated single copy orthologs present among predicted genes, indicating a high level of completeness of the genome. Conclusions We report the first de novo assembly of the Mongolian gerbil genome enhanced by assembly of transcriptome data from several tissues. Sequencing of this genome and transcriptome increases the utility of the gerbil as a model organism, opening the availability of now widely used genetic tools.


Background
The Mongolian gerbil is a small rodent that is native to Mongolia, southern Russia, and northern China. Laboratory gerbils used as model organisms originated from 20 founders captured in Mongolia in 1935 [1]. Gerbils have been used as model organisms for sensory systems (visual and auditory) and pathologies (aging, epilepsy, irritable bowel syndrome and stroke/ischemia). The gerbil's hearing range covers the human audiogram while also extending into ultrasonic frequencies, making gerbils a better model than rats or mice to study lower frequency human-like hearing [2]. In addition to the auditory system, the gerbil has also been used as a model for the visual system because gerbils are diurnal and therefore have more cone receptors than mice or rats making them a closer model to the human visual system [3]. The gerbil has also been used as a model for aging due to its ease of handling, prevalence of tumors, and experimental stroke manipulability [1,4]. Interestingly, the gerbil has been used as a model for stroke and ischemia due to variations in the blood supply to the brain due to an anatomical region known as the "Circle of Willis" [5].
In addition, the gerbil is a model for epileptic activity as a result of its natural minor and major seizure propensity when exposed to novel stimuli [6,7]. Lastly, the gerbil has been used as model for inflammatory bowel disease, colitis, and gastritis due to the similarity in the pathology of these diseases between humans and gerbils [8,9]. Despite its usefulness as a model for all these systems and medical conditions, the utility of the gerbil as a model organism has been limited due to a lack of a sequenced genome to manipulate. This is especially the case with the increased use of genetic tools to manipulate model organisms.
Here we describe a de novo assembly and annotation of the Mongolian gerbil genome and transcriptome. Recently, a separate group has sequenced the gerbil genome, however our work is further supported by comparisons with an in-depth transcriptome analysis, which was not performed by the previous group [10]. RNA-seq data were produced from 27 tissues that were used in the genome annotation and deposited in the China National GeneBank CNSA repository under the project CNP0000340 and NCBI Bioproject # SRP198569, SRA887264, PRJNA543000. This Transcriptome Shotgun Assembly project has been deposited in DDBJ/ENA/ GenBank under the accession GHNW00000000. The version described in this paperis the first version, GHNW01000000. The genome annotation data is available through Figshare, https://figshare.com/articles/ Mongolian_gerbil_genome_annotation/9978788. These data provide a draft genome sequence to facilitate the continued use of the Mongolian gerbil as a model organism and to help broaden the genetic rodent models available to researchers.

Genome sequencing
Insert library sequencing generated a total of 322.13 Gb in raw data, from which a total of 287.4 Gb of 'clean' data was obtained after removal of duplicates, contaminated reads, and low-quality reads.

Genome assembly
The gerbil genome was estimated to be approximately 2.48 Gbp using a k-mer-based approach. The final assembly had a total length of 2.54 Gb and was comprised of 31,769 scaffolds assembled from 114,522 contigs. The N50 sizes for contigs and scaffolds were 31.4 Kbp and 500.0 Kbp, respectively (Table 1). Given the genome size estimate of 2.48 Gbp, genome coverage by the final assembly was likely complete and is consistent with the previously published gerbil genome, which had a total length of 2.62 Gbp [10]. Completeness of the genome assembly was confirmed by successful mapping of the RNA-seq assembly back to the genome showing that 98% of the RNA-seq sequences can be mapped to the genome with > 50% sequence in one scaffold. In addition, 91% of the RNA-seq sequences can be mapped to the genome with > 90% sequence in one scaffold, further confirming genome completeness.

Transcriptome sequencing and assembly
Gene expression data were produced to aid in the genome annotation process. Transcriptome sequencing from the 27 tissues generated 131,845 sequences with a total length of 130,734,893 bp. The RNA-seq assembly resulted in 19,737 protein-coding genes with a total length of 29.4 Mbp, which is available in the China National GeneBank CNSA repository, Accession ID: CNP0000340 and this Transcriptome Shotgun Assembly project has been deposited at DDBJ/ENA/GenBankunder the accession GHNW00000000. The version described in this paperis the first version, GHNW01000000. The transcriptome data was also used to support the annotation and gene predictions as outlined below in the methods section (Tables 5 and 6).
A total of 22,998 protein-coding genes were predicted from the genome and transcriptome with an average transcript length of 23,846.58 bp. There was an average of 7.76 exons per gene with an average length of 197.9

Discussion
In this study, we show a complete sequencing, assembly, and annotation of the Mongolian gerbil genome and transcriptome. This is not the first paper to sequence the Mongolian gerbil, however our results are consistent with theirs (similar genome size of 2.62 Gbp compared to our results of 2.54 Gbp) [10] and further enhanced by transcriptomic analysis. The gerbil genome consists of 40% repetitive sequences which is consistent with the mouse genome [11] and rat genomes [12] (~40%) and is slightly larger than the previously published gerbil genome (34%) [10].
In addition to measuring standard assembly quality metrics, genome assembly and annotation quality were further assessed by comparison with closely related species, gene family construction, evaluation of housekeeping genes, and Benchmarking Universal Single-Copy Orthologs (BUSCO) search. The assembled gerbil genome was compared with other closely related model organisms including mouse, rat, and hamster ( Table 3). The genomes from these species varied in size from 2.3 to 2.8 Gbp. The total number of predicted protein coding genes in gerbil (22,998) is most similar to mouse (22, 077), followed by rat (23,347), and then hamster (20,747) ( Table 3). Gene family construction analysis showed that single-copy orthologs in gerbil are similar to mouse and rat (Fig. 1). We found there were 2141 genes consistent between human and gerbil housekeeping genes (this is similar to rat (2153) and mouse (2146)). Of the 3023 mammalian groups searched through BUSCO, 86% complete BUSCO groups were detected in the final gene set. The presence of 86% complete mammalian BUSCO gene groups suggests a high level of completeness of this gerbil genome assembly. A BUSCO search was also performed for the gerbil transcriptome data resulting in detection of 82% complete BUSCO groups in the final transcriptome dataset ( Table 4). The CDS length in the gerbil genome was 1535, similar to mouse (1465) and rat (1337) ( Table 5). The gerbil genome contained an average of 7.76 exons per gene that were on average 197.9 in length, similar to mouse (8.02 exons per gene averaging 182.61 in length) and rat (7.42 exons per gene averaging  179.83 in length) ( Table 5). The average intron length in the gerbil genome was 3300.83, similar to the 3632.46 in mouse and 3455.8 in rat (Table 5). Based on the results from the quality metrics described above, we are confident of the quality of the data for this assembly of the gerbil genome and transcriptome.

Conclusions
In summary, we report a fully annotated Mongolian gerbil genome sequence assembly enhanced by transcriptome data from several different gerbils and tissues. The gerbil genome and transcriptome add to the availability of alternative rodent models that may be better models for diseases than rats or mice. Additionally, the gerbil is an interesting comparative rodent model to mouse and rat since it has many traits in common, but also differs in seizure susceptibility, low-frequency hearing, cone visual processing, stroke/ischemia susceptibility, gut disorders and aging. Sequencing of the gerbil genome and transcriptome opens these areas to molecular manipulation in the gerbil and therefore better models for specific disease states.

Animals and genome sequencing
All experiments complied with all applicable laws, NIH guidelines, and were approved by the University of Colorado and Ludwig-Maximilians-Universitaet Munich IACUC. Five young adult (postnatal day 65-71) gerbils (three males and two females) were used for tissue RNA transcriptome analysis and DNA genome assembly (these animals are maintained and housed at the University of Colorado with original animals obtained from Charles River (Wilmington, MA) in 2011). In addition, two old (postnatal day 1013 or 2.7 years) female gerbil's tissue was used for transcriptome analysis (these were obtained from a colony housed at the Ludwig-Maximilians-Universitaet Munich (which were also originally obtained from Charles River (Wilmington, MA)) and tissues were sent on dry ice to be processed at the University of Colorado Anschutz). All animals were euthanized with isoflurane inhalation followed by decapitation. Genomic DNA was extracted from young adult animal tail and ear snips using a commercial kit (DNeasy Blood and Tissue Kit, Qiagen, Venlo, Netherlands). We then used the extracted DNA to create different pairend insert libraries of 250 bp, 350 bp, 500 bp, 800 bp, 2 Kb, 4 Kb, 6 Kb, and 10 Kb. These libraries were then sequenced using an Illumina HiSeq2000 Genome Analyzer (Ilumina, San Diego, CA, USA) generating a total of 322.13 Gb in raw data, from which a total of 287.4 Gb of 'clean' data was obtained after removal of duplicates, contaminated reads, and low-quality reads.

Genome assembly
High-quality reads were used for genome assembly using the SOAPdenovo (version 2.04) package.

Quality assessment
Genome assembly and annotation quality were further assessed by comparison with closely related species, gene family construction, evaluation of housekeeping genes, and Benchmarking Universal Single-Copy Orthologs (BUSCO) search. Gene family construction was performed using Treefam (http://www.treefam.org/). To examine housekeeping genes we downloaded 2169 human housekeeping genes from (http://www.tau.ac.il/ẽ lieis/HKG/) and extracted corresponding protein sequences to align to the gerbil genome using blastp (v.2.2.26). Lastly, we employed BUSCO (v1.2) to search 3023 mammalian groups.