Enhancement of de novo sequencing, assembly and annotation of the Mongolian gerbil genome with transcriptome sequencing and assembly from several different tissues
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.
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.
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.
KeywordsGerbil genome Meriones unguiculatus Transcriptome Model organism
Benchmarking Universal Single-Copy Orthologs
Long interspersed elements
Long terminal repeats
National Center for Biotechnology Information
RNA integrity number
High-throughput messenger RNA sequencing
Short interspersed elements
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 . 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 . 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 . 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” . 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 . 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.
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.
Global statistics of the Mongolian gerbil genome
Scaffold number (> 2000 bp)
Scaffold N50 (Kbp)
Contig number (> 2000 bp)
Contig N50 (Kbp)
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).
Summary of mobile element types
Percentage of the genome (%)
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 bp and average intron length of 3300.83 bp (Table 5). The 22,998 protein-coding genes were aligned to several protein databases, along with the RNA sequences, to identify their possible function, which resulted in 20,760 protein-coding genes that had a functional annotation, or 90.3% of the total gene set (Table 6). Annotation data is available through Figshare, https://figshare.com/articles/Mongolian_gerbil_genome_annotation/9978788
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)  and further enhanced by transcriptomic analysis. The gerbil genome consists of 40% repetitive sequences which is consistent with the mouse genome  and rat genomes  (~ 40%) and is slightly larger than the previously published gerbil genome (34%) .
Genome annotation comparisons with other model organisms
Protein coding genes
Divergence time to gerbils, Myr
RefSeq/Genbank assembly accession
Annotation release ID
Completeness of gerbil genome and transcriptome assembly as assessed by BUSCO
Total BUSCO groups searched
General statistics of predicted protein-coding genes
Average transcript length (bp)
Average CDS length (bp)
Average exon per gene
Average exon length (bp)
Average intron length (bp)
Meriones unguiculatus (10)
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 pair-end 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.
High-quality reads were used for genome assembly using the SOAPdenovo (version 2.04) package.
Transcriptome sequencing and assembly
Samples from 27 tissues were collected from the seven gerbils described above (Additional file 1: Table S1). The tissues were collected after the animals were euthanized with isoflurane (followed by decapitation) and stored on liquid nitrogen until homogenized with a pestle. RNA was prepared using the RNeasy mini isolation kit (Qiagen, Venlo, Netherlands). RNA integrity was analyzed using a Nanodrop Spectrophotometer (Thermo Fisher Waltham, MA, USA) followed by analysis with an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and samples with an RNA integrity number (RIN) value greater than 7.0 were used to prepare libraries which were sequenced using an Ilumina Hiseq2000 Genome Analyzer (Ilumina, San Diego, CA, USA). The sequenced libraries were assembled with Trinity (v2.0.6 parameters: “--min_contig_length 150 --min_kmer_cov 3 --min_glue 3 --bfly_opts ‘-V 5 --edge-thr=0.1 --stderr’”). Quality of the RNA assembly was assessed by filtering RNA-seq reads using SOAPnuke (v1.5.2 parameters: “-l 10 -q 0.1 -p 50 -n 0.05 -t 5,5,5,5”) followed by mapping of clean reads to the assembled genome using HISAT2 (v2.0.4) and StringTie (v1.3.0). The initial assembled transcripts were then filtered using CD-HIT (v4.6.1) with sequence identity threshold of 0.9 followed by a homology search (human, rat, mouse proteins) and TransDecoder (v2.0.1) open reading frame (ORF) prediction.
Genomic repeat elements of the genome assembly were also identified and annotated using RepeatMasker (v4.0.5 RRID:SCR_012954)  and RepBase library (v20.04) . In addition, we constructed a de novo repeat sequence database using LTR-FINDER (v1.0.6)  and RepeatModeler (v1.0.8)  to identify any additional repeat elements using RepeatMasker.
Protein-coding genes were predicted and annotated by a combination of homology searching, ab initio prediction (using AUGUSTUS (v3.1), GENSCAN (1.0), and SNAP (v2.0)), and RNA-seq data (using TopHat (v1.2 with parameters: “-p 4 --max-intron-length 50000 -m 1 –r 20 --mate-std-dev 20 --closure-search --coverage-search --microexon-search”) and Cufflinks (v2.2.1 http://cole-trapnell-lab.github.io/cufflinks/)) after repetitive sequences in the genome were masked using known repeat information detected by RepeatMasker and RepeatProteinMask. Homology searching was performed using protein data from Homo sapiens (human), Mus musculus (mouse), and Rattus norvegicus (rat) from Ensembl (v80) aligned to the masked genome using BLAT. Genewise (v2.2.0) was then used to improve the accuracy of alignments and to predict gene models. The de novo gene predictions and homology-based search were then combined using GLEAN. The GLEAN results were then integrated with the transcriptome dataset using an in-house program (Table 5).
Functional annotation of the final gene set
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/~elieis/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.
The authors would like to thank Hilde Wohlfrom for sending tissues from Germany. We would also like to thank Ziheng Huang and Huan Liu from BGI and Dr. Laura Saba and Dr. Karen Rossmassler (University of Colorado Anschutz) for assisting with NCBI upload and Dr. Rossmassler for assisting with manuscript revisions. We would also like to thank NIH NIDCD R01 DC017924.
SC, EAM, and AK developed the ideas, methods, and, wrote and revised the manuscript. BG, YF, YZ, WX, HW, XL, and XX advised and revised the manuscript. BG provided the old animal tissues from Munich, Germany. SC, YF, YZ, WX, HW, XL, and XX performed the analysis and annotation of the genome and transcriptome. EAM prepared the DNA and RNA samples for sequencing. All authors have read and approved the manuscript.
The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. EAM’s salary is supported by NIH 3T32DC012280-05S1. AK was supported by NIH R01 DC 11582 which provided reagents for DNA/RNA extraction and gerbil housing costs.
Ethics approval and consent to participate
All experiments, including those regarding collection of tissues from gerbils, complied with all applicable laws, NIH guidelines, and were approved by the University of Colorado and Ludwig-Maximilians-Universitaet Munich IACUC.
Consent for publication
The authors declare that they have no competing interests.
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