DOI: 10.5061/dryad.pzgmsbcvt
title: Four new genome sequences of the Pallas's cat (Otocolobus manul): an insight into the patterns of within-species variability

description: Manul (Otocolobus manul) is the only representative of the genus
 Otocolobus belonging to the Leopard Cat lineage. Their habitat is
 characterized by harsh environmental conditions. Although their
 populations are probably more stable than previously thought, it is still
 the case that their population size is declining. The main cause of their
 decline is the destruction of their natural habitat, which together with
 their natural behavior results in geographically fragmented populations
 and a potential threat of loss of genetic variability. Conservation
 programs exist to protect manuls, but those based on captive breeding are
 often unsuccessful due to their increased susceptibility to diseases. The
 manul is therefore a suitable model species for evolutionary and diversity
 studies as well as for studying mechanisms of adaptation to harsh
 environment and mechanisms of susceptibility to diseases. Whole genome
 sequencing (WGS) is an important tool for such studies, providing
 base-by-base view of the genome. Recently, the genome of the Otocolobus
 manul based on nanopore long-range sequencing has been published. Using
 whole genome resequencing via the Illumina platform, we obtained
 information on the genomes of four other manuls aiming to better
 understand inter-and intraspecific variation of the species. The
 parameters characterizing the quality of sequencing were within the
 standard range, all four genomes analyzed were similar in most
 characteristics. On average, we detected a total of 3,668,327 polymorphic
 variants. Information on different types of structural varinats not
 available from the reference genome was retrieved. The average
 whole-genome heterozygosity detected was almost identical to that found in
 the Otocolobus manul reference genome. In this context, we performed a
 more detailed analysis of the candidate gene EPAS1 potentially related to
 adaptation to the hypoxic environment. This analysis revealed both
 inter-and intraspecific variation, confirmed the presence of a previously
 described non-synonymous substitution in exon 15 unique to manuls and
 identified three additional unique non-synonymous substitutions located in
 so far not analyzed EPAS1 exonic sequences.
description: Tissues of four different manul individuals (one female and three
 males) obtained from Czech ZOOs (ZOO Jihlava, ZOO Brno, ZOO Prague) were
 selected based on available information about their origin and
 relatedness. The samples were obtained either post-mortem or as part of
 veterinary procedures performed for other reasons. DNA
 was extracted from available tissue samples (either blood, spleen or
 colon) using Qiagen (Germany) MagAttract HMW DNA isolation kit. The kit
 was used according to the manufacturer’s recommendations. Two isolations
 were made for each individual. DNA samples were evaluated in terms of
 purity (absorbance) and concentration using Tecan (Switzerland) Infinite
 200 Pro plate reader. DNA samples were stored at 4°C for 5 days and then
 transported on dry ice to the Novogene sequencing facility in Germany.
 Samples were checked prior to library construction using Agilent 5400
 fragment analyzer. All samples passed QC (quantity ≥ 200 ng;
 OD260/280=1.8-2.0, no degradation). Sequencing was
 performed using the Illumina NovaSeq X Plus platform as a service provided
 by Novogene (China).  A total amount of 0.2 μg DNA per
 sample was used as input material for the DNA library preparations. The
 genomic DNA sample was randomly fragmented by sonication to a size of 350
 bp. Then DNA fragments were endpolished, A-tailed, and ligated with the
 full-length adapter for Illumina sequencing. The fragments with adapters
 were size selected, PCR amplified, and purified by AMPure XP system
 (Beverly, USA). Subsequently, library quality was assessed on the Agilent
 5400 system (Agilent, USA) and quantified by QPCR (1.5 nM). The qualified
 libraries were pooled and sequenced on Illumina NovaSeq X Plus platform
 with PE150 chemistry. Distribution of sequencing
 quality along reads and sequencing error rate were evaluated, low-quality
 reads and adaptors were filtered using Fastp (v.0.20.0) with parameters -g
 -q 30 -u 50 -n 15 -l 150. The OtoMan_p1.0 genome
 (GCA_028564725.2) was used as reference. The filtered sequencing data were
 mapped to the reference sequence through BWA (Li et al., 2009a) software
 (parameters: mem -t 4 -k 32 -M). Resulting alignments were sorted using
 SAMtools (v1.13) with parameters sort -@ 6 -m 2G and merged for each
 sample using Picard (v1.111). Single nucleotide
 polymorphisms (SNP) and indels were called for entire cohort (joint
 calling) using Haplotypecaller from GATK (v4.0.5.1) (DePristo et al.,
 2011) with the following parameters --pair-hmm-gap-continuation-penalty 10
 -ERC GVCF --genotyping-mode DISCOVERY -stand-call-conf 30. Polymorphisms
 detected were annotated using ANNOVAR (v2015Dec14) (Wang et al., 2010) and
 their characteristics (e.g. quality, total numbers and distribution in
 different genomic regions) were evaluated. The original annotation record
 for reference genome was kindly provided by the authors (Flack et al.
 2023). As for structural variants (SVs), BreakDancer
 (v1.4.4) (Chen et al., 2009) software was used with default parameters to
 detect indels, inversions, intra-chromosomal translocations and
 inter-chromosomal translocations. The SVs detected were filtered by
 removing those with less than 2 supporting reads; indels and inversions
 were further annotated by ANNOVAR. Characteristics of SVs such as their
 total numbers, distribution across genome and length were
 assessed. Based on the genome reads depth, CNVnator
 (v0.3) (Abyzov et al., 2011) was used to detect Copy Number Variants
 (CNVs) of potential deletions and duplications with the following
 parameter -call 100. The CNVs detected were further annotated by ANNOVAR
 and their characteristics determined. The distribution
 of all types of variants across the whole genomes were visualized by
 Circos (Krzywinski et al., 2009). The genomic data were
 submitted to the NCBI under the BioProject ID PRJNA1098449. The individual
 BioSample IDs for each genome are SAMN40907891, SAMN40907892, SAMN40907893
 and SAMN40907894.
description: # Four new genome sequences of the Pallas´s cat (Otocolobus manul): an
 insight into the patterns of within-species variability
 [https://doi.org/10.5061/dryad.pzgmsbcvt](https://doi.org/10.5061/dryad.pzgmsbcvt) This dataset contains results of genome analyses performed on 4 individual genomes of Otocolobus manul. The files contained in this dataset are as provided by the sequencing facility (Novogene). This dataset serves as a full primary data repository for a peer-reviewed manuscript with the same name. ## Description of the data and file structure **Report** * *X204SC23111270-Z01-F003_Otocolobus_manul_Report*: *contains graphical overview of all collected statistical data in html format. * *src*: structured elements used in the html report above (images, fonts, methods, etc.) **Results** contains subfolders of analysis results as presented in the graphical report above. Each subfolder contains readme file with further details. * *01.OriginalData:* examples and explanation of input sequencing data - *02.QualityControl*: quality control metrics for each individual using Fastp software * *CleanData_QCsummary*: tabular representation of basic sequencing data (Raw bases (bp), Clean bases (bp), Error rate (%), Q20 (%), Q30 (%), GC content (%)) for each sequenced animal * *ErrorRate*: graphical representation of sequencing error rate for every sequencing run for each sequenced animal both in pdf and png formats * *Quality distribution*: graphical representation of quality distribution as the sequencing runs progressed, pdf and png formats * *ReadsClassification*: graphical representation of different classes of reads (Clean reads, Adapter related, Containing N, Low quality) in each sequencing run, pdf and png formats * *03.Mapping*: reference genome and mapping statistics, tools used - BWA, SAMtools, Picard * *MapStat*: tabular and graphical representation of various coverage statistics (average sequencing depth, 1X coverage (%), 4X coverage (%), coverage of individual reference chromosomes, xls, pdf and png formats * *Reference* * genome.fa: reference manul genome used for mapping * genes.gfff3: annotation record for reference genome * fa.stat: statistics of reference genome * genome.fa.fai: index file for reference genome * *04.SNP_VarDetect*: SNP variation files (GATK) and SNP statistics and annotation, tools used - GATK and ANNOVAR * *picture_in_reports*: contains graphical representations of variation statistics used in the report, pdf and png formats * *raw_variants.snp.avinput.exonic_variant_function*: bgzip-compressed annotation results produced by ANNOVAR. The first, second and third column annotate variant line number in input file, the variant effects on coding sequences and the gene/transcript being affected, yet the other columns are reproduced from input file * *raw_variants.snp.avinput.variant_function*: bgzip-compressed annotation results produced by ANNOVAR. The second and third column annotate variant effects on gene structure and the genes that are affected, yet the other columns are reproduced from vcf file * *raw_variants.snp.vcf*: variants in bgzip-compressed VCF format * *SNP.frequency.xls*: frequency in which substitutions between different base pairs occurs * *SNP_Annotation_Statistics.xls*: statistics of SNP detection and annotation - 05.InDel_VarDetect: InDel variation files and InDel statistics and annotation, tools used - GATK and ANNOVAR * *picture_in_reports*: contains graphical representations of variation statistics used in the report, pdf and png formats * *raw_variants.indel.avinput.exonic_variant_function*: bgzip-compressed annotation results produced by ANNOVAR. The first, second and third column annotate variant line number in input file, the variant effects on coding sequences and the gene/transcript being affected, yet the other columns are reproduced from input file * *raw_variants.indel.avinput.variant_function*: bgzip-compressed annotation results produced by ANNOVAR. The second and third column annotate variant effects on gene structure and the genes that are affected, yet the other columns are reproduced from vcf file * *raw_variants.indel.vcf*: indel variants in bgzip-compressed VCF format * *InDel.CDSpercentage.xls*: Length Distribution of CDS-located InDels * *InDel.GENOMEpercentage.xls*: Length Distribution of GENOME-located InDels * *InDel_Annotation_Statistics.xls*: Statistics of indel distribution based on the indel classification * *06.SV_VarDetect*: Structural variants variation files and SV statistics and annotation, tools used - Breakdancer and ANNOVAR * *Manul_x*: each individual has a folder containing - * *Manul_x.filted.ctx.gz*: bgzip-compressed results file of CTX variation * *Manul_x.sv.avinput.variant_function.gz*: bgzip-compressed result of SV annotations * *picture_in_reports*: contains graphical representations of SV length distribution, pdf and png formats * *SV.len.rate.xls*: Length Distribution of SVs * *SV_Annotation_Statistics.xls*: contains Statistics of SV detection and annotation - *07.CNV_VarDetect*: Copy number variants variation files and CNV statistics and annotation, tools used - CNVnator and ANNOVAR * *Manul_x*: each individual has a folder containing - * *Manul_x.cnv.vcf.gz*: The CNV variations detection result in bgzip-compressed format * *Manul_x.cnv.avinput.variant_function.gz*: CNV annotation results in bgzip-compressed format * *picture_in_reports*: contains graphical representation of CNV Annotation Statistics, pdf and png formats * *CNV_Annotation_Statistics.xls*: Statistics of CNV detection and annotation * 08.VarDetect_Visualisation: Visualisation of different detected variant types across studied genomes, each individual has separate files indicated by the ID, tools used - Circos * Manul_x.Circos: graphical representation of whole genome variations distribution, pdf and png formats * Manul_x.snpDensity: graphical representation of SNP density of whole genome, pdf and png formats * Manul_x.indeDensity: graphical representation of indel density of whole genome, pdf and png formats ## Sharing/Access information Raw genomic data were uploaded to the SRA and are available under BioProject number PRJNA1098449.