全基因组测序及其在遗传性疾病研究及诊断中的应用

最近,随着测序成本的不断降低,数据分析策略的不断提升,全基因组测序（whole-genome sequencing,WGS）已经在癌症、孟德尔遗传病、复杂疾病的致病基因检测中得到了一定运用,并逐步走向了临床诊断。全基因组测序不但可以检测编码区和非编码区的点突变（SNVs）和插入缺失（InDels）,还可以在全基因组范围内检测拷贝数变异（copy number variation,CNV）以及结构变异（structure variation,SV）。本文详细地介绍了全基因组测序的标准生物信息分析流程与方法,及其在疾病研究、临床诊断中的应用,并对全基因组测序在医学遗传学中的应用与研究进展,以及数据分析方面面临的挑战进行了概述。     

U87MG Decoded: The Genomic Sequence of a Cytogenetically Aberrant Human Cancer Cell Line

U87MG is a commonly studied grade IV glioma cell line that has been analyzed in at least 1,700 publications over four decades. In order to comprehensively characterize the genome of this cell line and to serve as a model of broad cancer genome sequencing, we have generated greater than 30× genomic sequence coverage using a novel 50-base mate paired strategy with a 1.4kb mean insert library. A total of 1,014,984,286 mate-end and 120,691,623 single-end two-base encoded reads were generated from five slides. All data were aligned using a custom designed tool called BFAST, allowing optimal color space read alignment and accurate identification of DNA variants. The aligned sequence reads and mate-pair information identified 35 interchromosomal translocation events, 1,315 structural variations (>100 bp), 191,743 small (<21 bp) insertions and deletions (indels), and 2,384,470 single nucleotide variations (SNVs). Among these observations, the known homozygous mutation in PTEN was robustly identified, and genes involved in cell adhesion were overrepresented in the mutated gene list. Data were compared to 219,187 heterozygous single nucleotide polymorphisms assayed by Illumina 1M Duo genotyping array to assess accuracy: 93.83% of all SNPs were reliably detected at filtering thresholds that yield greater than 99.99% sequence accuracy. Protein coding sequences were disrupted predominantly in this cancer cell line due to small indels, large deletions, and translocations. In total, 512 genes were homozygously mutated, including 154 by SNVs, 178 by small indels, 145 by large microdeletions, and 35 by interchromosomal translocations to reveal a highly mutated cell line genome. Of the small homozygously mutated variants, 8 SNVs and 99 indels were novel events not present in dbSNP. These data demonstrate that routine generation of broad cancer genome sequence is possible outside of genome centers. The sequence analysis of U87MG provides an unparalleled level of mutational resolution compared to any cell line to date.     

Development of Genetic Markers in Eucalyptus Species by Target Enrichment and Exome Sequencing

The advent of next-generation sequencing has facilitated large-scale discovery, validation and assessment of genetic markers for high density genotyping. The present study was undertaken to identify markers in genes supposedly related to wood property traits in three Eucalyptus species. Ninety four genes involved in xylogenesis were selected for hybridization probe based nuclear genomic DNA target enrichment and exome sequencing. Genomic DNA was isolated from the leaf tissues and used for on-array probe hybridization followed by Illumina sequencing. The raw sequence reads were trimmed and high-quality reads were mapped to the E. grandis reference sequence and the presence of single nucleotide variants (SNVs) and insertions/ deletions (InDels) were identified across the three species. The average read coverage was 216X and a total of 2294 SNVs and 479 InDels were discovered in E. camaldulensis, 2383 SNVs and 518 InDels in E. tereticornis, and 1228 SNVs and 409 InDels in E. grandis. Additionally, SNV calling and InDel detection were conducted in pair-wise comparisons of E. tereticornis vs. E. grandis, E. camaldulensis vs. E. tereticornis and E. camaldulensis vs. E. grandis. This study presents an efficient and high throughput method on development of genetic markers for family– based QTL and association analysis in Eucalyptus.     

Identification and characterization of nucleotide variations in the genome of Ziziphus jujuba (Rhamnaceae) by next generation sequencing

In this study, single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) in the genome of Ziziphus jujuba were identified using sequences generated by the Roche 454 GS-FLX sequencer. A total of, 573,141 reads were produced with an average read length of 360 bp. After quality control, 258,754 of the filtered reads were assembled into 23,864 contigs, and 293,458 remained as singletons. Using the contig assemblies as a reference, 17,160 SNPs and 478 InDels were identified. Among the SNPs, transitions occurred three times more frequently than transversions. In transitions, the number of C/T and G/A transitions was similar. Among the transversions, A/T was the most abundant, and C/G was much rarer than any of the other types of transversions, accounting for only about half the numbers of A/C, A/T and G/T transversions. For the InDels, mononucleotide changes amounted to 64.4 % of the total number of InDels. In general, the frequency of detected InDels decreased as the length of the InDels increased. This study provides valuable marker resources for future genetic studies of Ziziphus spp.     

Large structural variations in the haplotype-resolved African cassava genome

Cassava (Manihot esculenta Crantz, 2n = 36) is a global food security crop. It has a highly heterozygous genome, high genetic load, and genotype-dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi-C to resolve each haplotype of the genome of a farmer-preferred cassava line, TME7 (Oko-iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON-Phase and Hi-C read data. The ultralong-range information from Hi-C sequencing was also used for scaffolding. Comparison of the two phases revealed >5000 large haplotype-specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele-specific expression was further explored. RNA-sequencing data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy-to-use web-based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype-specific structural variation, gene hemizygosity, and allele-specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.     

A large‐scale gene discovery for the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

The red palm weevil (RPW; Rhynchophorus ferrugineus) is a devastating pest of palms, prevalent in the Middle East as well as many other regions of the world. Here, we report a large‐scale de novo complementary DNA (cDNA) sequencing effort that acquired ～5 million reads and assembled them into 26 765 contigs from 12 libraries made from samples of different RPW developmental stages based on the Roche/454 GS FLX platform. We annotated these contigs based on the publically available known insect genes and the Tribolium castaneum genome assembly. We find that over 80% of coding sequences (CDS) from the RPW contigs have high‐identity homologs to known proteins with complete CDS. Gene expression analysis shows that the pupa and larval stages have the highest and lowest expression levels, respectively. In addition, we also identified more than 60 000 single nucleotide polymorphisms and 1 200 simple sequence repeat markers. This study provides the first large‐scale cDNA dataset for RPW, a much‐needed resource for future molecular studies.     

Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly

Here we use whole-genome de novo assembly of second-generation sequencing reads to map structural variation (SV) in an Asian genome and an African genome. Our approach identifies small- and intermediate-size homozygous variants (1-50 kb) including insertions, deletions, inversions and their precise breakpoints, and in contrast to other methods, can resolve complex rearrangements. In total, we identified 277,243 SVs ranging in length from 1-23 kb. Validation using computational and experimental methods suggests that we achieve overall <6% false-positive rate and <10% false-negative rate in genomic regions that can be assembled, which outperforms other methods. Analysis of the SVs in the genomes of 106 individuals sequenced as part of the 1000 Genomes Project suggests that SVs account for a greater fraction of the diversity between individuals than do single-nucleotide polymorphisms (SNPs). These findings demonstrate that whole-genome de novo assembly is a feasible approach to deriving more comprehensive maps of genetic variation.     

Decoding the oak genome: public release of sequence data,assembly, annotation and publication strategies

The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole‐genome shotgun (WGS) approach, without the use of costly and time‐consuming methods, such as fosmid or BAC clone‐based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS‐FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired‐end and mate‐pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired‐end reads and contaminants detected, resulting in a total of 17 910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.     

Relationship between homoeologous regulatory and structural genes in allopolyploid genome – A case study in bread wheat

Background

Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed.

Results

The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome.

Conclusion

Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.     

Finding Nemo’s Genes: A chromosome‐scale reference assembly of the genome of the orange clownfish Amphiprion percula

The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome‐scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single‐molecule real‐time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi‐C‐based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein‐coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes.     

GCAT|Panel,a comprehensive structural variant haplotype map of the Iberian population from high-coverage whole-genome sequencing

The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization, and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression Models (LRMs), we present a catalogue of 35 431 441 variants, including 89 178 SVs (≥50 bp), 30 325 064 SNVs and 5 017 199 indels, across 785 Illumina high coverage (30x) whole-genomes from the Iberian GCAT Cohort, containing a median of 3.52M SNVs, 606 336 indels and 6393 SVs per individual. The haplotype panel is able to impute up to 14 360 728 SNVs/indels and 23 179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with Mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies.     

A comprehensive biomedical variant catalogue based on whole genome sequences of 582 dogs and eight wolves

The domestic dog serves as an excellent model to investigate the genetic basis of disease. More than 400 heritable traits analogous to human diseases have been described in dogs. To further canine medical genetics research, we established the Dog Biomedical Variant Database Consortium (DBVDC) and present a comprehensive list of functionally annotated genome variants that were identified with whole genome sequencing of 582 dogs from 126 breeds and eight wolves. The genomes used in the study have a minimum coverage of 10× and an average coverage of ~24×. In total, we identified 23 133 692 single‐nucleotide variants (SNVs) and 10 048 038 short indels, including 93% undescribed variants. On average, each individual dog genome carried ～4.1 million single‐nucleotide and ~1.4 million short‐indel variants with respect to the reference genome assembly. About 2% of the variants were located in coding regions of annotated genes and loci. Variant effect classification showed 247 141 SNVs and 99 562 short indels having moderate or high impact on 11 267 protein‐coding genes. On average, each genome contained heterozygous loss‐of‐function variants in 30 potentially embryonic lethal genes and 97 genes associated with developmental disorders. More than 50 inherited disorders and traits have been unravelled using the DBVDC variant catalogue, enabling genetic testing for breeding and diagnostics. This resource of annotated variants and their corresponding genotype frequencies constitutes a highly useful tool for the identification of potential variants causative for rare inherited disorders in dogs.     

Discovering single nucleotide variants and indels from bulk and single-cell ATAC-seq

Genetic variants and de novo mutations in regulatory regions of the genome are typically discovered by whole-genome sequencing (WGS), however WGS is expensive and most WGS reads come from non-regulatory regions. The Assay for Transposase-Accessible Chromatin (ATAC-seq) generates reads from regulatory sequences and could potentially be used as a low-cost ‘capture’ method for regulatory variant discovery, but its use for this purpose has not been systematically evaluated. Here we apply seven variant callers to bulk and single-cell ATAC-seq data and evaluate their ability to identify single nucleotide variants (SNVs) and insertions/deletions (indels). In addition, we develop an ensemble classifier, VarCA, which combines features from individual variant callers to predict variants. The Genome Analysis Toolkit (GATK) is the best-performing individual caller with precision/recall on a bulk ATAC test dataset of 0.92/0.97 for SNVs and 0.87/0.82 for indels within ATAC-seq peak regions with at least 10 reads. On bulk ATAC-seq reads, VarCA achieves superior performance with precision/recall of 0.99/0.95 for SNVs and 0.93/0.80 for indels. On single-cell ATAC-seq reads, VarCA attains precision/recall of 0.98/0.94 for SNVs and 0.82/0.82 for indels. In summary, ATAC-seq reads can be used to accurately discover non-coding regulatory variants in the absence of whole-genome sequencing data and our ensemble method, VarCA, has the best overall performance.     

Genome-wide DNA polymorphisms in elite indica rice inbreds discovered by whole-genome sequencing

Advances in next-generation sequencing technologies have aided discovery of millions of genome-wide DNA polymorphisms, single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels), which are an invaluable resource for marker-assisted breeding. Whole-genome resequencing of six elite indica rice inbreds (three cytoplasmic male sterile and three restorer lines) resulted in the generation of 338?million 75-bp paired-end reads, which provided 85.4% coverage of the Nipponbare genome. A total of 2?819?086 nonredundant DNA polymorphisms including 2?495?052 SNPs, 160?478 insertions and 163?556 deletions were discovered between the inbreds and Nipponbare, providing an average of 6.8 SNPs/kb across the genome. Distribution of SNPs and InDels in the chromosome was nonrandom with SNP-rich and SNP-poor regions being evident across the genome. A contiguous 4.3-Mb region on chromosome 5 with extremely low SNP density was identified. Overall, 83?262 nonsynonymous SNPs spanning 16?379 genes and 3620 nonsynonymous InDels in 2625 genes have been discovered which provide valuable insights into the basis underlying performance of the inbreds and the hybrids between these inbred combinations. SNPs and InDels discovered from this diverse set of indica rice inbreds not only enrich SNP resources for molecular breeding but also enable the study of genome-wide variations on hybrid performance.     

Mining single nucleotide polymorphisms from EST data of silkworm, Bombyx mori, inbred strain Dazao

We made use of 81,635 expressed sequence tags (ESTs) derived from 12 different cDNA libraries of the silkworm, Bombyx mori, inbred strain Dazao (P50), to identify high-quality candidate single nucleotide polymorphisms (SNPs). By PHRAP assembling, 12,980 contigs containing 11,537 contigs assembled by more than one read were obtained, and 101 candidate SNPs and 27 single base insertions/deletions were identified from 117 contigs assembled from 1576 high-quality reads base-called with PHRED and screened on the basis of the neighborhood quality standard (NQS). Simultaneously, we also predicted 40 SNPs in coding regions (cSNPs), of which 26 were predicted to lead to amino acid non-synonymous variations and 14 synonymous substitutions. Also, the 1.66:1 ratio of transition/transversion is different from that of other insects. As the first SNP analysis of a Lepidoptera, B. mori, the single nucleotide polymorphic density is estimated to be 1.3 x 10(-3) by sequence diversity. This analysis shows that expressed sequences from multiple libraries may provide an abundant source of comparative reads to mine for cSNPs from the silkworm genome.     

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole‐genome shotgun sequencing of the nuclear genome of flax. Seven paired‐end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep‐coverage (approximately 94× raw, approximately 69× filtered) short‐sequence reads (44–100 bp), produced a set of scaffolds with N₅₀ = 694 kb, including contigs with N₅₀ = 20.1 kb. The contig assembly contained 302 Mb of non‐redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole‐genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis‐assembly of regions at the genome scale. A total of 43 384 protein‐coding genes were predicted in the whole‐genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K_s) observed within duplicate gene pairs was consistent with a recent (5–9 MYA) whole‐genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam‐A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole‐genome shotgun short‐sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.     

Detection and characterization of genome-wide mutations in M1 vegetative cells of gamma-irradiated Arabidopsis

Radiation-induced mutations have been detected by whole-genome sequencing analyses of self-pollinated generations of mutagenized plants. However, large DNA alterations and mutations in non-germline cells were likely missed. In this study, in order to detect various types of mutations in mutagenized M1 plants, anthocyanin pigmentation was used as a visible marker of mutations. Arabidopsis seeds heterozygous for the anthocyanin biosynthetic genes were irradiated with gamma-rays. Anthocyanin-less vegetative sectors resulting from a loss of heterozygosity were isolated from the gamma-irradiated M1 plants. The whole-genome sequencing analysis of the sectors detected various mutations, including structural variations (SVs) and large deletions (≥100 bp), both of which have been less characterized in the previous researches using gamma-irradiated plant genomes of M2 or later generations. Various types of rejoined sites were found in SVs, including no-insertion/deletion (indel) sites, only-deletion sites, only-insertion sites, and indel sites, but the rejoined sites with 0–5 bp indels represented most of the SVs. Examinations of the junctions of rearrangements (SVs and large deletions), medium deletions (10–99 bp), and small deletions (2–9 bp) revealed unique features (i.e., frequency of insertions and microhomology) at the rejoined sites. These results suggest that they were formed preferentially via different processes. Additionally, mutations that occurred in putative single M1 cells were identified according to the distribution of their allele frequency. The estimated mutation frequencies and spectra of the M1 cells were similar to those of previously analyzed M2 cells, with the exception of the greater proportion of rearrangements in the M1 cells. These findings suggest there are no major differences in the small mutations (<100 bp) between vegetative and germline cells. Thus, this study generated valuable information that may help clarify the nature of gamma-irradiation-induced mutations and their occurrence in cells that develop into vegetative or reproductive tissues.     

Genome-wide single nucleotide polymorphism and Insertion-Deletion discovery through next-generation sequencing of reduced representation libraries in common bean

Single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels) are valuable molecular markers for genomics and genetics studies and molecular breeding. The advent of next-generation sequencing techniques has enabled researchers to approach high-throughput and cost-effective SNP and InDel discovery on a genomic scale. In this report, 36 common bean genotypes grown in Canada were used to construct reduced representation libraries for next-generation sequencing. Using 76 million sequence reads generated by the Illumina HiSeq 2000 Sequencing System, we identified a total of 43,698 putative SNPs and 1,267 putative InDels. Of the SNPs, 43,504 were bi-allelic and 194 were tri-allelic, and the InDels comprised 574 insertions and 693 deletions. The putative bi-allelic SNPs were distributed across all 11 chromosomes with the highest number of SNPs observed in chromosome 2 (4,788), and the lowest in chromosome 10 (2,941). With the aid of the recent release of the first chromosome-scale version of Phaseolus vulgaris, 24,907 bi-allelic SNPs, 79 tri-allelic SNPs, 315 insertions, and 377 deletions were located in 8,758, 77, 273, and 364 genes, respectively. Among these 24,907 bi-allelic SNPs, 7,168 nonsynonymous bi-allelic SNPs were identified within 36 common bean genotypes that were located in 4,303 genes. A total of 113 putative SNPs were randomly chosen for validation using high-resolution melt analysis. Of the 113 candidate SNPs, 105 (92.9 %) contained the predicted SNPs.     

SARS-CoV Genome Polymorphism： A Bioinformatics Study

A dataset of 103 SARS-CoV isolates （101 human patients and 2 palm civets） was investigated on different aspects of genome polymorphism and isolate classification. The number and the distribution of single nucleotide variations （SNVs） and insertions and deletions, with respect to a “profile”, were determined and discussed （“profile” being a sequence containing the most represented letter per position）. Distribution of substitution categories per codon positions, as well as synonymous and non-synonymous substitutions in coding regions of annotated isolates, was determined, along with amino acid （a.a.） property changes. Similar analysis was performed for the spike （S） protein in all the isolates （55 of them being predicted for the first time）. The ratio Ka/Ks confirmed that the S gene was subjected to the Darwinian selection during virus transmission from animals to humans. Isolates from the dataset were classified according to genome polymorphism and genotypes. Genome polymorphism yields to two groups, one with a small number of SNVs and another with a large number of SNVs, with up to four subgroups with respect to insertions and deletions. We identified three basic nine-locus genotypes： TTTT/TTCGG, CGCC/TTCAT, and TGCC/TTCGT, with four subgenotypes. Both classifications proposed are in accordance with the new insights into possible epidemiological spread, both in space and time.