Reference Genome Research Articles

Genome Sequence Resource of Neopestalotiopsis cubana, a Pathogen to Guava

The pestalotiopsis-like taxa, inclusive of Pseudopestalotiopsis, Pestalotiopsis, and Neopestalotiopsis, constitute a well-recognized fungal family. Recently, species within the Neopestalotiopsis genus have been identified as plant pathogens affecting crops and fruits. However, there is currently a paucity of reference genome data for Neopestalotiopsis cubana in germplasm resource evaluation research. In this study, we present a chromosome-level reference genome of N. cubana, constructed using DNBSEQ platform and PacBio RS II platform. The assembled genome spans a total length of 50,399,148 bp, with a GC content of 50.17%. Furthermore, the reference genome contains annotations for a total of 15,709 protein-coding genes and 657 non-coding RNAs (ncRNAs). The availability of this genome sequence data enhances our understanding of the lifestyle strategy and pathogenesis of Neopestalotiopsis species. Our study significantly contributes to the species richness of Neopestalotiopsis spp. and provides a foundation for future research.

A 6.49-Mb inversion associated with the purple embryo spot trait in potato

Abstract The embryo spot trait leads to a deep purple or reddish coloration at the base of the cotyledons of the embryo, visible on both sides of flat potato (Solanum tuberosum) seeds. This trait has long been used by potato researchers and breeders as a morphological marker during dihaploid induction. The formation of embryo spots reflects the accumulation of anthocyanins, but the genetic basis of this trait remains unclear. In this study, we mapped the embryo spot trait to a 6.78-Mb region at the end of chromosome 10 using an F2 population derived from a cross between spotted and spotless plants. The recombination rate in the candidate region is severely suppressed, posing challenges for the map-based cloning of the underlying gene and suggesting large-scale rearrangements in this region. A de novo genome assembly of the spotted individual and a comparative genomic analysis to the reference genome of spotless potato revealed a 6.49-Mb inversion present in the spotted plant genome. The left breakpoint of this inversion occurred in the promoter region of an R2R3 MYB transcription factor gene that is highly expressed in the cotyledon base of spotted embryos but is not expressed in that of spotless embryos. This study elucidated the genetic basis for embryo spot formation in potato and provides a foundation for future cloning of the causative gene.

Incomplete human reference genomes can drive false sex biases and expose patient-identifying information in metagenomic data

As next-generation sequencing technologies produce deeper genome coverages at lower costs, there is a critical need for reliable computational host DNA removal in metagenomic data. We find that insufficient host filtration using prior human genome references can introduce false sex biases and inadvertently permit flow-through of host-specific DNA during bioinformatic analyses, which could be exploited for individual identification. To address these issues, we introduce and benchmark three host filtration methods of varying throughput, with concomitant applications across low biomass samples such as skin and high microbial biomass datasets including fecal samples. We find that these methods are important for obtaining accurate results in low biomass samples (e.g., tissue, skin). Overall, we demonstrate that rigorous host filtration is a key component of privacy-minded analyses of patient microbiomes and provide computationally efficient pipelines for accomplishing this task on large-scale datasets.

Telomere-to-telomere Phragmites australis reference genome assembly with a B chromosome provides insights into its evolution and polysaccharide biosynthesis

Phragmites australis is a globally distributed grass species (Poaceae) recognized for its vast biomass and exceptional environmental adaptability, making it an ideal model for studying wetland ecosystems and plant stress resilience. However, genomic resources for this species have been limited. In this study, we assembled a chromosome-level reference genome of P. australis containing one B chromosome. An explosion of LTR-RTs, centered on the Copia family, occurred during the late Pleistocene, driving the expansion of P. australis genome size and subgenomic differentiation. Comparative genomic analysis showed that P. australis underwent two whole gene duplication events, was segregated from Cleistogenes songorica at 34.6 Mya, and that 41.26% of the gene families underwent expansion. Based on multi-tissue transcriptomic data, we identified structural genes in the biosynthetic pathway of pharmacologically active Phragmitis rhizoma polysaccharides with essential roles in rhizome development. This study deepens our understanding of Arundinoideae evolution, genome dynamics, and the genetic basis of key traits, providing essential data and a genetic foundation for wetland restoration, bioenergy development, and plant stress.

Population Genomics Reveals Local Adaptation Related to Temperature Variation in Two Stream Frog Species: Implications for Vulnerability to Climate Warming.

Identifying populations at highest risk from climate change is a critical component of conservation efforts. However, vulnerability assessments are usually applied at the species level, even though intraspecific variation in exposure, sensitivity and adaptive capacity play a crucial role in determining vulnerability. Genomic data can inform intraspecific vulnerability by identifying signatures of local adaptation that reflect population-level variation in sensitivity and adaptive capacity. Here, we address the question of local adaptation to temperature and the genetic basis of thermal tolerance in two stream frogs (Ascaphus truei and A. montanus). Building on previous physiological and temperature data, we used whole-genome resequencing of tadpoles from four sites spanning temperature gradients in each species to test for signatures of local adaptation. To support these analyses, we developed the first annotated reference genome for A. truei. We then expanded the geographic scope of our analysis using targeted capture at an additional 11 sites per species. We found evidence of local adaptation to temperature based on physiological and genomic data in A. montanus and genomic data in A. truei, suggesting similar levels of sensitivity (i.e., susceptibility) among populations regardless of stream temperature. However, invariant thermal tolerances across temperatures in A. truei suggest that populations occupying warmer streams may be most sensitive. We identified high levels of evolutionary potential in both species based on genomic and physiological data. While further integration of these data is needed to comprehensively evaluate spatial variation in vulnerability, this work illustrates the value of genomics in identifying spatial patterns of climate change vulnerability.

Gene pseudogenization in fertility-associated genes in cheetah (Acinonyx jubatus), a species with long-term low effective population size.

We are witnessing an ongoing global biodiversity crisis, and an increasing number of mammalian populations are at risk of decline. Species that have survived severe historic bottlenecks, such as the cheetah (Acinonyx jubatus) exhibit symptoms of inbreeding depression including reproductive and developmental defects. Although it has long been suggested that such defects stem from an accumulation of weakly deleterious mutations, the implications of such mutations leading to pseudogenization has not been assessed. Here, we use comparative analysis of eight felid genomes to better understand the impacts of deleterious mutations in the cheetah. We find novel pseudogenization events specific to the cheetah. Through careful curation, we identify 65 genes with previously unreported premature termination codons that likely affect gene function. With the addition of population data (n=6) we find 22 of these premature termination codons in at least one resequenced individual, four of which (DEFB116, ARL13A, CFAP119 and NT5DC4) are also found in a more recent reference genome. Mutations within three of these genes are linked with sterility, including azoospermia, which is common in cheetahs. Our results highlight the power of comparative genomic approaches for the discovery of novel causative variants in declining species.

A Chromosome level assembly of pomegranate (Punica granatum L.) variety grown in arid environment

The pomegranate (Punica granatum L.) is an ancient fruit-bearing tree known for its nutritional and antioxidant properties. They originated from the Middle East in regions having large farms including mountainous regions of Al-Baha in Saudi Arabia. Pomegranates can tolerate arid climates and are considered among the fruits that will play a major role in food security. However, the genomics resources of pomegranate growing in arid regions are scarce. Here, we present a high-quality chromosome-level reference genome using PacBio HiFi long reads. The final assembly was 384.65 Mb with N50 contig size of 43.11 Mb, with 353.42 Mb being anchored on the eight pseudo chromosomes. Annotation revealed that 48.79% of the genome comprises repetitive elements and contains 21,620 protein-coding genes. The new reference genome will contribute to identifying stress resistance traits in pomegranates thriving in arid environments as well as new dietary antioxidants and antimicrobial peptides with pharmaceutical and therapeutic applications.

Detection of Hymenoscyphus fraxineus in Leaf Rachises from European Ash (Fraxinus excelsior) in Germany

The ash dieback disease caused by Hymenoscyphus fraxineus is widespread in Germany and is the subject of intensive research efforts. The fungus identification is based on the genomic internal transcribed spacer (ITS) region, which can also be the site of intragenomic variability. In Germany, despite intense research efforts, only a few numbers of H. fraxineus sequence data are recorded. Therefore, this study aims to characterize H. fraxineus isolates obtained from diseased ash leaves in Brandenburg (Germany). Fungal isolates from infected ash leaf tissue were analyzed molecularly using species-specific primers and based on sequencing the ITS region of rDNA. The analysis of the two identified sequences revealed two base substitutionscompared to the reference sequences. Thus, they show an identity of 98.8%–100% to the reference sequences and support the assumption that H. fraxineus has multiple copies of the ITS region. The phylogenetic grouping with reference sequences did not show a distinct cluster for the European, particularly the German, sequences. This indicates that the evolvement of the genetic variability of the ITS region is still an ongoing process.

Guidelines for Gene and Genome Assembly Nomenclature.

The rapid increase in the number of reference-quality genome assemblies presents significant new opportunities for genomic research. However, the absence of standardized naming conventions for genome assemblies and annotations across datasets creates substantial challenges. Inconsistent naming hinders the identification of correct assemblies, complicates the integration of bioinformatics pipelines, and makes it difficult to link assemblies across multiple resources. To address this, we developed a specification for standardizing the naming of reference genome assemblies, to improve consistency across datasets and facilitate interoperability. This specification was created with FAIR (Findable, Accessible, Interoperable, and Reusable) practices in mind, ensuring that reference assemblies are easier to locate, access, and reuse across research communities. Additionally, it has been designed to comply with primary genomic data repositories, including members of the International Nucleotide Sequence Database Collaboration (INSDC) consortium, ensuring compatibility with widely used databases. While initially tailored to the agricultural genomics community, the specification is adaptable for use across different taxa. Widespread adoption of this standardized nomenclature would streamline assembly management, better enable cross-species analyses, and improve the reproducibility of research. It would also enhance natural language processing applications that depend on consistent reference assembly names in genomic literature, promoting greater integration and automated analysis of genomic data. This is a good time to consider more consistent genomic data nomenclature as many research communities and data resources are now finding themselves juggling multiple datasets from multiple data providers.

Chromosome-Level Genome Assembly and Annotation of the Highly Heterozygous Phallus echinovolvatus Provide New Insights into Its Genetics

Phallus echinovolvatus is a well-known edible and medicinal fungus with significant economic value. However, the available whole-genome information is lacking for this species. The chromosome-scale reference genome (Monop) and two haploid genomes (Hap1 and Hap2) of P. echinovolvatus, each assembled into 11 pseudochromosomes, were constructed using Illumina, PacBio-HiFi long-read sequencing, and Hi-C technology. The Monop had a size of 36.54 Mb, with 10,251 predicted protein-coding genes and including 433 carbohydrate-active enzyme genes, 385 cytochrome P450 enzyme genes, and 42 gene clusters related to secondary metabolite synthesis. Phylogenetic and collinearity analysis revealed a close evolutionary relationship between P. echinovolvatus and Clathrus columnatus in the core Phallales clade. Hap1 and Hap2 had sizes of 35.46 Mb and 36.11 Mb, respectively. Collinear relationships were not observed for 15.38% of the genes in the two haplotypes. Hap1 had 256 unique genes, and Hap2 had 370 unique genes. Our analysis of the P. echinovolvatus genome provides insights into the genetic basis of the mechanisms underlying the metabolic effects of bioactive substances and will aid ongoing breeding efforts and studies of genetic mechanisms.

Genetic profile of the whole genome sequence of African swine fever virus from the first outbreak in Malaysian Borneo.

African swine fever (ASF), a severe and highly contagious haemorrhagic viral disease of pigs, is becoming a major threat not only in Malaysia but around the world. The first confirmed case of ASF in Malaysia was reported in February 2021. Despite the emergence of ASF in Malaysia, genetic information on this causative pathogen for the local livestock is still limited. This study aimed to genetically characterize the African swine fever virus (ASFV) responsible for the 2021 outbreak in Malaysia. The genome of the ASFV isolated during the first outbreak in Malaysia was analysed as ASFV/Sabah/Malaysia/1160/21 which has 190,594 base pairs, with a nitrogenous bases (GC) content of 40.33% and 195 predicted Open Reading Frames (ORF). The complete genome sequence was compared with other annotated ASFV genomes retrieved from database of National Center for Biotechnology Information (NCBI) to obtain information based on target gene B646L, E183L, intergenic region (IGR) between I73R and I329L (IGRI73R-I329L), EP402R and B602L. The ASFV/Sabah/Malaysia/1160/21 genome had a high similarity percentage to the reference genome, Georgia 2007 and all Southeast Asian strains. Phylogenetic analysis revealed that the ASFV strain belonged to genotype II, serogroup 8, CVR1 and showed high characteristics of IGR variant II based on IGRI73R-I329L. This study expands our understanding of genetic diversity and provides significant insights into the genomic characteristics and variation of ASFV strains that are circulating in Malaysia.

From Lesions to Lessons: Two Decades of Filamentous Plant Pathogen Genomics.

Filamentous plant pathogens pose a severe threat to food security. Current estimates suggest up to 23% yield losses to pre- and post-harvest diseases and these losses are projected to increase due to climate change (Singh et al. 2023; Chaloner et al. 2021; Stukenbrock and Gurr 2023; Fisher et al. 2012; Steinberg and Gurr 2020). Understanding how filamentous plant pathogens emerge, spread and adapt to their hosts and new environmental niches is crucial to address their devastating impact on global agriculture. Over the past two decades, genomics has emerged as a central technology to advance our understanding of the population dynamics and coevolution of filamentous plant pathogens. The first genome of a filamentous plant pathogen, the blast fungus Magnaporthe oryzae, was sequenced 20 years ago. Since then, plant pathogen genomics has not only provided a deep understanding of the genomic makeup of plant pathogen populations, but has emerged as a fundamental technology for gene discovery, disease diagnostics, and has accelerated molecular biology. In merely two decades, we have witnessed several revolutions in the field, including the first reference genomes of many filamentous plant pathogens, population scale genomics of thousands of individuals, long-read and chromosome conformation sequencing for highly continuous assemblies and recent intersections of genomics and structural biology. Here, we commemorate the 20th anniversary by presenting important technological advances (Fig. 1) and lessons we learned from the past two decades of filamentous plant pathogen genomics, with a focus on cereal diseases.

Comparison of whole genome sequencing performance from fish swabs and fin clips

ObjectiveFin clipping is the standard DNA sampling technique for whole genome sequencing (WGS) of small fish. The collection of fin clips requires anaesthesia or even euthanisation of the individual. Swabbing may be a less invasive, non-lethal alternative to fin-clipping. Whether skin and gill swabs are comparable to fin clips in terms of DNA extraction quality and sequence read mapping performance from WGS was tested here on Eurasian minnows (Phoxinus phoxinus).ResultsOf 49 fin clips, all met the DNA concentration threshold of 20 ng/μl, whereas 43 of 88 swabs met this requirement. Preserving swabs in ATL buffer and treatment with Proteinase K during DNA extraction consistently raised skin swab DNA concentrations above the cut-off. All samples passed the A260/A280 absorbance ratio cut-off of 1.3. Ultimately, 93.88% of the fin clips, 30.61% of the skin, and 7.69% of the gill swabs were suitable for sequencing. Mapping performances of all three tissues were comparable in reads passing quality filtering, percentage of reads mapping to the P. phoxinus reference genome, and coverage. Overall, skin swabs treated with Proteinase K during extraction, can match fin clips in WGS performance and represent a viable non-invasive DNA sampling alternative.

MeStanG—Resource for High-Throughput Sequencing Standard Data Sets Generation for Bioinformatic Methods Evaluation and Validation

Metagenomics analysis has enabled the measurement of the microbiome diversity in environmental samples without prior targeted enrichment. Functional and phylogenetic studies based on microbial diversity retrieved using HTS platforms have advanced from detecting known organisms and discovering unknown species to applications in disease diagnostics. Robust validation processes are essential for test reliability, requiring standard samples and databases deriving from real samples and in silico generated artificial controls. We propose a MeStanG as a resource for generating HTS Nanopore data sets to evaluate present and emerging bioinformatics pipelines. MeStanG allows samples to be designed with user-defined organism abundances expressed as number of reads, reference sequences, and predetermined or custom errors by sequencing profiles. The simulator pipeline was evaluated by analyzing its output mock metagenomic samples containing known read abundances using read mapping, genome assembly, and taxonomic classification on three scenarios: a bacterial community composed of nine different organisms, samples resembling pathogen-infected wheat plants, and a viral pathogen serial dilution sampling. The evaluation was able to report consistently the same organisms, and their read abundances as provided in the mock metagenomic sample design. Based on this performance and its novel capacity of generating exact number of reads, MeStanG can be used by scientists to develop mock metagenomic samples (artificial HTS data sets) to assess the diagnostic performance metrics of bioinformatic pipelines, allowing the user to choose predetermined or customized models for research and training.

Molecular Markers for the Phylogenetic Reconstruction of Trypanosoma cruzi: A Quantitative Review

Trypanosoma cruzi is the parasite responsible for Chagas disease, which has a significant amount of genetic diversification among the species complex. Many efforts are routinely made to characterize the genetic lineages of T. cruzi circulating in a particular geographic area. However, the genetic loci used to typify the genetic lineages of T. cruzi have not been consistent between studies. We report a quantitative analysis of the phylogenetic power that is acquired from the commonly used genetic loci that are employed for the typification of T. cruzi into its current taxonomic nomenclature. Based on three quantitative criteria (the number of phylogenetic informative characters, number of available reference sequences in public repositories, and accessibility to DNA sequences for their use as outgroup sequences), we examine and discuss the most appropriate genetic loci for the genetic typification of T. cruzi. Although the mini-exon gene is by far the locus that has been most widely used, it is not the most appropriate marker for the typification of T. cruzi based on the construction of a resolved phylogenetic tree. Overall, the mitochondrial COII-NDI locus stands out as the best molecular marker for this purpose, followed by the Cytochrome b and the Lathosterol oxidase genes.

Interactive visualization and interpretation of pangenome graphs by linear-reference-based coordinate projection and annotation integration.

With the increasing availability of high-quality genome assemblies, pangenome graphs emerged as a new paradigm in the genomics field for identifying, encoding, and presenting genomic variation at both population and species levels. However, it remains challenging to truly dissect and interpret pangenome graphs via biologically informative visualization. To facilitate better exploration and understanding of pangenome graphs towards novel biological insights, here we present a web-based interactive Visualization and interpretation framework for linear-Reference-projected Pangenome Graphs (VRPG). VRPG provides efficient and intuitive supports for exploring and annotating pangenome graphs along a linear-genome-based coordinate system (e.g., that of a primary linear reference genome). Moreover, VRPG offers many unique features such as in-graph path highlighting for graph-constituent input assemblies, copy number characterization for graph-embedding nodes, graph-based mapping for query sequences, all of which are highly valuable for researchers working with pangenome graphs. Additionally, VRPG enables side-by-side visualization between the graph-based pangenome representation and the conventional primary-linear-reference-genome-based feature annotations, therefore seamlessly bridging the graph and linear genomic contexts. To further demonstrate its functionality and scalability, we applied VRPG to the cutting-edge yeast and human reference pangenome graphs derived from hundreds of high-quality genome assemblies via a dedicated web portal and examined their local genome diversity in the graph contexts.

Comparative genomic analysis of Fusarium oxysporum f. sp. lycopersici reveals telomeric duplications of a lineage-specific region carrying SIX8 and PSL1 and genome-wide expansion of Foxy transposable elements.

Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, is a soil-borne, vascular-colonizing fungal pathogen that severely impacts tomato production in most growing regions worldwide. Despite the availability of over thirty Fol genome sequences in public databases, only one chromosome-scale assembly exists, comprising the low sequence-coverage Fol4287 reference genome generated using Sanger sequencing. Thus, genome structural variation and comparative genomics analyses of Fol remain largely unexplored. Here, using third generation Nanopore long-read sequencing in combination with high-throughput chromosome conformation capture (Hi-C) data, we have independently constructed a high-quality assembly and annotation of the Fol007 race 2 genome that consists of 15 pseudochromosomes with 25 telomeres, including 10 telomere-to-telomere chromosomes. The Fol007 genome is predicted to contain 29,148 protein-encoding genes, including all known SIX genes except for SIX4, which is absent in Fol race 2. Compared to the genome of Fusarium verticillioides, the Fol007 genome contains four complete lineage-specific (LS) chromosomes, including chromosome 5 (Chr5), chromosome 13 (Chr13), and two small chromosomes, Chr14 and Chr15. Comparative genomic analysis between the newly assembled Fol007 genome and Fol4287_2010 deposited in the GenBank database reveals that the completely assembled Chr13 of Fol007 carrying all known SIX genes (except SIX4 and SIX8) and three novel candidate effector genes is relatively stable and corresponds to pathogenicity chromosome Chr14 in Fol4287_2010. It also reveals a telomeric duplication of an LS region carrying SIX8 and PSL1 on core chromosomes Chr6, Chr7 and Chr11, and LS chromosome Chr15, as well as the absence of segmental duplications on LS chromosomes of the Fol007 genome. Furthermore, compared to the genomes of Fol race 1 and non-pathogenic Fo strains, an active and specific Foxy transposable element, responsible for the inactivation of a second copy of SIX13, was identified and found to have expanded in the genomes of Fol race 2 and 3 strains. This element may contribute to Fusarium oxysporum genome evolution and has potential as a genetic marker for studying phylogenetic relationships among formae speciales of Fusarium oxysporum. These findings provide a basis for further genetic and genomic understanding of Fol evolution and virulence mechanisms employed by Fol and contribute another reference genome for Fusarium study more broadly.

Prediction of transcript isoforms and identification of tissue-specific genes in cucumber

BackgroundIdentification of global transcriptional events is crucial for genome annotation, as accurate annotation enhances the efficiency and comparability of genomic information across species. However, the annotation of transcripts in the cucumber genome remains to be improved, and many transcriptional events have not been well studied.ResultsWe collected 1,904 high-quality public cucumber transcriptome samples from the National Center for Biotechnology Information (NCBI) to identify and annotate transcript isoforms in the cucumber genome. Over 44.26 billion Q30 clean reads were mapped to the cucumber genome with an average mapping rate of 92.75%. Transcriptome assembly identified 151,453 transcripts spanning 20,442 loci. Among these, 12.7% of transcripts exactly matched annotated genes in the cucumber reference genome. More than 80% of the transcripts were classified as novel isoforms. Approximately 96.6% of these isoforms originated from known gene loci, while around 3.3% were derived from novel gene loci. Coding potential prediction identified 4,543 long non-coding RNAs (lncRNAs) across 3,376 loci. Building on these results, we identified tissue-specific transcripts in 10 tissues. Among that, 1,655 annotated genes and 4,214 predicted transcripts were considered as tissue-specific. The root exhibited the highest number of tissue-specific transcripts, followed by shoot apex. Subsequent selective pressure analysis revealed that tissue-specific regions experienced stronger directional selection compared to non-specific regions.ConclusionsBy analyzing thousands of published transcriptome data, we identified abundant transcriptional events and tissue-specific transcripts in cucumbers. This study presented here adds the great value to the public data and offers insights for further exploration of a more comprehensive tissue regulatory network in cucumber.

Nuclear and mitochondrial genome assemblies for the endangered wood-decaying fungus Somion occarium.

Somion occarium is a wood-decaying bracket fungus belonging to an order known to be rich in useful chemical compounds. Despite its widespread distribution, S. occarium has been assessed as endangered on at least one national Red List, presumably due to loss of old-growth forest habitat. Here, we present a near-complete, annotated nuclear genome assembly for S. occarium consisting of 31 Mbp arranged in 11 pseudochromosomes - 9 of which are telomere-to-telomere - as well as a complete mitochondrial genome assembly of 112.9 Kbp. We additionally performed phylogenomic analysis and annotated carbohydrate-active enzymes (CAZymes) to compare gene and CAZyme content across closely related species. This genome was sequenced as the representative for Kingdom Fungi in the European Reference Genome Atlas (ERGA) Pilot Project.

Ribotyping Staphylococcus epidermidis Using Probabilistic Sequence Analysis and Levenshtein Distance Algorithm

Staphylococcus epidermidis (S. epidermidis) live in different human locations and natural environments. For ribotyping S. epidermidis sub-species, 2507 PCR-amplified reads of 16S rRNA genes of S. epidermidis in a public dataset were used for probabilistic sequence analysis. A sequence probability logo (sequence pLogo) as a reference sequence of 16S rRNA genes of S. epidermidis was constructed. Through implementation of Levenshtein Distance algorithm, two 20-base pairs (bp) motifs, commonly present in 2507 PCR-amplified reads, were identified. The top 38 S. epidermidis isolates, which carried 16S rRNA nucleotide domains that were made of different sequences but have high similarity scores to two 20-bp motifs, were found from 11 human, 8 animal, 9 plant and 10 environmental samples, indicating that these two 20-bp motifs were broadly present in diverse S. epidermidis isolates. Thirty-one PCR-amplified reads of 16S rRNA genes, which were currently not in the dataset, were utilized to verify the feasibility of using two 20-bp motifs for ribotyping S. epidermidis sub-species. S. epidermidis S1, S3, but not S2, isolates on the human scalp carried a 20-bp sequence domain with high similarities to a 20-bp motif in the sequence pLogo. The phylogenetic tree showed that S. epidermidis S1, S2 and S3 were not from a single common ancestor. Two newly identified 20-bp motifs here, thus, provided reference nucleotide residues for ribotyping S. epidermidis.