Genome Length Research Articles

Identification and Characterization of Novel SPHINX/BMMF-like DNA Sequences Isolated from Non-Bovine Foods.

Sixteen novel circular rep-encoding DNA sequences with high sequence homologies to previously described SPHINX and BMMF sequences were isolated for the first time from non-bovine foods (pork, wild boar, chicken meat, Alaska pollock, pangasius, black tiger shrimp, apple, carrot, and sprouts from alfalfa, radish, and broccoli). The phylogenetic analysis of the full-length circular genomes grouped these together with previously described representatives of SPHINX/BMMF group 1 and 2 sequences (eight in each group). The characterization of genome lengths, genes present, and conserved structures confirmed their relationship to the known SPHINX/BMMF sequences. Further analysis of iteron-like tandem repeats of SPHINX/BMMF group 1-related genomes revealed a correlation with both full-length sequence tree branches as well as Rep protein sequence tree branches and was able to differentiate subtypes of SPHINX/BMMF group 1 members. For the SPHINX/BMMF group 2 members, a distinct grouping of sequences into two clades (A and B) with subgroups could be detected. A deeper investigation of potential functional regions upstream of the rep gene of the new SPHINX/BMMF group 2 sequences revealed homologies to the dso and sso regions of known plasmid groups that replicate via the rolling circle mechanism. Phylogenetic analyses were accomplished by a Rep protein sequence analysis of different ssDNA viruses, pCRESS, and plasmids with the known replication mechanism, as this yielded deeper insights into the relationship of SPHINX/BMMF group 1 and 2 Rep proteins. A clear relation of these proteins to the Rep proteins of plasmids could be confirmed. Interestingly, for SPHINX/BMMF group 2 members, the relationship to rolling circle replication plasmids could also be verified. Furthermore, a relationship of SPHINX/BMMF group 1 Rep proteins to theta-replicating plasmid Reps is discussed.

The ricebean genome provides insight into Vigna genome evolution and facilitates genetic enhancement.

The ricebean genome provides insight into Vigna genome evolution and facilitates genetic enhancement.

Genome Sequence of a Clostridioides difficile Strain Isolated from Feces from a Patient in Southern Thailand.

Clostridioides difficile is a Gram-positive, obligate anaerobic, toxin-producing bacillus that is linked to antibiotic-associated diarrhea. Here, we report the whole-genome sequence of a C. difficile strain isolated from stool from a patient, using next-generation sequencing (MGISEG-2000). De novo assembly revealed a genome length of 4,208,266 bp. Multilocus sequence typing (MLST) results showed that the isolate belonged to sequence type 23 (ST23).

Whole-genome assembly of A02 bacteria involved in nitrogen fixation within cassava leaves.

The endophytic nitrogen (N)-fixing bacterium A02 belongs to the genus Curtobacterium (Curtobacterium sp.) and is crucial for the N metabolism of cassava ( Manihot esculenta Crantz). We isolated the A02 strain from cassava cultivar SC205 and used the 15N isotope dilution method to study the impacts of A02 on growth and accumulation of N in cassava seedlings. Furthermore, the whole genome was sequenced to determine the N-fixation mechanism of A02. Compared with low N control (T1), inoculation with the A02 strain (T2) showed the highest increase in leaf and root dry weight of cassava seedlings, and 120.3 nmol/(mL·h) was the highest nitrogenase activity recorded in leaves, which were considered the main site for colonization and N-fixation. The genome of A02 was 3,555,568 bp in size and contained a circular chromosome and a plasmid. Comparison with the genomes of other short bacilli revealed that strain A02 showed evolutionary proximity to the endophytic bacterium NS330 (Curtobacterium citreum) isolated from rice (Oryza sativa) in India. The genome of A02 contained 13 nitrogen fixation (nif) genes, including 4 nifB, 1 nifR3, 2 nifH, 1 nifU, 1 nifD, 1 nifK, 1 nifE, 1 nifN, and 1 nifC, and formed a relatively complete N fixation gene cluster 8-kb long that accounted for 0.22% of the whole genome length. The nifHDK of strain A02 (Curtobacterium sp.) is identical to the Frankia alignment. Function prediction showed high copy number of the nifB gene was related to the oxygen protection mechanism. Our findings provide exciting information about the bacterial genome in relation to N support for transcriptomic and functional studies for increasing N use efficiency in cassava.

The Complete Chloroplast Genome Sequence of Staphylea holocarpa (Staphyleaceae).

Staphylea holocarpa (Hemsley 1895) is an ornamental deciduous shrub or tree in the family Staphyleaceae. As the shortage of the wild resources, S. holocarpa is also a rare plant. The revelation of the species origin and evolution progress and the relation. Therefore, the S. holocarpa complete chloroplast genome sequence was completed and characterized by de novo assembly. The cp genome length of S. holocarpa was 160,461bp and it has a typical quadripartite structure, consisted of an 89,760bp large single-copy region and a 18,639bp small single-copy region, which were divided by two inverted repeat regions of 26,031bp. After genome annotation, it comes to 130 genes that were predicted, which includes 85, 8, and 37 encoded proteins, rRNA, and tRNA, respectively. A phylogenetic analysis has shown that the S. holocarpa cp genome is related to the Staphylea trifolia. This work will be useful for further population genomic and phylogenetic studies of S. holocarpa.

Complete Genome Assembly and Annotation of Escherichia coli Bacteriophage 55 from Rivière la Quint in Gonaïves, Haiti.

We present the structural and functional annotation of Escherichia coli bacteriophage 55, which has a genome length of 170,393 bp, with 219 predicted genes.

VirMAP for cancer: Characterization of the intratumoral virome in virally associated cancers and a resource for investigators.

2568 Background: A major barrier to understanding the role of viruses in cancer progression has been the lack of sensitivity of current tools to assemble viral genomes in an untargeted way. Here, we utilize our novel VirMAP algorithm to identify eukaryotic and prokaryotic viruses associated with cancers, using RNA sequencing data from The Cancer Genome Atlas (TCGA). Methods: VirMAP was run on 16 TCGA RNASeq datasets of primary tumor tissues, with a focus on cancers with known viral associations (including cervical, head and neck, liver, stomach, esophagus and ovarian). Previously published survival data were used for exploratory analysis. Overall survival (OS) was assessed using the Kaplan-Meier method, and p-values were calculated using log-rank test. Multivariate analysis (MVA) was done using Cox proportional hazards model to control for age, sex, stage, and histology. Results: Viral sequence recovery (among 537 billion sequencing reads from 6185 patients) was at least equal to previously published algorithms such as Kraken, but with higher taxonomic resolution; overall recovery was 0.0041% from 16 tumor types. Cancers with high viral loads such as cervical cancer demonstrated high recovery rates (0.012% of all reads mapped to viral taxa, followed by 0.004% for liver, 0.0018% head and neck, 0.0016% stomach, 0.0009% esophageal, and 0.0002% ovarian). There was a wide range of viral signature richness, with cervical cancer showing the highest viral read count predominated by 18 different HPV serotypes; in contrast, stomach cancer had lower abundance but contained over 300 unique viral species. Exploratory analysis revealed novel correlations between viral signatures and OS. In cervical cancer, presence of HPV45 predicted worse survival (median OS 837 days vs 4086, p=0.0043); MVA demonstrated hazard ratio (HR) of 5.7 [2.6-11.4, p<0.0001]. In stomach cancer, patients often had more than one viral taxon present (mean 3.7, range 0-18); presence of seven or more unique taxa predicted worse OS (median OS 403 days vs 1153, p=0.0001). MVA showed HR of 1.1 for every additional taxon (p=0.0004). Across all 16 tumor types, the detection of phage virus was associated with worse OS (1073 days vs. 2094 days for non-phage virus and 2131 days for no virus, p<0.0001). Conclusions: VirMAP performs deep characterization of the tumor-associated virome in both high and low abundance settings, with generation of partial genome reconstructions and strain level taxonomic classification including phages. Further work is ongoing to optimize viral read normalization based on genome length, as well as subset analysis with disease-specific modifiers such as histology, race, and exposures. Utilizing publicly available platforms such as TCGA, clinical and correlative published data can be leveraged to better understand the nuanced pleotropic effects of the tumor virome on cancer progression.

The First Complete Genome Resource for the Plant-Pathogenic Fungus Exobasidium rhododendri.

Exobasidium rhododendri is a parasite of Rhododendron and also infects apples. A chromosome-level genome of E. rhododendri was assembled using the PacBio and Illumina datasets. The complete genome length is 17.6 Mb, with N50 of 5.45 Mb, and its GC content is 50.08%. The assembly obtained contains four scaffolds, including three nuclear chromosomes and one mitochondrion. There are a total of 6,982 predicted protein-encoding genes containing 406 fungal secretion proteins and 249 candidate effectors in the E. rhododendri genome. This high-quality genome will help understand the pathogenic mechanism of this fungus.

Rapid construction of infectious clones for distinct Newcastle disease virus genotypes.

The reverse genetics system of the Newcastle disease virus (NDV) has provided investigators with a powerful approach to understand viral molecular biology and vaccine development. It has been impressively improved with modified strategies since its first report, but it still poses some challenges. Most noteworthy, the genome complexity and length made full-length error-free cDNA assembly the most challenging and time-consuming step of NDV rescue. In the present study, we report a rapid full-length NDV genome construction with only a two-step ligation-independent cloning (LIC) strategy, which could be applied to distinct genotypes. In this approach, the genome of NDV was divided into two segments, and the cDNA clones were generated by RT-PCR followed by LIC. Subsequently, the infectious NDVs were rescued by co-transfection of the full-length cDNA clones and supporting plasmids expressing the NP, P, and L proteins of NDV in BHK-21 cells. Compared with the conventional cloning approaches, the two-step cloning method drastically reduced the number of cloning steps and saved researchers a substantial amount of time for constructing NDV infectious clones, thus enabling a rapid rescue of different genotypes of NDVs in a matter of weeks. Therefore, this two-step LIC cloning strategy may have an application to the rapid development of NDV-vectored vaccines against emerging animal diseases and the generation of different genotypes of recombinant NDVs for cancer therapy.

Research Note: Isolation and complete genome analysis of a genotype II goose astrovirus in Sichuan Province, China

The emergence of Goose astrovirus (GoAstV) has led to the gout in geese. This study aimed to isolate and identify the GoAstV from diseased goslings in Sichuan Province, China, followed by performing whole genome phylogenetic analysis of the isolate. The GoAstV was successfully isolated by inoculating the diseased gosling liver and kidney homogenate into the 11-day-old goose embryo allantoic cavity for 3 passages, and the isolate was named as GoAstV-C2 strain. The virus particles were spherical, without capsule, and the size was about 28 nm under transmission electronic microscope. The complete genome length of GoAstV-C2 was 7.035 nt, and the whole genome sequence phylogenetic analysis revealed that it belongs to the GoAstV genotype II (GoAstV-II) subgenotype IIc. The isolated GoAstV-C2 strain was able to be stably passaged in the goose embryo and uric acid sedimentation was observed. The complete genome bioinformation of GoAstV-C2 determined the evolutionary characteristics of the GoAstV isolated from Sichuan, China. This finding lays a foundation for the development of preventive measures, effective vaccines, and therapeutic drugs.

Label-free detection of synthetic, full genomic length HIV-1 RNA at the few-copy level

Oligonucleotide-functionalized graphene biosensors show immense promise for use as label-free point of care devices for detection of nucleic acid biomarkers at clinically relevant levels. Graphene-based nucleic acid sensors can be fabricated at low cost and have been shown to reach limits of detection in the attomolar range. Here we demonstrate devices functionalized with 22mer or 80mer DNA probes are capable of detecting full length genomic HIV-1 subtype B RNA, with a limit of detection below 1 aM in nuclease free water. We also show that these sensors are suitable for detection directly in Qiazol lysis reagent, again with a limit of detection below 1 aM for both 22mer and 80mer probes.

Dynamic Grammar Pruning for Program Size Reduction in Symbolic Regression

Grammar is a key input in grammar-based genetic programming. Grammar design not only influences performance, but also program size. However, grammar design and the choice of productions often require expert input as no automatic approach exists. This research work discusses our approach to automatically reduce a bloated grammar. By utilizing a simple Production Ranking mechanism, we identify productions which are less useful and dynamically prune those to channel evolutionary search towards better (smaller) solutions. Our objective in this work was program size reduction without compromising generalization performance. We tested our approach on 13 standard symbolic regression datasets with Grammatical Evolution. Using a grammar embodying a well-defined function set as a baseline, we compare effective genome length and test performance with our approach. Dynamic grammar pruning achieved significantly better genome lengths for all datasets, while significantly improving generalization performance on three datasets, although it worsened in five datasets. When we utilized linear scaling during the production ranking stages (the first 20 generations) the results dramatically improved. Not only were the programs smaller in all datasets, but generalization scores were also significantly better than the baseline in 6 out of 13 datasets, and comparable in the rest. When the baseline was also linearly scaled as well, the program size was still smaller with the Production Ranking approach, while generalization scores dropped in only three datasets without any significant compromise in the rest.

Complete Genome Assembly and Annotation of Escherichia coli Bacteriophage 107.

We present the annotated genome sequence of Escherichia coli bacteriophage 107, a T4-like bacteriophage. Phage 107 has a genome length of 167,509 bp and 287 predicted genes.

Small cardamom genome: development and utilization of microsatellite markers from a draft genome sequence of Elettaria cardamomum Maton.

Small cardamom (Elettaria cardamomum Maton), the queen of spices, is the third most expensive spice in the world after saffron and vanilla, valued highly for its aroma and taste. This perennial herbaceous plant is a native of coastal parts of Southern India and displays a significant amount of morphological diversity. Its genetic potential has not been exploited due to lack of genomic resources limiting our understanding of the genome and important metabolic pathways which give it the economic advantage in the spice industry. Here, we report upon the de novo assembled, draft whole genome sequence of cardamom variety, Njallani Green Gold. We used a hybrid assembly strategy using the reads from the Oxford Nanopore, Illumina and 10x Genomics GemCode sequencing chemistries. The assembled genome length was 1.06 Gb (gigabases) which is close to the estimated genome size of cardamom. More than 75% of the genome was captured in 8000 scaffolds with a N50 of 0.15 Mb. The genome appears to have a high repeat content and 68055 gene models were predicted. The genome is close to Musa species and displays an expansion and contraction in different gene families. The draft assembly was used for in silico mining of simple sequence repeats (SSRs). A total of 2,50,571 SSRs were identified of which 2,18,270 were perfect SSRs and 32,301 were compound SSRs. Among the perfect SSRs, trinucleotides were most abundant (1,25,329) and hexanucleotide repeats appear least (2,380). From the 2,50,571 SSRs mined, 2,27,808 primer pairs were designed based on flanking sequence information. Wet lab validation was performed for 246 SSR loci and based on their amplification profiles, 60 SSR markers were used for diversity analysis of a set of 60 diverse cardamom accessions. The average number of alleles detected per locus were 14.57 with a minimum of 4 and maximum of 30 alleles. Population structure analysis revealed the presence of high degree of admixtures which could primarily be due to cross-pollination prevalent in this species. The SSR markers identified would help in the development of gene or trait-linked markers which can be subsequently used for marker-assisted breeding for crop improvement in cardamom. The information on utilization of the SSR loci for generation of markers has been developed into a public database, 'cardamomSSRdb' that is freely available for use by the cardamom community.

Newly Characterized Porcine Epidemic Diarrhea Virus GII Subtype Strain

Diarrhea outbreaks in piglets on pig farms are commonly attributed to porcine epidemic diarrhea virus (PEDV) infection. This research analyzed the S gene prevalence variation and recombination patterns in PEDV GII strains. Throughout the previous two years, 172 clinical samples of piglet diarrhea have been collected, from which 24 PEDV isolates have been isolated. Analysis of the evolutionary relationships among all 24 S genes revealed that 21 were most closely related to strains within the GII-a subgroup. The 2 isolates grouped into one clade with the GII-b subgroup. According to the mutation analysis of the amino acids (aa) that encode the S protein, 43 of the common aa in strains of the GII subtype were found to have undergone a change in polarity or charge, and 36 of these aa had a mutation frequency of more than 90%. Three different aa mutation sites were identified as exclusive to GII-a subtype strains. The genomes of three PEDV isolates were sequenced, and the resulting range in genome length was 28,035−28,041 nt. The results of recombination analysis showed that the SD1 isolate is a novel strain recombinant from the foreign S-INDEL strain and a domestic GII subtype strain. Based on the findings, the PEDV GII-a strain has been the most circulating strain in several parts of China during the previous two years. Our study reveals for the first time the unique change of aa mutations in the S protein of the GII-a subtype strain and the new characteristics of the recombination of foreign strains and domestic GII subtype strains, indicating that it is crucial to monitor the epidemic dynamics of PEDV promptly to prevent and control the occurrence of PED effectively.

The complete chloroplast genome sequence of Sedum bulbiferum (Crassulaceae)

Sedum bulbiferum is a traditional medicinal plant in China, with few reports on its chloroplast genome. In this study, the chloroplast genome of Sedum bulbiferum was characterized, and its phylogenetic position among other closely related species was studied. The results showed that the full length of the chloroplast genome was 150,074 bp, containing a large single-copy (LSC) region and a small single-copy (SSC) region of 81,730 and 16,726 bp, respectively, as well as two inverted repeat regions (IRs) of 25,809 bp like other plants. A total of 128 genes were found, including 83 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis showed that Sedum bulbiferum is closely related to Sedum emarginatum, Sedum alfredii, Sedum tricarpum, Sedum plumbizincicola, and Sedum sarmentosum.

First record of the complete chloroplast genome of Polygonatum infundiflorum (Asparagaceae), a Korean endemic species

Polygonatum infundiflorum Y.S. Kim, B.U. Oh & C.G. Jang et al. 1998 is a Korean endemic species. This is first report on the complete chloroplast genome sequence of P. infundiflorum. The complete chloroplast genome length was 154,578 bp with a GC content of 37.7%. The large single-copy (LSC) region was 83,527 bp long, and the small single-copy (SSC) region was 18,457 bp long. The paired inverted repeats (IRs) were 26,297 bp and separated the LCS and SSC regions. There were 113 genes, comprising 78 protein-coding genes, four rRNA genes, 30 tRNA genes, and one pseudogene (infA). In total, 16 genes contained one intron, and two genes contained two introns. Phylogenetic analysis suggested that Polygonatum was divided into three sections, each forming a monophyletic group. P. infundiflorum was sister to P. macropodum and formed a monophyletic group with P. inflatum. This study provides basic information for future research and contributes to taxonomic and genetic studies on Polygonatum.

Characteristics of the Complete Genome of Ageratum Yellow Vein China Virus Infecting Sonchus oleraceus.

Sonchus (Sonchus oleraceus) originated from Europe and is now cultivated worldwide. The wild resources of sonchus are very abundant, and it has rich nutritional and medicinal value. In this study, 15 sonchus samples with typical symptoms showing leaf curling, vein thickening, and enations were collected from Guigang and Baise City of Guangxi, China. Diseased sonchus were identified by PCR detection, whole genome sequence amplification, and phylogenetic and recombination analysis. The results showed that all the samples were confirmed infected by begomoviruses, and three full-length viral genomes were obtained from 15 sonchus, named GG7-13, GG8-6, and BS63-5. The full genome lengths were 2,584, 2,735, and 2,746 nt, respectively. The nucleotide identities among the three isolates ranged from 92.67 to 99.93%. All of them shared the highest identities (greater than 91.69%) with other isolates of ageratum yellow vein China virus (AYVCNV) (available on GenBank). According to the guidelines of classification of begomoviruses, the virus isolates obtained in this study are different isolates of AYVCNV; a phylogenetic tree analysis showed that these isolates formed a large branch with three other Guangxi isolates of AYVCNV, indicating their close evolution. The genome structures of GG8-6 and BS63-5 are consistent with the monopartite genome virus of the begomoviruses, and both have six open reading frames (ORFs), while GG7-13 has a 151-nt deletion between C2 and C3, resulting in a mutant strain of only five ORFs. This study is the first report on S. oleraceus infected by ageratum yellow vein China virus.

Sex-typing of ingested human blood meal in Anopheles stephensi mosquito based on the amelogenin gene.

Identifying the sex of human hosts of insect disease vectors, using PCR amplification of the amelogenin gene (AMEL) from the ingested blood meal is an increasingly useful technique for epidemiological studies of vector-borne diseases, as well as within the criminal justice system. Detection of DNA from ingested blood is influenced by the choice of DNA extraction method, genomic target region, type and length of PCR, and rate of degradation in the DNA samples over time. Here, we have tested two types of PCR (i.e. conventional and nested), producing differently-sized PCR products, in time-course assays targeting the human AMEL gene in Anopheles stephensi mosquitoes that were fed on human male and female blood. The fed female mosquitoes were allowed to digest at 28°C for times ranging from 0 to 120h. Three AMEL primer pairs were used to amplify three sequences that were 977, 539, and 106 bp for the X chromosome and 790, 355, and 112 bp for Y. We found that time since feeding had a significant negative effect on the success of PCR amplification. The shortest fragments (106 and 112 bp) were amplified for the longest time after blood feeding (up to 60h), whereas the medium and longest loci were not amplified by conventional PCR even at 0h. However, the nested PCR protocol, targeting the medium sequence, could detect small amounts of human DNA up to 36h (1.5 days) after the blood meal. The shortest PCR assay standardized herein successfully detected small amounts of human DNA in female mosquitoes up to 60h after the blood meal. This assay represents a promising tool for identifying the sex of the human host from the blood meal in field-collected female mosquitoes.

Dasania phycosphaerae sp. nov., isolated from phytoplankton sample from the south coast of the Republic of Korea.

A novel bacterial strain, designated as GY-18T, was isolated from particulate material sampled from the Korean south coast. The bacterium was Gram-stain-negative, aerobic and rod-shaped. Oxidase and catalase were positive. Optimal growth was observed at 30 °C, pH 7.0, in the presence of 1 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene and whole-genome sequences suggested that strain GY-18T was a member of the genus Dasania , but represented a separate lineage. The 16S rRNA gene sequence of strain GY-18T showed high similarities to Dasania marina KCTC 12566T (98.4 %), Halioglobus japonicus KCTC 23429T (92.2 %), Marimicrobium arenosum KCTC 42300T (92.4 %) and Seongchinamella unica KCTC 62383T (92.2 %), respectively. The genome length of strain GY-18T was 3 808 450 bp with a DNA G+C content of 47.8 mol %. The major cellular fatty acids of strain GY-18T were summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0 and C10 : 0 3-OH (>10 %). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phospholipids were shown to be the major polar lipids. The predominant isoprenoid quinone was Q-8. Based on phylogenetic, phenotypic, chemotaxonomic and genomic features, we propose that strain GY-18T represents a novel species in the genus Dasania of the family Spongiibacteraceae , with the proposed name Dasania phycosphaerae sp. nov. The type strain is GY-18T (=KCTC 92290T=GDMCC 1.3586T).