The complete genome sequences of 5 species of Syngnathidae (Syngnathiformes, Actinopteri).

Biotechnology and Biological Sciences Research Council (BBSRC). Grant Numbers: BB/H016120/1, BB/I024631/1, BB/I025956/1, BB/K003240/2, BB/L012499/1

Background Sweet potato chlorotic stunt virus (family Closteroviridae, genus Crinivirus) features a large bipartite, single-stranded, positive-sense RNA genome. To date, only three complete genomic sequences of SPCSV can be accessed through GenBank. SPCSV was first detected from China in 2011, only partial genomic sequences have been determined in the country. No report on the complete genomic sequence and genome structure of Chinese SPCSV isolates or the genetic relation between isolates from China and other countries is available.Methodology/Principal FindingsThe complete genomic sequences of five isolates from different areas in China were characterized. This study is the first to report the complete genome sequences of SPCSV from whitefly vectors. Genome structure analysis showed that isolates of WA and EA strains from China have the same coding protein as isolates Can181-9 and m2-47, respectively. Twenty cp genes and four RNA1 partial segments were sequenced and analyzed, and the nucleotide identities of complete genomic, cp, and RNA1 partial sequences were determined. Results indicated high conservation among strains and significant differences between WA and EA strains. Genetic analysis demonstrated that, except for isolates from Guangdong Province, SPCSVs from other areas belong to the WA strain. Genome organization analysis showed that the isolates in this study lack the p22 gene.Conclusions/SignificanceWe presented the complete genome sequences of SPCSV in China. Comparison of nucleotide identities and genome structures between these isolates and previously reported isolates showed slight differences. The nucleotide identities of different SPCSV isolates showed high conservation among strains and significant differences between strains. All nine isolates in this study lacked p22 gene. WA strains were more extensively distributed than EA strains in China. These data provide important insights into the molecular variation and genomic structure of SPCSV in China as well as genetic relationships among isolates from China and other countries.

The Viral Genomes Project aims to provide molecular standards for viral genomic research. The project has produced over 1,600 records for more than 1,200 different species. The National Center for Biotechnology Information (NCBI) provides access to this data through the Entrez search and retrieval engine and offers visualization of the sequence information at various levels of detail. Taxonomically organized displays, precomputed sequence comparison data, and direct access to analytical tools provide researchers with the ability to analyze and compare viral genomes and proteomes in a fast and convenient manner. The Viral Genomes Project is a collaborative effort between NCBI staff and many dedicated scientists worldwide. The URL for the database is http://www.ncbi.nlm.nih.gov/genomes/VIRUSES/viruses.html. As the number of viral records in the public sequence databases (GenBank, EMBL, and DDBJ) grows, retrieving a viral genomic sequence of interest with associated information is becoming increasingly complex. High redundancy in the databases is a common problem for all organisms; in the case of viruses, however, the large number of available strains, isolates, and mutants further exacerbates the problem. For example, a search of Entrez Nucleotide currently retrieves more than 95,500 records for Human immunodeficiency virus 1 (HIV-1) and more than 22,500 records for Hepatitis C virus (HCV) alone; the total number of viral nucleotide records exceeds 220,000. Among these are both partial and complete genomic sequences, including partial sequences marked as a complete genome by submitters. Historically, sequence databases were merely archives of sequences directly submitted by users. Although a stricter submission procedure has been applied in recent years and therefore the quality of sequence records has greatly improved, a significant number of records are still underannotated, and the information in the old sequence records is often outdated. Furthermore, viral genomes are remarkably variable, consisting of either single-stranded or double-stranded DNA or RNA in either linear or circular form and comprising one or more segments. This variability makes viral records especially prone to inaccuracies in molecular information annotation. To cope with these problems, NCBI has created the Viral Genomes Project as a part of the NCBI Genomes Project (19). Only complete or, occasionally, nearly complete viral genomic sequences missing only nontranslated portions (usually the ends of a genomic molecule) are being collected for this project, thereby greatly reducing redundancy. All available complete viral genomic sequences are being collected in order to faithfully represent the great genome variability found in many viruses. For example, 314 complete genome sequences of HIV-1 from various strains and isolates are included in the Entrez Genome collection. But only one sequence ({type:entrez-nucleotide,attrs:{text:NC_001802,term_id:9629357}}NC_001802) has been selected as a reference (RefSeq) to serve as a molecular standard. RefSeq records are manually curated to correct and update content in the original sequence records, which often involves consultations with the original submitters and/or other outside experts. The collection of preselected reference sequences greatly facilitates comparison of the genomes of different viruses. As of December 2003, the Viral Genomes Project contained 1,677 viral reference genomic sequences representing 1,223 virus species, which make a significant contribution to the NCBI RefSeq collection (13). Figure ​Figure11 shows the growth of the viral RefSeq collection during the past 3 years. FIG. 1. The growth of NCBI's Viral Genomes Project. The bars represent the numbers of new and all viral genome reference sequences in each quarter. While a number of databases provide information on viral sequences, most of them are limited to certain families or groups (reviewed in references 4, 3, 6, and 12; http://www.dpvweb.net/index.php). The most comprehensive and well-established viral database, ICTVdB, provides “searchable descriptions of virus isolates, species, genera, families, orders; images of many viruses; and links to genomic and protein databanks” (2). ICTVdB has been a primary resource for information about biological properties of viruses. It plays a major role in viral taxonomic classification, on which our project relied heavily. ICTVdB does present links to viral sequences, but these sequences are original records from public sequence databases and therefore may contain inaccurate or outdated information. The Viral Genomes Project described here is the first comprehensive resource that provides access to the curated set of complete viral genomes in an easily navigable way and offers a collection of tools and precomputed results which greatly facilitate viral genome analysis. These precomputed analyses and tools include the global alignment of genome neighbors, available in both text and graphical forms; viral protein clusters (VOGs), (putative) functional and evolutionary groups of viral proteins derived from RefSeq genomes (which eliminates redundancy) and classified by sequence similarity; convenient VOG displays, including those integrated with the Conserved Domain Database (CDD), an NCBI collection of conserved protein domains; and a BLAST search against a selected set of viral proteins. To start exploring the Viral Genomes resources, go to http://www.ncbi.nlm.nih.gov/genomes/VIRUSES/viruses.html.

Newborn screening: adapting to advancements in whole-genome sequencing.

<i>Newborn Screening:</i> Adapting to Advancements in Whole-Genome Sequencing

Hepatitis viruses are the leading causes of chronic liver disease resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma in the world and also in Turkey. Although Turkey has an intermediate rate of hepatitis B virus (HBV) infection with a prevalence reported as 5%, a complete HBV genome sequence has not been published. In this study, the molecular characterization and phylogenetic analysis are described of 11 complete HBV genomes isolated from 11 naïve patients (5 male, 6 female; ages: 18--54 years old, median 35 years old) with chronic HBV infection. Of 11 patients, 7 and 4 were HBeAg positive/anti-HBe negative and HBeAg negative/anti-HBe positive, respectively. All patients had no co-infection with HCV, HDV, or HIV. HBV DNA was extracted from the sera of the patients. The complete genome was amplified by PCR and cloned into a TA vector. The PCR products were sequenced directly and the complete HBV genome sequences were determined. Ten HBV genomes were 3182 base pairs in length. There was a 183 bp deletion (between nucleotides 2987--3169) in pre-S region in one HBeAg positive patient. There were two pre-core stop codons (G1896A) in two HBeAg negative and three core promoter dual mutations (T1762/A1764) in one HBeAg positive and two HBeAg negative patients' HBV genomes. Phylogenetic analysis of all complete genomes yielded that all Turkish sequences were clustered in genotype D branch (ten in subgenotype D1 and one in subgenotype D2). The analysis of S gene amino acid sequences revealed that surface gene subtypes of one and ten HBV strains were subtype ayw3 and ayw2, respectively. This study indicates that Turkish patients with chronic hepatitis B infection show very little genotypic heterogeneity. Genotype D of HBV DNA and subtype ayw2 of surface gene represent almost the whole Turkish patient population infected with HBV.

Fundamental improvement was made for genome sequencing since the next‐generation sequencing (NGS) came out in the 2000s. The newer technologies make use of the power of massively‐parallel short‐read DNA sequencing, genome alignment and assembly methods to digitally and rapidly search the genomes on a revolutionary scale, which enable large‐scale whole genome sequencing (WGS) accessible and practical for researchers. Nowadays, whole genome sequencing is more and more prevalent in detecting the genetics of diseases, studying causative relations with cancers, making genome‐level comparative analysis, reconstruction of human population history, and giving clinical implications and instructions. In this review, we first give a typical pipeline of whole genome sequencing, including the lab template preparation, sequencing, genome assembling and quality control, variants calling and annotations. We compare the difference between whole genome and whole exome sequencing (WES), and explore a wide range of applications of whole genome sequencing for both mendelian diseases and complex diseases in medical genetics. We highlight the impact of whole genome sequencing in cancer studies, regulatory variant analysis, predictive medicine and precision medicine, as well as discuss the challenges of the whole genome sequencing.

To the Editor: Porcine deltacoronavirus (PDCoV) was discovered in 2012, during a study to identify new coronaviruses in mammals and birds in Hong Kong (1).In February 2014, this novel porcine coronavirus was detected in pigs in Ohio, United States (2), and has since been reported in at least 17 US states (3-5).Concern regarding the epidemiology, evolution, and pathogenicity of this emerging virus is increasing.Recently, PDCoV was identified in South Korea (6).We report PDCoV in mainland China.Since December 2010, a large-scale outbreak of diarrhea in suckling piglets has occurred on swine farms in mainland China (7).The causative agent was considered to be a variant of porcine epidemic diarrhea virus (PEDV) (8), and the role of PDCoV in the outbreak was not investigated at that time.Using 2 pairs of specific primers to detect PDCoV, as described by Wang et al. (2), we tested 215 intestinal or fecal samples collected at various times during 2004-2014 from piglets with clinical diarrhea in Anhui, Guangxi, Hubei, and Jiangsu provinces, mainland China (online Technical Appendix Table 1, http://wwwnc.cdc.gov/EID/article/21/12/15-0283-Techapp1.pdf).All samples were submitted from commercial pig farms to our laboratory for enteropathogen detection.Of these samples, 165 (124 from Hubei, 41 from Jiangsu) had been collected in 2014, and 50 (40 from Jiangsu, 6 from Anhui, 4 from Guangxi) had been collected during 2004-2013 and preserved in our laboratory.The 215 samples were simultaneously tested for PEDV and transmissible gastroenteritis virus (TGEV) by using the primers listed in online Technical Appendix Table 2. Of the samples tested, 14 (6.51%) were positive for PDCoV,

Relatively high prevalence and mortality rates of bovine ephemeral fever (BEF) have been reported in recent epidemics in some countries, including Turkey, when compared with previous outbreaks. A limited number of complete genome sequences of BEF virus (BEFV) are available in the GenBank Database. In this study, the complete genome of highly pathogenic BEFV isolated during an outbreak in Turkey in 2012 was analyzed for genetic characterization. The complete genome of the Turkish BEFV isolate was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. It was found that the complete genome of the Turkish BEFV isolate was 14,901 nt in length. The complete genome sequence obtained from the study showed 91-92% identity at nucleotide level to Australian (BB7721) and Chinese (Bovine/China/Henan1/2012) BEFV isolates. Phylogenetic analysis of the glycoprotein gene of the Turkish BEFV isolate also showed that Turkish isolates were closely related to Israeli isolates. Because of the limited number of complete BEFV genome sequences, the results from this study will be useful for understanding the global molecular epidemiology and geodynamics of BEF.

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel or next generation [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein-coding genes in the genome (≈1.1% of genome), and an exome test for a single patient generates ≈6 gigabases (109 bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ≈27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. Decreasing sequencing costs and broad dissemination of next-generation (next-gen) equipment and expertise are increasing availability of massively parallel sequencing of patient DNA samples (short read massively parallel or next-gen sequencing).1,2 Most rapidly expanding is exome sequencing, where all protein-coding sequences (exons) are selected from total genomic DNA and selectively sequenced.3 Alternative approaches to next-gen sequencing include targeted sequencing (TS) and whole genome (complete genome) sequencing. Currently, marketed targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel next-gen approaches are beginning to supplant …

In August 2014, an outbreak of enterovirus D68 (EV-D68) occurred in North America, causing severe respiratory disease in children. Due to a lack of complete genome sequence data, there is only a limited understanding of the molecular evolution and epidemiology of EV-D68 during this outbreak, and it is uncertain whether the differing clinical manifestations of EV-D68 infection are associated with specific viral lineages. We developed a high-throughput complete genome sequencing pipeline for EV-D68 that produced a total of 59 complete genomes from respiratory samples with a 95% success rate, including 57 genomes from Kansas City, MO, collected during the 2014 outbreak. With these data in hand, we performed phylogenetic analyses of complete genome and VP1 capsid protein sequences. Notably, we observed considerable genetic diversity among EV-D68 isolates in Kansas City, manifest as phylogenetically distinct lineages, indicative of multiple introductions of this virus into the city. In addition, we identified an intersubclade recombination event within EV-D68, the first recombinant in this virus reported to date. Finally, we found no significant association between EV-D68 genetic variation, either lineages or individual mutations, and a variety of demographic and clinical variables, suggesting that host factors likely play a major role in determining disease severity. Overall, our study revealed the complex pattern of viral evolution within a single geographic locality during a single outbreak, which has implications for the design of effective intervention and prevention strategies. Until recently, EV-D68 was considered to be an uncommon human pathogen, associated with mild respiratory illness. However, in 2014 EV-D68 was responsible for more than 1,000 disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak. Here, we doubled the number of publicly available complete genome sequences of EV-D68 by performing high-throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them. Overall, these results will help inform the design of intervention strategies for EV-D68.

Determining the complete genome sequence data of adenoviruses has recently become greatly important due to their use by scientists as vectors in cancer studies and other fields, including vaccine development. However, the GenBank database currently has few complete genome sequences of adenoviruses, which are known for their large genomes. To address this gap, we analysed next-generation sequencing data obtained from our previous study to provide the complete genome sequence of the canine adenovirus-2 strain. For the obtained canine adenovirus-2 strain (OQ596341), comparative genomics, recombination and phylogenetic analysis were conducted. This sequence was compared and phylogenetically analysed with the 20 complete genome sequences of canine adenovirus previously reported in GenBank worldwide, as well as partial E3 ORFA sequences obtained from Türkiye. The nucleotide similarity rates of the sequence obtained from this study with other CAdV-2 whole genomes are over 99.04%. The gene alignment results reveal that the OQ596341 was found to be closely related to the AC000020 reference genome and LC557011. There are two recombination events related to the genome in this study. Comparisons with other complete genome sequences revealed several previously unseen mutations. These mutations include H34Y in the E1A gene; P55A in the E1B 55K gene; D13N and D202N in the IVa2 gene; K679R, V934I and K989N in the Pol gene; E205K in the pTP gene; T455A in the pIIIa gene; A310V in the V gene; G151R in the protease gene; E268K in the 100K gene; G66S and G141S in the 33K gene; T14A, E250K, D287N and I293T in the E3 ORFA gene; and L193F in the E434K gene. Moreover, a comparison with partial sequences obtained from Türkiye revealed the E250K mutation in the E3 ORFA gene, which we report for the first time in Türkiye. The complete CAdV-2 genome sequence obtained in the present study is the first sequence from Europe. Comparative analysis with other genomes revealed some unique mutations. This study is the first to report the E250K amino acid change in the E3 ORFA gene in Türkiye. We anticipate that this data can be used in future CAdV-2 vaccine development studies. Further studies are recommended to evaluate the impact of these mutations on viral tropism and other host interactions.

By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study's findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.

Next Generation Technology Edges Genome Sequencing toward the Clinic