Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome.
Mycobacterium abscessus is an environmental bacterium with increasing clinical relevance. Here, we report the annotated whole-genome sequence of M. abscessus strain M152.
Pantoea sp. strain A4 is a Gram-negative bacterium isolated from the Rafflesia flower. We present here, for the first time, the genome sequence of Rafflesia-associated Pantoea sp. strain A4, which exhibited quorum-sensing activity.
Mycobacterium abscessus is a ubiquitous, rapidly growing species of nontuberculous mycobacteria that colonizes organic surfaces and is frequently associated with opportunistic infections in humans. We report here the draft genome sequence of Mycobacterium abscessus strain M139, which shows genomic features reported to be characteristic of both Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. massiliense.
Enterobacter sp. strain SST3 is an endophytic bacterium isolated from Saccharum spp. Here we present its annotated draft genome that may shed light on its role as a bacterial endophyte of sugarcane. To our knowledge, this is the first genome announcement of a sugarcane-associated bacterium from the genus Enterobacter.
Roseomonas sp. strain B5 was isolated from Malaysian tropical soil that showed N-acylhomoserine lactone degradation. This is the first genome announcement of a member from the genus of Roseomonas and the first report on the quorum-quenching activity of Roseomonas spp.
Mycobacterium massiliense is a rapidly growing mycobacterial species. The pathogenicity of this subspecies is not well known. We report here the annotated genome sequence of M. massiliense strain M18, which was isolated from a lymph node biopsy specimen from a Malaysian patient suspected of having tuberculous cervical lymphadenitis.
Methylobacterium sp. strain GXF4 is an isolate from grapevine. Here we present the sequence, assembly, and annotation of its genome, which may shed light on its role as a grapevine xylem inhabitant. To our knowledge, this is the first genome announcement of a plant xylem-associated strain of the genus Methylobacterium.
Ralstonia sp. strain PBA was isolated from textile wastewater in a coculture with Hydrogenophaga sp. strain PBC. Here we present the assembly and annotation of its genome, which may provide further insights into the mechanism of its interaction with strain PBC during 4-aminobenzenesulfonate degradation.
Salmonella enterica serovar Typhi is the causative agent of typhoid fever, which causes nearly 21.7 million illnesses and 217,000 deaths globally. Herein, we describe the whole-genome sequence of the Salmonella Typhi strain ST0208, isolated from a sporadic case of typhoid fever in Kuala Lumpur, Malaysia. The whole-genome sequence and comparative genomics allow an in-depth understanding of the genetic diversity, and its link to pathogenicity and evolutionary dynamics, of this highly clonal pathogen that is endemic to Malaysia.
The long-read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource-limited settings, such as field conditions or low- and middle-income countries. For this purpose, protocols for extracting high-molecular-weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram-negative bacteria. However, we found that without modification, these protocols are unsuitable for gram-positive Streptococcus spp., a major threat to fish farming and food security in low- and middle-income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high-molecular-weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish-pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high-molecular-weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low-copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource-limited settings-adapted protocol for high-molecular-weight DNA extraction could be implemented successfully for similarly recalcitrant-to-lysis gram-positive bacteria, and it represents a method of choice for MinION-based disease diagnostics in low- and middle-income countries.
This paper reported on the various filamentous fungi strains that were isolated from a wild grown Catharanthus roseus. Based on the morphological characteristics and molecular technique through a Polymerase Chain Reaction and DNA sequencing method using internal transcribed spacer (ITS), these fungi had been identified as a Colletotrichum sp., Macrophomina phaseolina, Nigrospora sphaerica and Fusarium solani. The ultrastructures of spores and hyphae were observed under a Scanning Electron Microscope. The hydrolytic enzyme test showed that all strains were positive in secreting cellulase. Colletotrichum sp. and F. solani strains also gave a positive result for amylase while only F. solani was capable to secrete protease. These fungi were putatively classified as endophytic fungi since they produced extracellular enzymes that allow them to penetrate plant cell walls and colonize with symbiotic properties.
Mycobacterium abscessus group subsp., such as M. massiliense, M. abscessus sensu stricto and M. bolletii, are an environmental organism found in soil, water and other ecological niches, and have been isolated from respiratory tract infection, skin and soft tissue infection, postoperative infection of cosmetic surgery. To determine the unique genetic feature of M. massiliense, we sequenced the complete genome of M. massiliense type strain JCM 15300 (corresponding to CCUG 48898). Comparative genomic analysis was performed among Mycobacterium spp. and among M. abscessus group subspp., showing that additional ß-oxidation-related genes and, notably, the mammalian cell entry (mce) operon were located on a genomic island, M. massiliense Genomic Island 1 (MmGI-1), in M. massiliense. In addition, putative anaerobic respiration system-related genes and additional mycolic acid cyclopropane synthetase-related genes were found uniquely in M. massiliense. Japanese isolates of M. massiliense also frequently possess the MmGI-1 (14/44, approximately 32%) and three unique conserved regions (26/44; approximately 60%, 34/44; approximately 77% and 40/44; approximately 91%), as well as isolates of other countries (Malaysia, France, United Kingdom and United States). The well-conserved genomic island MmGI-1 may play an important role in high growth potential with additional lipid metabolism, extra factors for survival in the environment or synthesis of complex membrane-associated lipids. ORFs on MmGI-1 showed similarities to ORFs of phylogenetically distant M. avium complex (MAC), suggesting that horizontal gene transfer or genetic recombination events might have occurred within MmGI-1 among M. massiliense and MAC.
The complete mitochondrial genome of the Bass yabby Trypaea australiensis was obtained from a partial genome scan using the MiSeq sequencing system. The T. australiensis mitogenome is 16,821 bp in length (70.25% A + T content) made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs and a putative 1977 bp non-coding AT-rich region. This Trypaea mitogenome sequence is the 5th for the family Callianassidae and represents a new gene order for the Decapoda involving protein-coding, rRNA and tRNA genes and the control region.
This study used morphological characterization and phylogenetic analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA to investigate the phylogeny of Passiflora species. The samples were collected from various regions of East Malaysia, and discriminant function analysis based on linear combinations of morphological variables was used to classify the Passiflora species. The biplots generated five distinct groups discriminated by morphological variables. The group consisted of cultivars of P. edulis with high levels of genetic similarity; in contrast, P. foetida was highly divergent from other species in the morphological biplots. The final dataset of aligned sequences from nine studied Passiflora accessions and 30 other individuals obtained from GenBank database (NCBI) yielded one most parsimonious tree with two strongly supported clades. Maximum parsimony (MP) tree showed the phylogenetic relationships within this subgenus Passiflora support the classification at the series level. The constructed phylogenic tree also confirmed the divergence of P. foetida from all other species and the closeness of wild and cultivated species. The phylogenetic relationships were consistent with results of morphological assessments. The results of this study indicate that ITS region analysis represents a useful tool for evaluating genetic diversity in Passiflora at the species level.
Congracilaria babae was first reported as a red alga parasitic on the thallus of Gracilaria salicornia based on Japanese materials. It was circumscribed to have deep spermatangial cavities, coloration similar to its host and the absence of rhizoids. We observed a parasitic red alga with morphological and anatomical features suggestive of C. babae on a Hydropuntia species collected from Sabah, East Malaysia. We addressed the taxonomic affinities of the parasite growing on Hydropuntia sp. based on the DNA sequence of molecular markers from the nuclear, mitochondrial and plastid genomes (nuclear ITS region, mitochondrial cox1 gene and plastid rbcL gene). Phylogenetic analyses based on all genetic markers also implied the monophyly of the parasite from Hydropuntia sp. and C. babae, suggesting their conspecificity. The parasite from Hydropuntia sp. has a DNA signature characteristic to C. babae in having plastid rbcL gene sequence identical to G. salicornia. C. babae is likely to have evolved directly from G. salicornia and subsequently radiated onto a secondary host Hydropuntia sp. We also recommend the transfer of C. babae to the genus Gracilaria and propose a new combination, G. babae, based on the anatomical observations and molecular data.
A total of 30 specimens belonging to five species, namely; Cryptozona siamensis, Sarika resplendens and Sarika sp. from the family Ariophantidae as well as Quantula striata and Quantula sp. from the family Dyakiidae were collected from the Langkawi Island in Northern Peninsular Malaysia. All specimens were identified through comparisons of shell morphology and amplification of a 500 bp segment of the 16S rRNA mtDNA gene. To assess phylogenetic insights, the sequences were aligned using ClustalW and phylogenetic trees were constructed. The analyses showed two major lineages in both Maximum Parsimony and Neighbour Joining phylogenetic trees. Each putative taxonomic group formed a monophyletic cluster. Our study revealed low species and intraspecies genetic diversities based on the 16S rRNA gene sequences. Thus, this study has provided an insight of land snail diversity in populations of an island highly influenced by anthropogenic activities through complementary use of shell morphological and molecular data.
This study examines the population genetic structure of Tor tambroides, an important freshwater fish species in Malaysia, using fifteen polymorphic microsatellite loci and sequencing of 464 base pairs of the mitochondrial cytochrome c oxidase I (COI) gene. A total of 152 mahseer samples were collected from eight populations throughout the Malaysia river system. Microsatellites results found high levels of intrapopulation variations, but mitochondrial COI results found high levels of interpopulations differentiation. The possible reasons for their discrepancies might be the varying influence of genetic drift on each marker or the small sample sizes used in most of the populations. The Kelantan population showed very low levels of genetic variations using both mitochondrial and microsatellite analyses. Phylogenetic analysis of the COI gene found a unique haplotype (ER8∗), possibly representing a cryptic lineage of T. douronensis, from the Endau-Rompin population. Nevertheless, the inclusion of nuclear microsatellite analyses could not fully resolve the genetic identity of haplotype ER8∗ in the present study. Overall, the findings showed a serious need for more comprehensive and larger scale samplings, especially in remote river systems, in combination with molecular analyses using multiple markers, in order to discover more cryptic lineages or undescribed "genetic species" of mahseer.
Although it is possible to recover the complete mitogenome directly from shotgun sequencing data, currently reported methods and pipelines are still relatively time consuming and costly. Using a sample of the Australian freshwater crayfish Engaeus lengana, we demonstrate that it is possible to achieve three-day turnaround time (four hours hands-on time) from tissue sample to NCBI-ready submission file through the integration of MiSeq sequencing platform, Nextera sample preparation protocol, MITObim assembly algorithm and MITOS annotation pipeline.
This study was done to detect and diagnose beta-thalassemia (beta-Thal) gene quickly. We applied sequence specific Amplification (SSA) method to the analysis. 13 kinds of beta-Thal and two kinds of hemoglobin variants were able to detect under the same PCR condition. These mutations were found frequently in ten countries of Asian region (the southern part of China, Vietnam, Cambodia, Thailand, Myanmar, Malaysia, Singapore, Indonesia, Pakistan, India), and 15 kinds in total (-28CapA-->G, CD5-CT, CD8/9+-G, CD15G-->A, CD17A-->T, IVSI-1G-->T, CD41/42-4del, CD16-C, CD26G-->A(betaE), IVSI-5G-->C, CD35C-->A, CD71/72 +A, CD6A-->T (betaS), -619del, IVSII-654C-->T). More than 80% of patients are included in these mutations. To make the reagents a kit, the procedure became simple and rapid. DNA was extracted by salting out method. The PCR product was detected by polyacrylamide gel electrophoresis and silver staining. The confirmation of the variant was done by the PCR-direct sequencing method. It took approximately six hours for PCR reaction, electrophoresis and staining. This method made us to detect and diagnose beta-Thal in one day.