Displaying publications 1 - 20 of 34 in total

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  1. Diversity of microbiota associated with symptomatic and non-symptomatic bacterial wilt-diseased banana plants determined using 16S rRNA metagenome sequencing
    Suhaimi NSM, Goh SY, Ajam N, Othman RY, Chan KG, Thong KL
    World J Microbiol Biotechnol, 2017 Aug 21;33(9):168.
    PMID: 28828756 DOI: 10.1007/s11274-017-2336-0
    Banana is one of the most important fruits cultivated in Malaysia, and it provides many health benefits. However, bacterial wilt disease, which attacks bananas, inflicts major losses on the banana industry in Malaysia. To understand the complex interactions of the microbiota of bacterial wilt-diseased banana plants, we first determined the bacterial communities residing in the pseudostems of infected (symptomatic) and diseased-free (non-symptomatic) banana plants. We characterized the associated microorganisms using the targeted 16S rRNA metagenomics sequencing on the Illumina MiSeq platform. Taxonomic classifications revealed 17 and nine known bacterial phyla in the tissues of non-symptomatic and symptomatic plants, respectively. Cyanobacteria and Proteobacteria (accounted for more than 99% of the 16S rRNA gene fragments) were the two most abundant phyla in both plants. The five major genera found in both plant samples were Ralstonia, Sphingomonas, Methylobacterium, Flavobacterium, and Pseudomonas. Ralstonia was more abundant in symptomatic plant (59% out of the entire genera) as compared to those in the non-symptomatic plant (only 36%). Our data revealed that 102 bacterial genera were only assigned to the non-symptomatic plant. Overall, this study indicated that more diverse and abundant microbiota were associated with the non-symptomatic bacterial wilt-diseased banana plant as compared to the symptomatic plant. The higher diversity of endophytic microbiota in the non-symptomatic banana plant could be an indication of pathogen suppression which delayed or prevented the disease expression. This comparative study of the microbiota in the two plant conditions might provide caveats for potential biological control strategies.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods
  2. Fulltext High-Throughput Sequencing-Based Investigation of Viruses in Human Cancers by Multienrichment Approach
    Mollerup S, Asplund M, Friis-Nielsen J, Kjartansdóttir KR, Fridholm H, Hansen TA, et al.
    J Infect Dis, 2019 09 13;220(8):1312-1324.
    PMID: 31253993 DOI: 10.1093/infdis/jiz318
    BACKGROUND: Viruses and other infectious agents cause more than 15% of human cancer cases. High-throughput sequencing-based studies of virus-cancer associations have mainly focused on cancer transcriptome data.

    METHODS: In this study, we applied a diverse selection of presequencing enrichment methods targeting all major viral groups, to characterize the viruses present in 197 samples from 18 sample types of cancerous origin. Using high-throughput sequencing, we generated 710 datasets constituting 57 billion sequencing reads.

    RESULTS: Detailed in silico investigation of the viral content, including exclusion of viral artefacts, from de novo assembled contigs and individual sequencing reads yielded a map of the viruses detected. Our data reveal a virome dominated by papillomaviruses, anelloviruses, herpesviruses, and parvoviruses. More than half of the included samples contained 1 or more viruses; however, no link between specific viruses and cancer types were found.

    CONCLUSIONS: Our study sheds light on viral presence in cancers and provides highly relevant virome data for future reference.

    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  3. Fulltext A Sensitive, Colorimetric, High-Throughput Loop-Mediated Isothermal Amplification Assay for the Detection of Plasmodium knowlesi
    Britton S, Cheng Q, Grigg MJ, William T, Anstey NM, McCarthy JS
    Am J Trop Med Hyg, 2016 07 06;95(1):120-2.
    PMID: 27162264 DOI: 10.4269/ajtmh.15-0670
    The simian parasite Plasmodium knowlesi is now the commonest cause of malaria in Malaysia and can rapidly cause severe and fatal malaria. However, microscopic misdiagnosis of Plasmodium species is common, rapid antigen detection tests remain insufficiently sensitive and confirmation of P. knowlesi requires polymerase chain reaction (PCR). Thus available point-of-care diagnostic tests are inadequate. This study reports the development of a simple, sensitive, colorimetric, high-throughput loop-mediated isothermal amplification assay (HtLAMP) diagnostic test using novel primers for the detection of P. knowlesi. This assay is able to detect 0.2 parasites/μL, and compared with PCR has a sensitivity of 96% for the detection of P. knowlesi, making it a potentially field-applicable point-of-care diagnostic tool.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  4. Fulltext De novo sequencing, assembly and analysis of eight different transcriptomes from the Malayan pangolin
    Mohamed Yusoff A, Tan TK, Hari R, Koepfli KP, Wee WY, Antunes A, et al.
    Sci Rep, 2016 09 13;6:28199.
    PMID: 27618997 DOI: 10.1038/srep28199
    Pangolins are scale-covered mammals, containing eight endangered species. Maintaining pangolins in captivity is a significant challenge, in part because little is known about their genetics. Here we provide the first large-scale sequencing of the critically endangered Manis javanica transcriptomes from eight different organs using Illumina HiSeq technology, yielding ~75 Giga bases and 89,754 unigenes. We found some unigenes involved in the insect hormone biosynthesis pathway and also 747 lipids metabolism-related unigenes that may be insightful to understand the lipid metabolism system in pangolins. Comparative analysis between M. javanica and other mammals revealed many pangolin-specific genes significantly over-represented in stress-related processes, cell proliferation and external stimulus, probably reflecting the traits and adaptations of the analyzed pregnant female M. javanica. Our study provides an invaluable resource for future functional works that may be highly relevant for the conservation of pangolins.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  5. Fulltext In-depth tanscriptomic analysis on giant freshwater prawns
    Mohd-Shamsudin MI, Kang Y, Lili Z, Tan TT, Kwong QB, Liu H, et al.
    PLoS One, 2013;8(5):e60839.
    PMID: 23734171 DOI: 10.1371/journal.pone.0060839
    Gene discovery in the Malaysian giant freshwater prawn (Macrobrachium rosenbergii) has been limited to small scale data collection, despite great interest in various research fields related to the commercial significance of this species. Next generation sequencing technologies that have been developed recently and enabled whole transcriptome sequencing (RNA-seq), have allowed generation of large scale functional genomics data sets in a shorter time than was previously possible. Using this technology, transcriptome sequencing of three tissue types: hepatopancreas, gill and muscle, has been undertaken to generate functional genomics data for M. rosenbergii at a massive scale. De novo assembly of 75-bp paired end Ilumina reads has generated 102,230 unigenes. Sequence homology search and in silico prediction have identified known and novel protein coding candidate genes (∼24%), non-coding RNA, and repetitive elements in the transcriptome. Potential markers consisting of simple sequence repeats associated with known protein coding genes have been successfully identified. Using KEGG pathway enrichment, differentially expressed genes in different tissues were systematically represented. The functions of gill and hepatopancreas in the context of neuroactive regulation, metabolism, reproduction, environmental stress and disease responses are described and support relevant experimental studies conducted previously in M. rosenbergii and other crustaceans. This large scale gene discovery represents the most extensive transcriptome data for freshwater prawn. Comparison with model organisms has paved the path to address the possible conserved biological entities shared between vertebrates and crustaceans. The functional genomics resources generated from this study provide the basis for constructing hypotheses for future molecular research in the freshwater shrimp.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  6. Transcriptome-seq provides insights into sex-preference pattern of gene expression between testis and ovary of the crucifix crab ( Charybdis feriatus)
    Zhang Y, Miao G, Fazhan H, Waiho K, Zheng H, Li S, et al.
    Physiol Genomics, 2018 05 01;50(5):393-405.
    PMID: 29570432 DOI: 10.1152/physiolgenomics.00016.2018
    The crucifix crab, Charybdis feriatus, which mainly inhabits Indo-Pacific region, is regarded as one of the most high-potential species for domestication and incorporation into the aquaculture sector. However, the regulatory mechanisms of sex determination and differentiation of this species remain unclear. To identify candidate genes involved in sex determination and differentiation, high throughput sequencing of transcriptome from the testis and ovary of C. feriatus was performed by the Illumina platform. After removing adaptor primers, low-quality sequences and very short (<50 nt) reads, we obtained 80.9 million and 66.2 million clean reads from testis and ovary, respectively. A total of 86,433 unigenes were assembled, and ~43% (37,500 unigenes) were successfully annotated to the NR, NT, Swiss-Prot, KEGG, COG, GO databases. By comparing the testis and ovary libraries, we obtained 27,636 differentially expressed genes. Some candidate genes involved in the sex determination and differentiation of C. feriatus were identified, such as vasa, pgds, vgr, hsp90, dsx-f, fem-1, and gpr. In addition, 88,608 simple sequence repeats were obtained, and 61,929 and 77,473 single nucleotide polymorphisms from testis and ovary were detected, respectively. The transcriptome profiling was validated by quantitative real-time PCR in 30 selected genes, which showed a good consistency. The present study is the first high-throughput transcriptome sequencing of C. feriatus. These findings will be useful for future functional analysis of sex-associated genes and molecular marker-assisted selections in C. feriatus.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  7. Fulltext Biases in small RNA deep sequencing data
    Raabe CA, Tang TH, Brosius J, Rozhdestvensky TS
    Nucleic Acids Res, 2014 Feb;42(3):1414-26.
    PMID: 24198247 DOI: 10.1093/nar/gkt1021
    High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA (small RNA-seq or sRNA-seq), such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. Given the central importance of RNA-seq to molecular biology and personalized medicine, we review recent findings that challenge small non-protein coding RNA-seq data and suggest approaches and precautions to overcome or minimize bias.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  8. Noncoding CGG repeat expansions in neuronal intranuclear inclusion disease, oculopharyngodistal myopathy and an overlapping disease
    Ishiura H, Shibata S, Yoshimura J, Suzuki Y, Qu W, Doi K, et al.
    Nat Genet, 2019 08;51(8):1222-1232.
    PMID: 31332380 DOI: 10.1038/s41588-019-0458-z
    Noncoding repeat expansions cause various neuromuscular diseases, including myotonic dystrophies, fragile X tremor/ataxia syndrome, some spinocerebellar ataxias, amyotrophic lateral sclerosis and benign adult familial myoclonic epilepsies. Inspired by the striking similarities in the clinical and neuroimaging findings between neuronal intranuclear inclusion disease (NIID) and fragile X tremor/ataxia syndrome caused by noncoding CGG repeat expansions in FMR1, we directly searched for repeat expansion mutations and identified noncoding CGG repeat expansions in NBPF19 (NOTCH2NLC) as the causative mutations for NIID. Further prompted by the similarities in the clinical and neuroimaging findings with NIID, we identified similar noncoding CGG repeat expansions in two other diseases: oculopharyngeal myopathy with leukoencephalopathy and oculopharyngodistal myopathy, in LOC642361/NUTM2B-AS1 and LRP12, respectively. These findings expand our knowledge of the clinical spectra of diseases caused by expansions of the same repeat motif, and further highlight how directly searching for expanded repeats can help identify mutations underlying diseases.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  9. Fulltext Multi-platform discovery of haplotype-resolved structural variation in human genomes
    Chaisson MJP, Sanders AD, Zhao X, Malhotra A, Porubsky D, Rausch T, et al.
    Nat Commun, 2019 04 16;10(1):1784.
    PMID: 30992455 DOI: 10.1038/s41467-018-08148-z
    The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50 bp) and 27,622 SVs (≥50 bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods
  10. Suppression Subtractive Hybridization Versus Next-Generation Sequencing in Plant Genetic Engineering: Challenges and Perspectives
    Sahebi M, Hanafi MM, Azizi P, Hakim A, Ashkani S, Abiri R
    Mol Biotechnol, 2015 Oct;57(10):880-903.
    PMID: 26271955 DOI: 10.1007/s12033-015-9884-z
    Suppression subtractive hybridization (SSH) is an effective method to identify different genes with different expression levels involved in a variety of biological processes. This method has often been used to study molecular mechanisms of plants in complex relationships with different pathogens and a variety of biotic stresses. Compared to other techniques used in gene expression profiling, SSH needs relatively smaller amounts of the initial materials, with lower costs, and fewer false positives present within the results. Extraction of total RNA from plant species rich in phenolic compounds, carbohydrates, and polysaccharides that easily bind to nucleic acids through cellular mechanisms is difficult and needs to be considered. Remarkable advancement has been achieved in the next-generation sequencing (NGS) field. As a result of progress within fields related to molecular chemistry and biology as well as specialized engineering, parallelization in the sequencing reaction has exceptionally enhanced the overall read number of generated sequences per run. Currently available sequencing platforms support an earlier unparalleled view directly into complex mixes associated with RNA in addition to DNA samples. NGS technology has demonstrated the ability to sequence DNA with remarkable swiftness, therefore allowing previously unthinkable scientific accomplishments along with novel biological purposes. However, the massive amounts of data generated by NGS impose a substantial challenge with regard to data safe-keeping and analysis. This review examines some simple but vital points involved in preparing the initial material for SSH and introduces this method as well as its associated applications to detect different novel genes from different plant species. This review evaluates general concepts, basic applications, plus the probable results of NGS technology in genomics, with unique mention of feasible potential tools as well as bioinformatics.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  11. Genome survey and development of polymorphic microsatellite loci for Sillago sihama based on Illumina sequencing technology
    Qiu B, Fang S, Ikhwanuddin M, Wong L, Ma H
    Mol Biol Rep, 2020 Apr;47(4):3011-3017.
    PMID: 32124169 DOI: 10.1007/s11033-020-05348-z
    In this study, we first conducted a genome survey assay for Sillago sihama by Illumina sequencing platform, and then developed 15 polymorphic microsatellite loci in a wild population. A total of 129.46 Gb raw data were obtained, of which 115.07 Gb were clean data, with a sequencing depth of 179.3-folds. This genome was estimated to be 522.6 Mb in size, with the heterozygosity, repeat content and GC content being 0.63%, 21% and 44%. A total of 630,028 microsatellites were identified from the genome, of which, dinucleotide repeat was the most abundant (56.80%), followed by mononucleotide repeat (30.23%). Furthermore, 60 pairs of primers were designed and synthesized based on microsatellite sequences, of which 15 were polymorphic in a wild population. A total of 91 alleles were found, with an average of 6.07 per locus. Number of alleles, observed and expected heterozygosity per locus ranged from two to 13, from 0.250 to 0.862, and from 0.396 to 0.901, respectively. Twelve loci were highly informative (PIC > 0.5), and the others were medium informative (0.25 
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods
  12. Fulltext Complete mitochondrial genome of Dacus vijaysegarani and phylogenetic relationships with congeners and other tephritid fruit flies (Insecta: Diptera)
    Yong HS, Chua KO, Song SL, Liew YJ, Eamsobhana P, Chan KG
    Mol Biol Rep, 2021 Aug;48(8):6047-6056.
    PMID: 34357549 DOI: 10.1007/s11033-021-06608-2
    BACKGROUND: Tephritid fruit flies of the genus Dacus are members of the tribe Dacini, subfamily Dacinae. There are some 274 species worldwide, distributed in Africa and the Asia-Pacific. To date, only five complete mitochondrial genomes (mitogenomes) of Dacus fruit flies have been published and are available in the GenBank.

    METHODS AND RESULTS: In view of the lack of study on their mitogenome, we sequenced (by next generation sequencing) and annotated the complete mitogenome of D. vijaysegarani from Malaysia to determine its features and phylogenetic relationship. The whole mitogenome of D. vijaysegarani has identical gene order with the published mitogenomes of the genus Dacus, with 13 protein-coding genes, two rRNA genes, 22 tRNAs, a non-coding A + T rich control region, and intergenic spacer and overlap sequences. Phylogenetic analysis based on 15 mitochondrial genes (13 PCGs and two rRNA genes), reveals Dacus, Zeugodacus and Bactrocera forming a distinct clade. The genus Dacus forms a monophyletic group in the subclade containing also the Zeugodacus group; this Dacus-Zeugodacus subclade is distinct from the Bactrocera subclade. D. (Mellesis) vijaysegarani forms a lineage with D. (Mellesis) trimacula in the subcluster containing also the lineage of D. (Mellesis) conopsoides and D. (Callantra) longicornis. D. (Dacus) bivittatus and D. (Didacus) ciliatus form a distinct subcluster. Based on cox1 sequences, the Malaysia and Vietnam taxa of D. vijaysegarani may not be conspecific.

    CONCLUSIONS: Overall, the mitochondrial genome of D. vijaysegarani provided essential molecular data that could be useful for further studies for species diagnosis, evolution and phylogeny research of other tephritid fruit flies in the future.

    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods
  13. The complete mitogenome of the endangered freshwater crayfish Cherax tenuimanus (Smith 1912) (Crustacea: Decapoda: Parastacidae)
    Austin CM, Tan MH, Gan HY, Gan HM
    Mitochondrial DNA A DNA Mapp Seq Anal, 2016 11;27(6):4176-4177.
    PMID: 25630729
    Next-Gen sequencing was used to recover the complete mitochondrial genome of Cherax tenuimanus. The mitogenome consists of 15,797 base pairs (68.14% A + T content) containing 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs, and a 779 bp non-coding AT-rich region. Mitogenomes have now been recovered for all six species of Cherax native to Western Australia.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  14. Next generation sequencing yields the complete mitochondrial genome of the Zebra moray, Gymnomuraena zebra (Anguilliformes: Muraenidae)
    Loh KH, Shao KT, Chen HM, Chen CH, Chong VC, Loo PL, et al.
    Mitochondrial DNA A DNA Mapp Seq Anal, 2016 11;27(6):4230-4231.
    PMID: 26000942
    In this study, the complete mitogenome sequence of the Zebra moray, Gymnomuraena zebra (Anguilliformes: Muraenidae) has been sequenced by the next-generation sequencing method. The assembled mitogenome consisting of 16,576 bp includes 13 protein coding genes, 22 transfer RNAs, and two ribosomal RNAs genes. The overall base composition of Zebra moray is 30.2% for A, 26.8% for C, 17.2% for G, and 25.8% for T and show 80% identities to Kidako moray, Gymnothorax kidako. The complete mitogenome of the Zebra moray provides an essential and important DNA molecular data for further phylogeography and evolutionary analysis for moray eel phylogeny.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods
  15. High-throughput terrestrial biodiversity assessments: mitochondrial metabarcoding, metagenomics or metatranscriptomics?
    Wilson JJ, Brandon-Mong GJ, Gan HM, Sing KW
    Mitochondrial DNA A DNA Mapp Seq Anal, 2019 01;30(1):60-67.
    PMID: 29591722 DOI: 10.1080/24701394.2018.1455189
    Consensus on the optimal high-throughput sequencing (HTS) approach to examine biodiversity in mixed terrestrial arthropod samples has not been reached. Metatranscriptomics could increase the proportion of taxonomically informative mitochondrial reads in HTS outputs but has not been investigated for terrestrial arthropod samples. We compared the efficiency of 16S rRNA metabarcoding, metagenomics and metatranscriptomics for detecting species in a mixed terrestrial arthropod sample (pooled DNA/RNA from 38 taxa). 16S rRNA metabarcoding and nuclear rRNA-depleted metatranscriptomics had the highest detection rate with 97% of input species detected. Based on cytochrome c oxidase I, metagenomics had the highest detection rate with 82% of input species detected, but metatranscriptomics produced a larger proportion of reads matching (Sanger) reference sequences. Metatranscriptomics with nuclear rRNA depletion may offer advantages over metabarcoding through reducing the number of spurious operational taxonomic units while retaining high detection rates, and offers natural enrichment of mitochondrial sequences which may enable increased species detection rates compared with metagenomics.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods
  16. Next generation sequencing yields the complete mitochondrial genome of the Clarion angelfish, Holacanthus clarionensis (Perciformes: Pomacanthidae)
    Shen KN, Chang CW, Loh KH, Chen CH, Hsiao CD
    Mitochondrial DNA A DNA Mapp Seq Anal, 2016 11;27(6):4118-4119.
    PMID: 25600747
    In this study, the complete mitogenome sequence of the Clarion angelfish, Holacanthus clarionensis (Perciformes: Pomacanthidae) has been sequenced by next-generation sequencing method. The length of the assembled mitogenome is 16,615 bp, including 13 protein coding genes, 22 transfer RNAs, and two ribosomal RNAs genes. The overall base composition of Clarion angelfish is 28.3% for A, 29.3% for C, 16.5% for G, 25.9% for T and show 85% identities to flame angelfish Centropyge loriculus. The complete mitogenome of the Clarion angelfish provides essential and important DNA molecular data for further phylogeography and evolutionary analysis for marine angelfish phylogeny.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods
  17. Evaluation of Y chromosomal SNP haplogrouping in the HID-Ion AmpliSeq™ Identity Panel
    Ochiai E, Minaguchi K, Nambiar P, Kakimoto Y, Satoh F, Nakatome M, et al.
    Leg Med (Tokyo), 2016 Sep;22:58-61.
    PMID: 27591541 DOI: 10.1016/j.legalmed.2016.08.001
    The Y chromosomal haplogroup determined from single nucleotide polymorphism (SNP) combinations is a valuable genetic marker to study ancestral male lineage and ethical distribution. Next-generation sequencing has been developed for widely diverse genetics fields. For this study, we demonstrate 34 Y-SNP typing employing the Ion PGM™ system to perform haplogrouping. DNA libraries were constructed using the HID-Ion AmpliSeq™ Identity Panel. Emulsion PCR was performed, then DNA sequences were analyzed on the Ion 314 and 316 Chip Kit v2. Some difficulties became apparent during the analytic processes. No-call was reported at rs2032599 and M479 in six samples, in which the least coverage was observed at M479. A minor misreading occurred at rs2032631 and M479. A real time PCR experiment using other pairs of oligonucleotide primers showed that these events might result from the flanking sequence. Finally, Y haplogroup was determined completely for 81 unrelated males including Japanese (n=59) and Malay (n=22) subjects. The allelic divergence differed between the two populations. In comparison with the conventional Sanger method, next-generation sequencing provides a comprehensive SNP analysis with convenient procedures, but further system improvement is necessary.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods
  18. Fulltext A generic assay for whole-genome amplification and deep sequencing of enterovirus A71
    Tan le V, Tuyen NT, Thanh TT, Ngan TT, Van HM, Sabanathan S, et al.
    J Virol Methods, 2015 Apr;215-216:30-6.
    PMID: 25704598 DOI: 10.1016/j.jviromet.2015.02.011
    Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  19. Fulltext Next-Generation Biomarkers in Multiple Myeloma: Understanding the Molecular Basis for Potential Use in Diagnosis and Prognosis
    Soliman AM, Das S, Teoh SL
    Int J Mol Sci, 2021 Jul 13;22(14).
    PMID: 34299097 DOI: 10.3390/ijms22147470
    Multiple myeloma (MM) is considered to be the second most common blood malignancy and it is characterized by abnormal proliferation and an accumulation of malignant plasma cells in the bone marrow. Although the currently utilized markers in the diagnosis and assessment of MM are showing promising results, the incidence and mortality rate of the disease are still high. Therefore, exploring and developing better diagnostic or prognostic biomarkers have drawn global interest. In the present review, we highlight some of the recently reported and investigated novel biomarkers that have great potentials as diagnostic and/or prognostic tools in MM. These biomarkers include angiogenic markers, miRNAs as well as proteomic and immunological biomarkers. Moreover, we present some of the advanced methodologies that could be utilized in the early and competent diagnosis of MM. The present review also focuses on understanding the molecular concepts and pathways involved in these biomarkers in order to validate and efficiently utilize them. The present review may also help in identifying areas of improvement for better diagnosis and superior outcomes of MM.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
  20. Fulltext Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation
    Lim CC, Choong YS, Lim TS
    Int J Mol Sci, 2019 Apr 15;20(8).
    PMID: 30991723 DOI: 10.3390/ijms20081861
    Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some 'fine tuning' may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.
    Matched MeSH terms: High-Throughput Nucleotide Sequencing/methods*
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