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PubMed2006

Global transcriptional profiling of the toxic dinoflagellate Alexandrium fundyense using Massively Parallel Signature Sequencing.

BACKGROUND: Dinoflagellates are one of the most important classes of marine and freshwater algae, notable both for their functional diversity and ecological significance. They occur naturally as free-living cells, as endosymbionts of marine invertebrates and are well known for their involvement in "red tides". Dinoflagellates are also notable for their unusual genome content and structure, which suggests that the organization and regulation of dinoflagellate genes may be very different from that of most eukaryotes. To investigate the content and regulation of the dinoflagellate genome, we performed a global analysis of the transcriptome of the toxic dinoflagellate Alexandrium fundyense under nitrate- and phosphate-limited conditions using Massively Parallel Signature Sequencing (MPSS). RESULTS: Data from the two MPSS libraries showed that the number of unique signatures found in A. fundyense cells is similar to that of humans and Arabidopsis thaliana, two eukaryotes that have been extensively analyzed using this method. The general distribution, abundance and expression patterns of the A. fundyense signatures were also quite similar to other eukaryotes, and at least 10% of the A. fundyense signatures were differentially expressed between the two conditions. RACE amplification and sequencing of a subset of signatures showed that multiple signatures arose from sequence variants of a single gene. Single signatures also mapped to different sequence variants of the same gene. CONCLUSION: The MPSS data presented here provide a quantitative view of the transcriptome and its regulation in these unusual single-celled eukaryotes. The observed signature abundance and distribution in Alexandrium is similar to that of other eukaryotes that have been analyzed using MPSS. Results of signature mapping via RACE indicate that many signatures result from sequence variants of individual genes. These data add to the growing body of evidence for widespread gene duplication in dinoflagellates, which would contribute to the transcriptional complexity of these organisms. The MPSS data also demonstrate that a significant number of dinoflagellate mRNAs are transcriptionally regulated, indicating that dinoflagellates commonly employ transcriptional gene regulation along with the post-transcriptional regulation that has been well documented in these organisms.

Animals↗
PubMed2026

Genetic Identification of Burned Human Remains: A Systematic Review.

Background/Objectives: DNA-based identification of degraded human remains represents a major challenge in forensic science, particularly in cases involving burned, fragmented, or commingled bodies. Advances in forensic genetics have expanded the analytical capabilities for such samples; however, the effectiveness of different approaches and their integration within Disaster Victim Identification (DVI) workflows remain heterogeneous. This systematic review aims to critically evaluate current evidence on DNA-based identification of degraded remains, focusing on methodological strategies, emerging genomic technologies, and DVI applications, while integrating laboratory evidence and operational forensic practice into a structured analytical framework. Methods: A systematic literature search was conducted in Scopus and Web of Science from database inception to 5 June 2026, following PRISMA 2020 guidelines. Eligible studies included original research addressing DNA analysis of degraded, thermally altered, or highly compromised human remains in forensic or DVI contexts. After a multistep screening process involving title/abstract and full-text evaluation, 37 studies were included. Data were extracted and organized into three thematic categories: (i) core DNA analysis, (ii) advanced molecular technologies, and (iii) DVI case applications. Results: The findings demonstrate that DNA recovery from degraded remains is influenced by thermal exposure, tissue type, and sampling strategy. Teeth and dense cortical bone consistently provide higher DNA yield. While autosomal STR profiling remains the primary analytical approach, its limitations in highly degraded samples are mitigated through the complementary use of mitochondrial DNA (mtDNA), Y-chromosome STRs (Y-STRs), and SNP markers, together with advanced sequencing technologies such as massively parallel sequencing (MPS). Emerging technologies, including rapid DNA systems and predictive models based on macroscopic indicators, significantly enhance efficiency and success rates. DVI studies report identification rates exceeding 90-95% when multidisciplinary and structured workflows are applied. The evidence further supports a flexible triage-based analytical strategy, in which marker selection is guided by tissue preservation and degradation level. Conclusions: DNA-based identification of degraded human remains has evolved into an adaptive, multi-level forensic process. Successful outcomes rely on the integration of optimized sampling, hierarchical genetic analysis, and coordinated DVI strategies. The findings support a triage-based framework that links tissue selection, degradation assessment, and analytical methodology to maximize identification success. Future developments should focus on predictive models, advanced genomic tools, and standardized workflows to further improve identification in challenging forensic scenarios.

Humans↗
PubMed2000

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

Cell Line↗
PubMed2026

Probability of Mitochondrial DNA heteroplasmy in different tissues from European populations.

Mitochondrial DNA (mtDNA) heteroplasmy complicates genetic analyses due to its variability across individuals and tissues. We analyzed over 400 Spanish blood samples and integrated published Massively Parallel Sequencing (MPS) data from ten additional European tissues. Heteroplasmy was tissue-specific, with skeletal muscle, kidney, and liver showing the highest levels, while the intestines, skin, and cerebellum had the lowest. Blood uniquely displayed more heteroplasmies in coding than non-coding regions. Several conserved positions not previously described as hotspots showed high frequencies. These results establish the first comprehensive tissue-specific heteroplasmic profile of the complete mitochondrial genome in a European population, improving the interpretation of mtDNA variation in forensic and biomedical contexts.

Humans↗
PubMed2024

Sequencing the orthologs of human autosomal forensic short tandem repeats provides individual- and species-level identification in African great apes.

BACKGROUND: Great apes are a global conservation concern, with anthropogenic pressures threatening their survival. Genetic analysis can be used to assess the effects of reduced population sizes and the effectiveness of conservation measures. In humans, autosomal short tandem repeats (aSTRs) are widely used in population genetics and for forensic individual identification and kinship testing. Traditionally, genotyping is length-based via capillary electrophoresis (CE), but there is an increasing move to direct analysis by massively parallel sequencing (MPS). An example is the ForenSeq DNA Signature Prep Kit, which amplifies multiple loci including 27 aSTRs, prior to sequencing via Illumina technology. Here we assess the applicability of this human-based kit in African great apes. We ask whether cross-species genotyping of the orthologs of these loci can provide both individual and (sub)species identification. RESULTS: The ForenSeq kit was used to amplify and sequence aSTRs in 52 individuals (14 chimpanzees; 4 bonobos; 16 western lowland, 6 eastern lowland, and 12 mountain gorillas). The orthologs of 24/27 human aSTRs amplified across species, and a core set of thirteen loci could be genotyped in all individuals. Genotypes were individually and (sub)species identifying. Both allelic diversity and the power to discriminate (sub)species were greater when considering STR sequences rather than allele lengths. Comparing human and African great-ape STR sequences with an orangutan outgroup showed general conservation of repeat types and allele size ranges. Variation in repeat array structures and a weak relationship with the known phylogeny suggests stochastic origins of mutations giving rise to diverse imperfect repeat arrays. Interruptions within long repeat arrays in African great apes do not appear to reduce allelic diversity. CONCLUSIONS: Orthologs of most human aSTRs in the ForenSeq DNA Signature Prep Kit can be analysed in African great apes. Primer redesign would reduce observed variability in amplification across some loci. MPS of the orthologs of human loci provides better resolution for both individual and (sub)species identification in great apes than standard CE-based approaches, and has the further advantage that there is no need to limit the number and size ranges of analysed loci.

Animals↗
PubMed2006

Patterns of nucleotide misincorporations during enzymatic amplification and direct large-scale sequencing of ancient DNA.

Whereas evolutionary inferences derived from present-day DNA sequences are by necessity indirect, ancient DNA sequences provide a direct view of past genetic variants. However, base lesions that accumulate in DNA over time may cause nucleotide misincorporations when ancient DNA sequences are replicated. By repeated amplifications of mitochondrial DNA sequences from a large number of ancient wolf remains, we show that C/G-to-T/A transitions are the predominant type of such misincorporations. Using a massively parallel sequencing method that allows large numbers of single DNA strands to be sequenced, we show that modifications of C, as well as to a lesser extent of G, residues cause such misincorporations. Experiments where oligonucleotides containing modified bases are used as templates in amplification reactions suggest that both of these types of misincorporations can be caused by deamination of the template bases. New DNA sequencing methods in conjunction with knowledge of misincorporation processes have now, in principle, opened the way for the determination of complete genomes from organisms that became extinct during and after the last glaciation.

Animals↗
PubMed2006

Analysis of the prostate cancer cell line LNCaP transcriptome using a sequencing-by-synthesis approach.

BACKGROUND: High throughput sequencing-by-synthesis is an emerging technology that allows the rapid production of millions of bases of data. Although the sequence reads are short, they can readily be used for re-sequencing. By re-sequencing the mRNA products of a cell, one may rapidly discover polymorphisms and splice variants particular to that cell. RESULTS: We present the utility of massively parallel sequencing by synthesis for profiling the transcriptome of a human prostate cancer cell-line, LNCaP, that has been treated with the synthetic androgen, R1881. Through the generation of approximately 20 megabases (MB) of EST data, we detect transcription from over 10,000 gene loci, 25 previously undescribed alternative splicing events involving known exons, and over 1,500 high quality single nucleotide discrepancies with the reference human sequence. Further, we map nearly 10,000 ESTs to positions on the genome where no transcription is currently predicted to occur. We also characterize various obstacles with using sequencing by synthesis for transcriptome analysis and propose solutions to these problems. CONCLUSION: The use of high-throughput sequencing-by-synthesis methods for transcript profiling allows the specific and sensitive detection of many of a cell's transcripts, and also allows the discovery of high quality base discrepancies, and alternative splice variants. Thus, this technology may provide an effective means of understanding various disease states, discovering novel targets for disease treatment, and discovery of novel transcripts.

Adenocarcinoma↗
PubMed2013

Zfp423 binds autoregulatory sites in p19 cell culture model.

Zfp423 is a 30 zinc finger transcription factor that forms regulatory complexes with EBF family members and factors targeted by canonical signaling pathways. Zfp423 mutations produce a range of developmental abnormalities in mice and humans related to the ciliopathies. Surprisingly, computational analysis of clustered Zfp423 and partner motifs in conserved genomic sequences predicts enrichment in Zfp423 and Ebf genes. In cell culture models selected for Zfp423 and EBF expression, we identify strong and reproducible occupancy of two Zfp423 intronic sites using chromatin immunoprecipitation with multiple independent antibodies. Both sites are significantly enriched in either quantitative PCR or massively parallel sequencing assays. A site in intron 5 acts as a classical enhancer in transient assays, but does not require the consensus motif for activity, suggesting a redundant or modulatory role for Zfp423 binding in this context. We speculate that Zfp423 may repress this enhancer as part of a developmental ratchet.

Animals↗
PubMed2005

Detection of oncogenic mutations in the EGFR gene in lung adenocarcinoma with differential sensitivity to EGFR tyrosine kinase inhibitors.

The complete sequencing of the human genome and the development of molecularly targeted cancer therapy have promoted efforts to identify systematically the genetic alterations in human cancer. By high-throughput sequencing of tyrosine kinase genes in human non-small-cell lung cancer, we identified somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene (EGFR) that are correlated with clinical response to EGFR tyrosine kinase inhibitors (TKIs). We have shown that these mutant forms of EGFR induce oncogenic transformation in different cellular systems. Cells whose growth depends on EGFR with mutations in exons 19 and 21 are sensitive to EGFR-TKIs, whereas cells expressing insertion mutations in exon 20 or the T790M point mutant, found in tumor biopsies from patients that relapsed after an initial response to EGFR-TKIs, are resistant. Furthermore, by applying a novel, massively parallel sequencing technology, we have shown that clinically relevant oncogene mutations can be detected in clinical specimens with very low tumor content, thereby enabling optimal patient selection for mutation-directed therapy. In summary, by applying high-throughput genomic resequencing, we have identified a novel therapeutic target, mutant EGFR, in lung cancer and evaluated its role in predicting response to targeted therapy.

Adenocarcinoma↗
PubMed2025

The Use of Next-Generation Sequencing in Personalized Medicine.

The revolutionary progress in development of next-generation sequencing (NGS) technologies has made it possible to deliver accurate genomic information in a timely manner. Over the past several years, NGS has transformed biomedical and clinical research and found its application in the field of personalized medicine. Here we discuss the rise of personalized medicine and the history of NGS. We discuss current applications and uses of NGS in medicine, including infectious diseases, oncology, genomic medicine, and dermatology. We provide a brief discussion of selected studies where NGS was used to respond to wide variety of questions in biomedical research and clinical medicine. Finally, we discuss the challenges of implementing NGS into routine clinical use.

Humans↗
PubMed2006

Diversity of microRNAs in human and chimpanzee brain.

We used massively parallel sequencing to compare the microRNA (miRNA) content of human and chimpanzee brains, and we identified 447 new miRNA genes. Many of the new miRNAs are not conserved beyond primates, indicating their recent origin, and some miRNAs seem species specific, whereas others are expanded in one species through duplication events. These data suggest that evolution of miRNAs is an ongoing process and that along with ancient, highly conserved miRNAs, there are a number of emerging miRNAs.

Animals↗
PubMed2026

Molecular analysis of individuals with suspected 46,XY differences of sex development in a homogenous and understudied population.

Differences of sex development (DSD) are a group of rare congenital conditions defined by atypical chromosomal, gonadal, and/or hormonal sex. Despite advances in massively parallel sequencing (MPS), more than half of DSD cases have an unknown genetic aetiology. We recruited and analysed 21 individuals with 46,XY DSD from the Greater Middle East population using chromosomal microarray and whole exome sequencing. Participants had DSD ranging from micropenis to anorchia (absence of testes) with extra-genital features reported in four individuals (19%). Using a combination of microarray and WES, a genetic diagnosis (variants curated as likely pathogenic or pathogenic) was identified in 12/21 (57%) individuals. Microarray analysis showed two DSD participants with extra genital features had chromosomal abnormalities (48,XXXY and mosaic Y chromosomal rearrangement). Microarray also indicated a high degree of consanguinity, with extensive long contiguous stretches of homozygosity (LCSH) (>3% of the genome) in 6/21 (28.6%) individuals, all of whom received a genetic diagnosis. WES analysis revealed variants in the NR5A1 (three individuals), SRD5A2 (three individuals), TALDO1 (one individual) and AR (two individuals) genes. This includes the novel frameshift variant, c.1309del (p.Leu437Cysfs*59), in NR5A1. This study contributes to the characterisation of clinical features and molecular findings in individuals with DSD in this understudied and homogenous population and highlights the challenges with DSD diagnosis in the region. The genetic diagnoses identified may contribute to improved patient care and management.

Humans↗
PubMed2019

Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE): a cloud-based platform for curating and classifying germline variants.

Variant interpretation in the era of massively parallel sequencing is challenging. Although many resources and guidelines are available to assist with this task, few integrated end-to-end tools exist. Here, we present the Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE), a web- and cloud-based platform for annotation, identification, and classification of variations in known or putative disease genes. Starting from a set of variants in variant call format (VCF), variants are annotated, ranked by putative pathogenicity, and presented for formal classification using a decision-support interface based on published guidelines from the American College of Medical Genetics and Genomics (ACMG). The system can accept files containing millions of variants and handle single-nucleotide variants (SNVs), simple insertions/deletions (indels), multiple-nucleotide variants (MNVs), and complex substitutions. PeCanPIE has been applied to classify variant pathogenicity in cancer predisposition genes in two large-scale investigations involving >4000 pediatric cancer patients and serves as a repository for the expert-reviewed results. PeCanPIE was originally developed for pediatric cancer but can be easily extended for use for nonpediatric cancers and noncancer genetic diseases. Although PeCanPIE's web-based interface was designed to be accessible to non-bioinformaticians, its back-end pipelines may also be run independently on the cloud, facilitating direct integration and broader adoption. PeCanPIE is publicly available and free for research use.

Child↗
PubMed2004

Tumor necrosis factor-like weak inducer of apoptosis-induced neurodegeneration.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) family of cytokines. It has proangiogenic and proinflammatory properties in vivo and induces cell death in tumor cell lines. TWEAK effects are mediated by the membrane receptor Fn14. In a systematic search for genes regulated in a murine stroke model with the tag-sequencing technique massively parallel signature sequencing, we have identified TWEAK as an induced gene. After 24 hr of focal cerebral ischemia in vivo or oxygen glucose deprivation in primary cortical neurons, both TWEAK and its receptor Fn14 were significantly upregulated. TWEAK induced cell death in primary neurons. Transfection of a nuclear factor (NF)-kappaB-luciferase fusion gene demonstrated that TWEAK stimulated transcriptional activity of NF-kappaB through Fn14 and the IkappaB kinase. Inhibition of NF-kappaB reduced TWEAK-stimulated neuronal cell death, suggesting that NF-kappaB mediates TWEAK-induced neurodegeneration at least in part. Intraperitoneal injection of a neutralizing anti-TWEAK antibody significantly reduced the infarct size after 48 hr of permanent cerebral ischemia. In summary, our data show that TWEAK induces neuronal cell death and is involved in neurodegeneration in vivo.

Animals↗
PubMed2024

The use of next-generation sequencing in personalized medicine.

The revolutionary progress in development of next-generation sequencing (NGS) technologies has made it possible to deliver accurate genomic information in a timely manner. Over the past several years, NGS has transformed biomedical and clinical research and found its application in the field of personalized medicine. Here we discuss the rise of personalized medicine and the history of NGS. We discuss current applications and uses of NGS in medicine, including infectious diseases, oncology, genomic medicine, and dermatology. We provide a brief discussion of selected studies where NGS was used to respond to wide variety of questions in biomedical research and clinical medicine. Finally, we discuss the challenges of implementing NGS into routine clinical use.

High-throughput sequencing↗
PubMed2005

Microarray expression profiling resources for plant genomics.

Large volumes of genomic data have been generated for several plant species over the past decade, including structural sequence data and functional annotation at the genome level. Various technologies such as expressed sequence tags (ESTs), massively parallel signature sequencing (MPSS) and microarrays have been used to study gene expression and to provide functional data for many genes simultaneously. This review focuses on recent advances in the application of microarrays in plant genomic research and in gene expression databases available for plants. Large sets of Arabidopsis microarray data are publicly available. Recently developed array platforms are currently being used to generate genome-wide expression profiles for several crop species. Coupled to these platforms are public databases that provide access to these large-scale expression data, which can be used to aid the functional discovery of gene function.

Arabidopsis↗
PubMed2006

Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing.

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.

Base Sequence↗
PubMed2004

Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes.

Summary To overcome the detection limits inherent to DNA array-based methods of transcriptome analysis, we developed a real-time reverse transcription (RT)-PCR-based resource for quantitative measurement of transcripts for 1465 Arabidopsis transcription factors (TFs). Using closely spaced gene-specific primer pairs and SYBR Green to monitor amplification of double-stranded DNA (dsDNA), transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products. The amplification efficiency of each PCR was determined from the log slope of SYBR Green fluorescence versus cycle number in the exponential phase, and was used to correct the readout for each primer pair and run. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001 and 100 copies per cell. Only 13% of TF transcripts were undetectable in these organs. For comparison, 22K Arabidopsis Affymetrix chips detected less than 55% of TF transcripts in the same samples, the range of transcript levels was compressed by a factor more than 100, and the data were less accurate especially in the lower part of the response range. Real-time RT-PCR revealed 35 root-specific and 52 shoot-specific TF genes, most of which have not been identified as organ-specific previously. Finally, many of the TF transcripts detected by RT-PCR are not represented in Arabidopsis EST (expressed sequence tag) or Massively Parallel Signature Sequencing (MPSS) databases. These genes can now be annotated as expressed.

Arabidopsis↗