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Genomics, Proteomics & Bioinformatics Oct 2016The revolution of genome sequencing is continuing after the successful second-generation sequencing (SGS) technology. The third-generation sequencing (TGS) technology,... (Review)
Review
The revolution of genome sequencing is continuing after the successful second-generation sequencing (SGS) technology. The third-generation sequencing (TGS) technology, led by Pacific Biosciences (PacBio), is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that promises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies (ONT). MinION identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MinION has thus generated much excitement and interest in the genomics community. While de novo genome assemblies can be cheaply produced from SGS data, assembly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in genome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.
Topics: Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Nanopores; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA
PubMed: 27646134
DOI: 10.1016/j.gpb.2016.05.004 -
Journal of Clinical Microbiology Dec 2019Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so... (Review)
Review
Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.
Topics: Clinical Laboratory Services; Clinical Laboratory Techniques; Communicable Diseases; High-Throughput Nucleotide Sequencing; Humans; Nanopore Sequencing
PubMed: 31619531
DOI: 10.1128/JCM.01315-19 -
Archiwum Medycyny Sadowej I Kryminologii 2015The DNA analysis is a cornerstone in contemporary forensic sciences. DNA sequencing technologies are powerful tools that enrich molecular sciences in the past based on... (Review)
Review
The DNA analysis is a cornerstone in contemporary forensic sciences. DNA sequencing technologies are powerful tools that enrich molecular sciences in the past based on Sanger sequencing and continue to glowing these sciences based on Next generation sequencing (NGS). Next generation sequencing has excellent potential to flourish and increase the molecular applications in forensic sciences by jumping over the pitfalls of the conventional method of sequencing. The main advantages of NGS compared to conventional method that it utilizes simultaneously a large number of genetic markers with high-resolution of genetic data. These advantages will help in solving several challenges such as mixture analysis and dealing with minute degraded samples. Based on these new technologies, many markers could be examined to get important biological data such as age, geographical origins, tissue type determination, external visible traits and monozygotic twins identification. It also could get data related to microbes, insects, plants and soil which are of great medico-legal importance. Despite the dozens of forensic research involving NGS, there are requirements before using this technology routinely in forensic cases. Thus, there is a great need to more studies that address robustness of these techniques. Therefore, this work highlights the applications of forensic sciences in the era of massively parallel sequencing.
Topics: Forensic Genetics; High-Throughput Nucleotide Sequencing; Humans; Microsatellite Repeats; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Sequence Analysis, DNA
PubMed: 27543959
DOI: 10.5114/amsik.2015.61029 -
Philosophical Transactions of the Royal... Jan 2015The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field's focus was previously limited to mitochondrial DNA and a few nuclear... (Review)
Review
The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field's focus was previously limited to mitochondrial DNA and a few nuclear markers, whole genome sequences from the deep past can now be retrieved. This breakthrough is tightly connected to the massive sequence throughput of next generation sequencing platforms and the ability to target short and degraded DNA molecules. Many ancient specimens previously unsuitable for DNA analyses because of extensive degradation can now successfully be used as source materials. Additionally, the analytical power obtained by increasing the number of sequence reads to billions effectively means that contamination issues that have haunted aDNA research for decades, particularly in human studies, can now be efficiently and confidently quantified. At present, whole genomes have been sequenced from ancient anatomically modern humans, archaic hominins, ancient pathogens and megafaunal species. Those have revealed important functional and phenotypic information, as well as unexpected adaptation, migration and admixture patterns. As such, the field of aDNA has entered the new era of genomics and has provided valuable information when testing specific hypotheses related to the past.
Topics: Animals; DNA; Genomics; High-Throughput Nucleotide Sequencing; History, Ancient; Humans
PubMed: 25487338
DOI: 10.1098/rstb.2013.0387 -
Nature Methods Apr 2017Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost...
Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.
Topics: 3T3 Cells; Animals; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Leukocytes, Mononuclear; Macrophages; Mice; Mycobacterium tuberculosis; RNA, Messenger; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 28192419
DOI: 10.1038/nmeth.4179 -
Genes Jan 2019The adoption of single molecule real-time (SMRT) sequencing [...].
The adoption of single molecule real-time (SMRT) sequencing [...].
Topics: Animals; High-Throughput Nucleotide Sequencing; Humans; Plants; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 30621217
DOI: 10.3390/genes10010024 -
Applied and Environmental Microbiology Sep 2013Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds...
Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
Topics: Animals; Biota; Computational Biology; Feces; High-Throughput Nucleotide Sequencing; Humans; Metagenome; Mice; Soil Microbiology
PubMed: 23793624
DOI: 10.1128/AEM.01043-13 -
GigaScience Aug 2018With the rapid development of next-generation sequencing technology, ever-increasing quantities of genomic data pose a tremendous challenge to data processing.... (Review)
Review
With the rapid development of next-generation sequencing technology, ever-increasing quantities of genomic data pose a tremendous challenge to data processing. Therefore, there is an urgent need for highly scalable and powerful computational systems. Among the state-of-the-art parallel computing platforms, Apache Spark is a fast, general-purpose, in-memory, iterative computing framework for large-scale data processing that ensures high fault tolerance and high scalability by introducing the resilient distributed dataset abstraction. In terms of performance, Spark can be up to 100 times faster in terms of memory access and 10 times faster in terms of disk access than Hadoop. Moreover, it provides advanced application programming interfaces in Java, Scala, Python, and R. It also supports some advanced components, including Spark SQL for structured data processing, MLlib for machine learning, GraphX for computing graphs, and Spark Streaming for stream computing. We surveyed Spark-based applications used in next-generation sequencing and other biological domains, such as epigenetics, phylogeny, and drug discovery. The results of this survey are used to provide a comprehensive guideline allowing bioinformatics researchers to apply Spark in their own fields.
Topics: Animals; Computational Biology; Genomics; High-Throughput Nucleotide Sequencing; Humans; Mice; Software
PubMed: 30101283
DOI: 10.1093/gigascience/giy098 -
Annual Review of Genomics and Human... Aug 2016The term next-generation sequencing is almost a decade old, but it remains the colloquial way to describe highly parallel or high-output sequencing methods that produce... (Review)
Review
The term next-generation sequencing is almost a decade old, but it remains the colloquial way to describe highly parallel or high-output sequencing methods that produce data at or beyond the genome scale. Since the introduction of these technologies, the number of applications and methods that leverage the power of genome-scale sequencing has increased at an exponential pace. This review highlights recent concepts, technologies, and methods from next-generation sequencing to illustrate the breadth and depth of the applications and research areas that are driving progress in genomics.
Topics: Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 27362342
DOI: 10.1146/annurev-genom-083115-022413 -
PLoS Computational Biology Mar 2020Life scientists are increasingly turning to high-throughput sequencing technologies in their research programs, owing to the enormous potential of these methods. In a...
Life scientists are increasingly turning to high-throughput sequencing technologies in their research programs, owing to the enormous potential of these methods. In a parallel manner, the number of core facilities that provide bioinformatics support are also increasing. Notably, the generation of complex large datasets has necessitated the development of bioinformatics support core facilities that aid laboratory scientists with cost-effective and efficient data management, analysis, and interpretation. In this article, we address the challenges-related to communication, good laboratory practice, and data handling-that may be encountered in core support facilities when providing bioinformatics support, drawing on our own experiences working as support bioinformaticians on multidisciplinary research projects. Most importantly, the article proposes a list of guidelines that outline how these challenges can be preemptively avoided and effectively managed to increase the value of outputs to the end user, covering the entire research project lifecycle, including experimental design, data analysis, and management (i.e., sharing and storage). In addition, we highlight the importance of clear and transparent communication, comprehensive preparation, appropriate handling of samples and data using monitoring systems, and the employment of appropriate tools and standard operating procedures to provide effective bioinformatics support.
Topics: Biomedical Research; Communication; Computational Biology; High-Throughput Nucleotide Sequencing; Humans; Research Design
PubMed: 32214318
DOI: 10.1371/journal.pcbi.1007531