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Offering long reads and rapidly improving accuracy, nanopore sequencing has the potential to upend the DNA sequencing market.

Sanger and his colleagues invented the DNA sequencing technology by cleverly applying the function of DNA polymerase to DNA sequence detection in 1977. Human Genome Project (HGP) launched in the early 1990s greatly facilitated the progress of the DNA sequencing technology and prompted the automation of DNA sequencing which conversely accelerate the human genome draft accomplished in 2001. For the past decade, great advancements in the speed and sequencing throughput in the next generation sequencing (NGS) technologies have dramatically reduced the cost of the whole genome sequencing to $1000. Although there is no great improvement in technique for NGS compared with the first generation sequencing in the basic principle of sequencing, the second generation sequencing system processes the quantified sequencing fragments in massive parallel way, which makes sequencing throughput increased significantly and at the same time greatly reduce the cost. Recently, the third generation sequencing technologies emerge with the advantages of very long sequencing fragment and no need for PCR amplification during the sequencing process, which significantly decrease the complex of assembling process and avoid the potential bias and false variations caused by PCR technology. Although the accuracy of the third generation sequencing technologies can only reach about 85% and the price is relatively high, the technologies still show their promising future with the gradual improvement in experiment processes and computational methods. When biomedical researches and clinical applications will benefit from the technology innovation, the enormous applications of NGS in precision medicine and biomedical research fields will further accelerate the progress and innovations in NGS. At the same time, with the vast accumulation of massive sequencing data, how to efficiently analyze and interpret those data becomes a big challenge, bioinformatics plays the key role in the process. Well trained bioinformaticians with good background of mathematics, statistics, computer skills and biology are in high demand. It can be anticipated that the continuous innovations in NGS technology will further significantly decrease the cost for the human genome sequencing and promise the advent of the era with the sequencing cost close to $100, which will push personal genome sequencing widely used in clinical applications and make the precision medicine come true in daily life.

Recent advances in the field of genomics have largely been due to the ability to sequence DNA at increasing throughput and decreasing cost. DNA sequencing was first introduced in 1977, and next-generation sequencing technologies have been available only during the past decade, but the diverse experiments and corresponding analyses facilitated by these techniques have transformed biological and biomedical research. Here, I review developments in DNA sequencing technologies over the past 10 years and look to the future for further applications.

We employed an in vivo assay system of Caenorhabditis elegans to determine if and which microRNAs (miRNAs) were dysregulated upon exposure to coal combustion related fine particulate matter (PM2.5) by profiling the miRNAs using SOLiD sequencing. From this, expression of 25 miRNAs was discovered to become dysregulated by exposure to PM2.5. Using the corresponding C. elegans deletion mutants, 5 miRNAs (mir-231, mir-232, mir-230, mir-251 and mir-35) were found to be involved in the control of PM2.5 toxicity. Furthermore, mutation of mir-231 or mir-232 induced a resistance to PM2.5 toxicity, whereas mutation of mir-230, mir-251, or mir-35 induced a susceptibility to PM2.5 toxicity. SMK-1, an ortholog of the mammalian SMEK protein, was identified as a molecular target for mir-231 in the regulation of PM2.5 toxicity. In addition, the genes of sod-3, sod-4 and ctl-3, which are necessary for protection against oxidative stress, were determined to be important downstream targets of smk-1 in the regulation of PM2.5 toxicity. The triggering of this mir-231-SMK-1-SOD-3/SOD-4/CTL-3 signaling pathway may be a critical molecular basis for the role of oxidative stress in the induction of coal combustion related PM2.5 toxicity.

In this review, we discuss the emergence of the fourth‐generation sequencing technologies that preserve the spatial coordinates of RNA and DNA sequences with up to subcellular resolution, thus enabling back mapping of sequencing reads to the original histological context. This information is used, for example, in two current large‐scale projects that aim to unravel the function of the brain. Also in cancer research, fourth‐generation sequencing has the potential to revolutionize the field. Cancer Research UK has named “Mapping the molecular and cellular tumor microenvironment in order to define new targets for therapy and prognosis” one of the grand challenges in tumor biology. We discuss the advantages of sequencing nucleic acids directly in fixed cells over traditional next‐generation sequencing (NGS) methods, the limitations and challenges that these new methods have to face to become broadly applicable, and the impact that the information generated by the combination of in situ sequencing and NGS methods will have in research and diagnostics.

Nearly after three decades of Sanger sequencing method dominating the field of nucleotide sequencing, the new age of novel sequencing techniques commenced with 454-pyrosequencing in the year 2005 and thus started the era of next generation sequencing techniques. Since then many other novel sequencing techniques with increased accuracy, simplicity and cost-effectiveness have come up and called next to next generation sequencing techniques or third generation sequencing techniques. The scientific knowledge generated from next generation sequencing techniques has transformed the field of structural and functional genomic studies in different crop plants. In this review, various next generation sequencing techniques are described, with their applications and future prospective.

In the present century sequencing is to the DNA science, what gel electrophoresis was to it in the last century. From 1977 to 2016 three generation of the sequencing technologies of various types have been developed. Second and third generation sequencing technologies referred commonly to as next generation sequencing technology, has evolved significantly with increase in sequencing speed, decrease in sequencing cost, since its inception in 2004. GS FLX by 454 Life Sciences/Roche diagnostics, Genome Analyzer, HiSeq, MiSeq and NextSeq by Illumina, Inc., SOLiD by ABI, Ion Torrent by Life Technologies are various type of the sequencing platforms available for second generation sequencing. The platforms available for the third generation sequencing are Helicos™ Genetic Analysis System by SeqLL, LLC, SMRT Sequencing by Pacific Biosciences, Nanopore sequencing by Oxford Nanopore's, Complete Genomics by Beijing Genomics Institute and GnuBIO by BioRad, to name few. The present article is an overview of the principle and the sequencing chemistry of these high throughput sequencing technologies along with brief comparison of various types of sequencing platforms available.

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48–96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5–6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Next generation sequencing transformed the field of evolutionary biology and high throughput sequencing platforms are routinely used in phylogenomic, population genomic or metagenomic studies. Here I review the recent technical advancements of third generation sequencing instruments, thereby covering nanopore sequencing and single molecule real-time (SMRT) sequencing. The output and error rates are compared with sequencing platforms of the second generation (454 pyrosequencing, Illumina and Ion Torrent). Third generation sequencers produce sequence reads in hitherto unprecedented lengths and will help to strongly increase the quality of genome assemblies. Moreover, the speed of sequencing and ease of sample preparation enables sequencing in the field. Even though the output and error rate of the new generation of sequencer remains to be improved, new possibilities for evolutionary research will open up in the near future by these new techniques.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone.

Research in evolutionary biology involving nonmodel organisms is rapidly shifting from using traditional molecular markers such as mtDNA and microsatellites to higher throughput SNP genotyping methodologies to address questions in population genetics, phylogenetics and genetic mapping. Restriction site associated DNA sequencing (RAD sequencing or RADseq) has become an established method for SNP genotyping on Illumina sequencing platforms. Here, we developed a protocol and adapters for double-digest RAD sequencing for Ion Torrent (Life Technologies; Ion Proton, Ion PGM) semiconductor sequencing. We sequenced thirteen genomic libraries of three different nonmodel vertebrate species on Ion Proton with PI chips: Arctic charr Salvelinus alpinus, European whitefish Coregonus lavaretus and common lizard Zootoca vivipara. This resulted in ~962 million single-end reads overall and a mean of ~74 million reads per library. We filtered the genomic data using Stacks, a bioinformatic tool to process RAD sequencing data. On average, we obtained ~11,000 polymorphic loci per library of 6-30 individuals. We validate our new method by technical and biological replication, by reconstructing phylogenetic relationships, and using a hybrid genetic cross to track genomic variants. Finally, we discuss the differences between using the different sequencing platforms in the context of RAD sequencing, assessing possible advantages and disadvantages. We show that our protocol can be used for Ion semiconductor sequencing platforms for the rapid and cost-effective generation of variable and reproducible genetic markers.

Next-generation sequencing (NGS) technologies have provided us with an opportunity to explore genomes with unprecedented detail. Alongside these advancements, NGS has its own challenges (much like all technology) creating a plethora of analytical and biological considerations before these methods can be standardized. Meanwhile, encouraging single-cell sequencing has become a recent trend among scientists who are optimistic that this approach will better characterize cancer (and other similarly complex diseases) [1,2]. The technology necessary to sequence a complete genome from a ‘single’ cell has been available, but now, combined with growing popularity and mainstream acceptance, these methods are soon to become pedestrian tools, among a list of many existing NGS technologies. Will this technique be the missing link to scaffold data between genome sequencing methods, which currently contribute volumes of data but with nominal insight into therapies? To answer these questions we have chosen to review current trends in sequencing technology and relative to novel therapies.

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.

The Illumina HiSeq X platform has helped to reduce the cost of whole genome sequencing substantially, but its application for bisulphite sequencing is not straightforward. We describe the optimization of a library preparation and sequencing approach that maximizes the yield and quality of sequencing, and the elimination of a previously unrecognized artefact affecting several percent of bisulphite sequencing reads.

Droplet-based single-cell sequencing pioneers Allon Klein and Evan Macosko summarise the growth of the technology and look ahead to future developments.

Today's short-read sequencing instruments can generate read lengths between 50 bp and 700 bp depending on the specific instrument. These high-throughput sequencing approaches have revolutionized genomic science, allowing hundreds of thousands of full genomes to be sequenced, and have become indispensable tools for many researchers. With greater insight has come the revelation that many genomes are much more complicated than originally thought and include many rearrangements and copy-number variations. Unfortunately, short-read sequencing technologies are not well suited for identifying many of these types of events. Long-read sequencing technologies can read contiguous fragments of DNA in excess of 10 kb and are much better suited for detecting large structural events. The newest long-read sequencing instrument is the MinION device from Oxford Nanopore. The rapid sequencing speed and low upfront instrument cost are features drawing interest in this device from the genomics community. This unit provides a representative protocol for carrying out human genome sequencing on the Oxford Nanopore MinION. (c) 2017 by John Wiley & Sons, Inc.

The first sequencing of a complete genome was published forty years ago by the double Nobel Prize in Chemistry winner Frederick Sanger. That corresponded to the small sized genome of a bacteriophage, but since then there have been many complex organisms whose DNA have been sequenced. This was possible thanks to continuous advances in the fields of biochemistry and molecular genetics, but also in other areas such as nanotechnology and computing. Nowadays, sequencing sensors based on genetic material have little to do with those used by Sanger. The emergence of mass sequencing sensors, or new generation sequencing (NGS) meant a quantitative leap both in the volume of genetic material that was able to be sequenced in each trial, as well as in the time per run and its cost. One can envisage that incoming technologies, already known as fourth generation sequencing, will continue to cheapen the trials by increasing DNA reading lengths in each run. All of this would be impossible without sensors and detection systems becoming smaller and more precise. This article provides a comprehensive overview on sensors for DNA sequencing developed within the last 40 years.

Improvement of next-generation sequencing (NGS) has revealed novel driver genes and new therapeutic targets for cancer patients. We summarize here the history of NGS development, current situation of typical analysis pipeline utilizing NGS – especially whole exome sequencing (WES) – whole transcriptome sequencing (WTS/RNA-seq), and useful public databases and tools for NGS data analysis. Large-scale sequencing projects such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) have sequenced hundreds of tumor samples of different subtypes to provide catalogues of cancer genome. Chromothripsis, kataegis, and chromoplexy were discovered from those studies using second-generation technologies. Recent novel findings, such as RET gene fusion in lung cancer using these NGS following clinical translation, are also noted here. Clinical sequencing to find personalized treatment strategies for cancer patients has also been done for lung cancer. Mutations of EGFR and ALK are widely applied to screening for administration of EGFR-TKI and crizotinib, respectively. WES – targeting >100s mutation sequencing using NGS – has rapidly become a common way of clinical sequencing. Actionable mutations are used not only as targets of molecular therapy but also as markers for better stratifications in clinical trials.

Sequencing is a process used for determining the array of biopolymers applicable in identifying microbial gene arrangements, phenotypes, evolutionary biology, metagenomics, potential drug targets, gene cloning, etc. Commercial sequencers are emerging all around the globe due to rapid development of recombinant DNA (rDNA) technology. For sequencing DNA, next-generation sequencing (NGS) methods provide faster, inexpensive, accurate sequencing of polymers than traditional approaches. The concept of NGS is not quite new; it started in the mid-late 1990s with the successful introduction of methods thereafter, namely, Roche 454 pyrosequencing, Illumina sequencing, SOLiD sequencing, etc. In this book chapter, we briefly elaborate on the above-stated methods and its advantages and disadvantages. Furthermore, we will be discussing the sequencing methods that are under development in the biological research.

The next generation sequencing technologies represent a major revolution in genome sequencing which has made large scale genome sequencing feasible. Consequently, they are currently sequencing platforms of choice for both research and clinical applications. These technologies use a wide variety of approaches for sequencing to achieve a common end-point which is massive parallel sequencing of the genome. A detailed description of these technologies and the molecular process they employ to sequence the DNA has been discussed here. The approaches for validating these technologies for clinical application and their utility in clinical genomics, drug discovery, and development have also been discussed.

Genome sequencing has become one of the most commonly used methods to understand inherent genomic function. Newer genome sequencing methods developed since 2005 have enabled a new arena in genome analysis. In this chapter, we summarize the “next-generation” methods for genome sequencing. Emphasis is placed on advanced sequencing methods such as massively parallel signature sequencing, polony sequencing, 454 sequencing, Illumina technology, ion torrent technology, SOLiD DNA sequencing technology, and DNA nanoball sequencing.

Next-generation sequencing (NGS) has been extensively used in genomic study and clinical diagnostic arena in the past decade. With the decreasing cost of NGS, family-based sequencing analysis has been increasingly used to identify causal genes for Mendelian disorders and to aid the rare variants association analysis for common complex traits. By incorporating relatedness among family members, several family-based variant calling algorithms have been developed to increase variant calling accuracy, and showed superior performance comparing with naive calling algorithms. Using parent-offspring trio sequencing, numerous causal genes and risk alleles for neuropsychiatric and other disorders have been successfully identified. In addition, family-based NGS can help to increase the statistic power for rare variants association analysis for common complex traits. Family-based NGS is also found to be particularly useful in clinical diagnostic laboratories. Comparing with proband-only sequencing, family-based NGS helps to quickly narrow down variants inheritance, increasing the diagnostic yield and decreasing the turnaround time of clinical tests. In this review, we will first discuss the methodology for family-based NGS analysis, focusing on variant calling, and then review the application of such design in research and clinical settings.

Progress in science depends on new ideas, new discoveries, and new techniques, and often the three are interdependent. Nowhere is this more apparent than in the two techniques, DNA sequencing and polymerase chain reaction (PCR), that were born out of creative ideas and rapidly gave rise to an explosion of fundamental and applied discoveries. Sanger developed the first practical DNA sequencing method. The basic idea of the PCR method is to take a very small amount of DNA and make billions of identical copies of it. The DNA sequencing and PCR methods are carefully explained.

Recently, the requirement for fast, low-cost, and highly reliable methods has triggered the development of next-generation DNA sequencing technologies. In addition to overcoming the restrictions in Sanger sequencing methods, large-scale genetic data have provided the development of different analysis methods. In this paper, widely used next-generation sequencing platforms, the analyses of the data obtained, and the applications of these technologies are discussed.

The molecular basis for in vivo graphene oxide (GO) toxicity is still largely unclear. We here used Caenorhabditis elegans to investigate the microRNAs (miRNAs) control of GO toxicity. With the aid of SOLiD sequencing, we identified 23 up-regulated and 8 down-regulated miRNAs in GO-exposed nematodes. Gene ontology and KEGG pathway database analysis implied that these identified miRNAs might be involved in control of many biological processes, and some of them suggest the possible new functions of GO. Functions of the identified miRNAs in regulating the GO toxicity on lifespan were confirmed in the available miRNAs mutants. Moreover, we provide the evidence to raise a hypothesis that GO may reduce lifespan through influencing the functions of insulin/IGF signaling, TOR signaling, and germline signaling pathways controlled by miRNAs. Our results will be helpful for understanding the molecular basis for GO toxicity, and finding clues for useful surface modifications to reduce GO toxicity. From the Clinical Editor In this study, toxicity of graphene oxide is studied in a Caenorhabditis elegans model via microRNA analysis. The authors report that multiple important pathways are influenced by GO and raise a hypothesis that GO may reduce lifespan through influencing the functions of insulin/IGF signaling, TOR signaling, and germline signaling pathways.

Deregulated microRNAs (miRNAs) have been reported to be of functional relevance for tumor biology. In this study, we analyzed the small RNA transcriptomes of 10 paired colorectal cancer samples using SOLiD next-generation sequencing, and generated a total of >15141000 reads. miRNA expression profiles obtained by SOLiD sequencing correlated well with quantitative PCR results. The results showed that the expression of 36 miRNAs was significantly different between the two groups of samples. Additionally, we explored the global miRNA-mRNA interactions using a statistical model. In-depth analysis reveals a diagram of extensive post-transcriptional miRNA regulations. Signaling pathways associated with the miRNA altered expression signature were identified using gene enrichment analysis. The results suggest that these microRNAs, in the aggregate, regulate signaling pathways, such as MAPK, Wnt and p53 pathways in cancer, which are known to be involved in the transformation of colorectal cancer. This evidence demonstrates that miRNAs can cooperatively regulate a given pathway and play a subtle role by regulating their target genes.

The world has now entered into a new era of genomics because of the continued advancements in the next generation high throughput sequencing technologies, which includes sequencing by synthesis-fluorescent in situ sequencing (FISSEQ), pyrosequencing, sequencing by ligation using polony amplification, supported oligonucleotide detection (SOLiD), sequencing by hybridization along with sequencing by ligation, and nanopore technology. Great impacts of these methods can be seen for solving the genome related problems of plant and animal kingdom that will open the door of a new era of genomics. This may ultimately overcome the Sanger sequencing that ruled for 30 years. NGS is expected to advance and make the drug discovery process more rapid.

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.

Sequencing technologies have placed a wide range of genomic analyses within the capabilities of many laboratories. However, sequencing costs often set limits to the amount of sequences that can be generated and, consequently, the biological outcomes that can be achieved from an experimental design. In this Review, we discuss the issue of sequencing depth in the design of next-generation sequencing experiments. We review current guidelines and precedents on the issue of coverage, as well as their underlying considerations, for four major study designs, which include de novo genome sequencing, genome resequencing, transcriptome sequencing and genomic location analyses (for example, chromatin immunoprecipitation followed by sequencing (ChIP-seq) and chromosome conformation capture (3C)).

Next-generation high throughput sequencing technologies became available at the onset of the 21st century. They provide a highly efficient, rapid, and low cost DNA sequencing platform beyond the reach of the standard and traditional DNA sequencing technologies developed in the late 1970s. They are continually improved to become faster, more efficient and cheaper. They have been used in many fields of biology since 2004. In 2009, next-generation sequencing (NGS) technologies began to be applied to several areas of plant virology including virus/viroid genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. Identification and characterization of known and unknown viruses and/or viroids in infected plants are currently among the most successful applications of these technologies. It is expected that NGS will play very significant roles in many research and non-research areas of plant virology.

Endometrial receptivity is crucial for implantation and establishment of a normal pregnancy. The shift from proliferative to receptive endometrium is still far from being understood. In this paper, ...

Deep sequencing technologies are currently cutting edge, and are opening fascinating opportunities in biomedicine, producing over 100-times more data compared to the conventional capillary sequencers based on the Sanger method. Next-generation sequencing (NGS) is now generally defined as the sequencing technology that, by employing parallel sequencing processes, producing thousands or millions of sequence reads simultaneously. Since the GS20 was released as the first NGS sequencer on the market by 454 Life Sciences, the competition in the development of the new sequencers has become intense. In this review, we describe the current deep sequencing systems and discuss the application of advanced technologies in the field of hepatology.

The development of next-generation sequencing (NGS) technologies has provided novel tools for genome analysis and expression profiling. A high throughput cDNA sequencing method using a bench top next-generation sequencing system, GS Junior, is now available. Here, we used an alternative protocol to the standard method for generating the cDNA library. This protocol can decrease the number of processing steps to manipulate RNA when constructing a cDNA library from an RNA sample, and does not require mRNA isolation from total RNA. Thus it can decrease the risk of RNA degradation and the cost for preparing a cDNA library. Also, the efficiency of sequencing data obtained with this approach is comparable to the standard method as verified by sequencing characteristics and expression levels of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Chain termination cycle sequencing, or "first-generation" DNA sequencing, was developed 3 decades ago but remains one of the most commonly used procedures for diagnostic analyses. Automated capillary-gel electrophoresis genetic analyzers greatly improved the efficiency of sequencing DNA templates between 100 and approximately 1,300 nucleotides long. Cycle sequencing may be completed the same day by using fast protocols for the initial amplification and cycle-sequencing reactions and by utilization of commercial sequence interpretation and analysis software. These changes allowed sequencing to become a routine tool for pathogen identification, discovery, and genotyping.

Nucleic acid sequencing is the mainstay of biological research. There are several generations of DNA sequencing technologies that can be well characterized through their nature and the kind of output they provide. Dideoxy terminator sequencing developed by Sanger dominated for 30 years and was the workhorse used for the Human Genome Project. In 2005 the first 2nd generation sequencer was presented with an output orders of magnitude higher than Sanger sequencing and dramatically decreased cost. We are now at the dawn of 3rd generation with nanopore systems that are being developed for DNA sequencing. Meanwhile the field is also broadening into applications that complement 1st, 2nd and 3rd generation sequencing systems to get high resolution genetic information. The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) consortium funded by the European Commission under FP7 has made great contributions to the development of new nucleic acid analysis methodology.

Retroviral particles are known to package specific host cell components such as RNA molecules in addition to the two copies of the viral RNA genome. The highly sensitive SOLiD sequencing technology was used to determine the cellular miRNA content of human immunodeficiency virus type 1 (HIV-1) particles. We determined the relative concentration of cellular miRNAs in a T cell line and several primary cell subsets before and after HIV-1 infection, and compared those values to the miRNA content of virion particles. A small subset of the cellular miRNAs is dramatically concentrated in the virions up to 115 fold, suggesting a biological function in HIV-1 replication. (C) 2012 Elsevier B.V. All rights reserved

The high cost of library preparation remains a major obstacle to sequencing large numbers of individual genomes. Illumina’s proprietary tagmentation technology allows for rapid and easy preparation of sequencing libraries, but remains prohibitively expensive for many users. Here we propose a modified version of the protocol, which uses Illumina reagents at 1/20th the scale. We show that the scaled-down protocol performs comparably to that of the manufacturer on a non-model insect genome. Surprisingly, the scaled-down protocol also produced 14% fewer PCR duplicates that the full-scale protocol. Since PCR duplicates effectively wasted redundant data, our protocol presented here can help save not just library preparation costs, but sequencing costs as well.

Background Nanopore sequencing provides a rapid, cheap and portable real-time sequencing platform with the potential to revolutionize genomics. However, several applications are limited by relatively high single-read error rates (>10 %), including RNA-seq, haplotype sequencing and 16S sequencing. Results We developed the Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) as a strategy for obtaining long and accurate nanopore reads, starting with low input DNA. Applying INC-Seq for 16S rRNA-based bacterial profiling generated full-length amplicon sequences with a median accuracy >97 %. Conclusions INC-Seq reads enabled accurate species-level classification, identification of species at 0.1 % abundance and robust quantification of relative abundances, providing a cheap and effective approach for pathogen detection and microbiome profiling on the MinION system. Electronic supplementary material The online version of this article (doi:10.1186/s13742-016-0140-7) contains supplementary material, which is available to authorized users.

The Oxford Nanopore MinION sequences DNA by sensing changes in electrical current flow in real-time as molecules traverse nanopores. Optionally, the voltage across specific nanopores can be reversed, ejecting the DNA molecule. This enables “Read Until”, the selection of specific DNA molecules for sequencing. We use dynamic time warping to match reads to reference, selecting regions of small genomes, individual amplicons, or normalization of the amplicon set. This first demonstration of direct selection of specific DNA molecules in real-time enables many novel future applications.

Background The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency. Results We compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits. Conclusion We describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2757-4) contains supplementary material, which is available to authorized users.

Second-generation sequencing technologies transformed the study of microbial transcriptomes. They helped reveal the transcription start sites and antisense transcripts of microbial species, improving the microbial genome annotation. Quantification of genome-wide gene expression levels allowed for functional studies of microbial research. Ever-evolving sequencing technologies are reshaping approaches to studying microbial transcriptomes. Recently, Oxford Nanopore Technologies delivered a sequencing platform called MinION, a third-generation sequencing technology, to the research community. We expect it to be the next sequencing technology that enables breakthroughs in life science fields. The studies of microbial transcriptomes will be no exception. In this paper, we review microbial transcriptomics studies using second- generation sequencing technology. We also discuss the prospect of microbial transcriptomics studies with thirdgeneration sequencing.

Since the completion of the human genome project in 2003, extraordinary progress has been made in genome sequencing technologies, which has led to a decreased cost per megabase and an increase in the number and diversity of sequenced genomes. An astonishing complexity of genome architecture has been revealed, bringing these sequencing technologies to even greater advancements. Some approaches maximize the number of bases sequenced in the least amount of time, generating a wealth of data that can be used to understand increasingly complex phenotypes. Alternatively, other approaches now aim to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions. These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.

Next-generation sequencing (NGS) technologies have revolutionized the biosciences and become invaluable to the discovery of gene function and its involvement in disease conditions. The fast pace of innovation in NGS technologies has enabled the production of huge volumes of sequence data at progressively lower cost. However, the increasing throughput combined with the growing accessibility of these technologies poses significant challenges for downstream data analysis. Here, we provide an overview of NGS methods and key secondary analysis pipelines with a focus on technology provided by Illumina. As a case study, we highlight potential applications in cancer research. © 2016 by John Wiley & Sons, Inc. Keywords: cloud computing; Next-Generation Sequencing (NGS); tumor normal analysis; Whole-Genome Sequencing (WGS); BaseSpace

Recent advances in sequencing technology, coupled with the relatively small genomes of viruses, make routine sequencing of entire genomes in clinical and public health settings increasingly feasible. The first two widely adopted DNA sequencing methodologies described, the chemical cleavage method of Maxam and Gilbert (1) and the chain termination method of Sanger et al., were both published in the 1970s (2, 3). Coincidental to the subject of this chapter, the first full genome to be sequenced by Sanger was that of a virus, albeit a bacteriophage, PhiX 174 (4). The Sanger method proved to be the more durable sequencing technology and, especially after the process was automated in 1996, was the most widely used method for DNA sequencing for more than a decade. Beginning in 2005, however, advances in sequencing technology, the so-called next generation sequencing (NGS) methodologies, resulted in a dramatic increase in the amount of sequence that can be generated and a concomitant dramatic decrease in the cost of sequencing. These factors have led to the widespread implementation of NGS in place of the Sanger method for typical sequencing applications and also for some novel purposes, for example, replacing microarrays to study gene expression. The increased use of NGS technologies has, not surprisingly, resulted in a rapid increase in the number of sequences submitted to the National Center for Biotechnology Information's (NCBI) Genbank database. In particular, the number of viral sequences submitted since 2012 has increased 22.9% as measured in nucleotide base pairs (5), and the number of publications based on NGS is increasing at an impressively rapid pace (6).

Third-generation long-range DNA sequencing and mapping technologies are creating a renaissance in high-quality genome sequencing. Unlike second-generation sequencing, which produces short reads a few hundred base-pairs long, third-generation single-molecule technologies generate over 10,000 bp reads or map over 100,000 bp molecules. We analyze how increased read lengths can be used to address longstanding problems in de novo genome assembly, structural variation analysis and haplotype phasing.

The full arrival and broader availability of next-generation sequencing (NGS) is transforming the practice of medicine, including neurology. Compared with the traditional one-gene-at-a-time Sanger sequencing, NGS, or massively parallel sequencing, is a radically different approach to genetic sequencing. NGS allows for a large number of genes to be captured and sequenced in parallel, creating an enormous amount of data in a relatively short period of time at much lower cost "per gene."

Background Illumina’s sequencing platforms are currently the most utilised sequencing systems worldwide. The technology has rapidly evolved over recent years and provides high throughput at low costs with increasing read-lengths and true paired-end reads. However, data from any sequencing technology contains noise and our understanding of the peculiarities and sequencing errors encountered in Illumina data has lagged behind this rapid development. Results We conducted a systematic investigation of errors and biases in Illumina data based on the largest collection of in vitro metagenomic data sets to date. We evaluated the Genome Analyzer II, HiSeq and MiSeq and tested state-of-the-art low input library preparation methods. Analysing in vitro metagenomic sequencing data allowed us to determine biases directly associated with the actual sequencing process. The position- and nucleotide-specific analysis revealed a substantial bias related to motifs (3mers preceding errors) ending in “GG”. On average the top three motifs were linked to 16 % of all substitution errors. Furthermore, a preferential incorporation of ddGTPs was recorded. We hypothesise that all of these biases are related to the engineered polymerase and ddNTPs which are intrinsic to any sequencing-by-synthesis method. We show that quality-score-based error removal strategies can on average remove 69 % of the substitution errors - however, the motif-bias remains. Conclusion Single-nucleotide polymorphism changes in bacterial genomes can cause significant changes in phenotype, including antibiotic resistance and virulence, detecting them within metagenomes is therefore vital. Current error removal techniques are not designed to target the peculiarities encountered in Illumina sequencing data and other sequencing-by-synthesis methods, causing biases to persist and potentially affect any conclusions drawn from the data. In order to develop effective diagnostic and therapeutic approaches we need to be able to identify systematic sequencing errors and distinguish these errors from true genetic variation. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-0976-y) contains supplementary material, which is available to authorized users.