Plant genome sequencing: applications for crop improvement

DNA sequencing technology is undergoing a revolution with the commercialization of second generation technologies capable of sequencing thousands of millions of nucleotide bases in each run. The data explosion resulting from this technology is likely to continue to increase with the further development of second generation sequencing and the introduction of third generation single‐molecule sequencing methods over the coming years. The question is no longer whether we can sequence crop genomes which are often large and complex, but how soon can we sequence them? Even cereal genomes such as wheat and barley which were once considered intractable are coming under the spotlight of the new sequencing technologies and an array of new projects and approaches are being established. The increasing availability of DNA sequence information enables the discovery of genes and molecular markers associated with diverse agronomic traits creating new opportunities for crop improvement. However, the challenge remains to convert this mass of data into knowledge that can be applied in crop breeding programs.     

Environmental biotechnology in the postgenomics era

The sequencing of the human genome and many microbial genomes has provided new opportunities to study the environmental impact on life processes, leading to development of new technologies that can be protected by patenting. Development of such new technologies has, however, led in some cases to judicial intervention because of their controversial nature. This article illustrates some of the trends in postgenomics biotechnology development and the attendant legal and ethical considerations.     

Utilizing field collected insects for next generation sequencing: Effects of sampling,storage, and DNA extraction methods

DNA sequencing technologies continue to advance the biological sciences, expanding opportunities for genomic studies of non‐model organisms for basic and applied questions. Despite these opportunities, many next generation sequencing protocols have been developed assuming a substantial quantity of high molecular weight DNA (>100 ng), which can be difficult to obtain for many study systems. In particular, the ability to sequence field‐collected specimens that exhibit varying levels of DNA degradation remains largely unexplored. In this study we investigate the influence of five traditional insect capture and curation methods on Double‐Digest Restriction Enzyme Associated DNA (ddRAD) sequencing success for three wild bee species. We sequenced a total of 105 specimens (between 7–13 specimens per species and treatment). We additionally investigated how different DNA quality metrics (including pre‐sequence concentration and contamination) predicted downstream sequencing success, and also compared two DNA extraction methods. We report successful library preparation for all specimens, with all treatments and extraction methods producing enough highly reliable loci for population genetic analyses. Although results varied between species, we found that specimens collected by net sampling directly into 100% EtOH, or by passive trapping followed by 100% EtOH storage before pinning tended to produce higher quality ddRAD assemblies, likely as a result of rapid specimen desiccation. Surprisingly, we found that specimens preserved in propylene glycol during field sampling exhibited lower‐quality assemblies. We provide recommendations for each treatment, extraction method, and DNA quality assessment, and further encourage researchers to consider utilizing a wider variety of specimens for genomic analyses.     

Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: a multidisciplinary development unifying molecular biology, chemical and electronics engineering

The current status and research trends of detection techniques for DNA-based analysis such as DNA finger printing, sequencing, biochips and allied fields are examined. An overview of main detectors is presented vis-à-vis these DNA operations. The biochip method is explained, the role of micro- and nanoelectronic technologies in biochip realization is highlighted, various optical and electrical detection principles employed in biochips are indicated, and the operational mechanisms of these detection devices are described. Although a diversity of biochips for diagnostic and therapeutic applications has been demonstrated in research laboratories worldwide, only some of these chips have entered the clinical market, and more chips are awaiting commercialization. The necessity of tagging is eliminated in refractive-index change based devices, but the basic flaw of indirect nature of most detection methodologies can only be overcome by generic and/or reagentless DNA sensors such as the conductance-based approach and the DNA-single electron transistor (DNA-SET) structure. Devices of the electrical detection-based category are expected to pave the pathway for the next-generation DNA chips. The review provides a comprehensive coverage of the detection technologies for DNA finger printing, sequencing and related techniques, encompassing a variety of methods from the primitive art to the state-of-the-art scenario as well as promising methods for the future.     

Comparing Apples and Oranges?: Next Generation Sequencing and Its Impact on Microbiome Analysis

Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number of methodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance in microbiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced with MiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10 million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.     

Can a tablet device alter undergraduate science students' study behavior and use of technology?

This article reports findings from a study investigating undergraduate biological sciences students' use of technology and computer devices for learning and the effect of providing students with a tablet device. A controlled study was conducted to collect quantitative and qualitative data on the impact of a tablet device on students' use of devices and technology for learning. Overall, we found that students made extensive use of the tablet device for learning, using it in preference to laptop computers to retrieve information, record lectures, and access learning resources. In line with other studies, we found that undergraduate students only use familiar Web 2.0 technologies and that the tablet device did not alter this behavior for the majority of tools. We conclude that undergraduate science students can make extensive use of a tablet device to enhance their learning opportunities without institutions changing their teaching methods or computer systems, but that institutional intervention may be needed to drive changes in student behavior toward the use of novel Web 2.0 technologies.     

DNA测序技术概述

DNA测序技术作为现代生命科学研究的核心技术之一,自上世纪70年代中期DNA发明以来发展迅速。我们简要综述现有的几代DNA测序技术的原理及其发展历程,并对未来可能出现的第三代测序进行预测。     

纳米孔测序信号处理及其在DNA数据存储的应用

随着高通量测序技术的不断更新,可以在单个分子水平读取核苷酸序列的第三代测序技术迅速发展,纳米孔测序技术是其具有代表性的单分子测序技术,该技术通过检测DNA单链分子穿过纳米孔时引起的跨膜电流信号的变化,实现碱基识别。纳米孔测序仪在便携性、碱基读取速度、测序读段长度等方面较传统的第一代与第二代测序技术都有明显优势。随着纳米孔测序技术的不断发展成熟,与其配套的各种信号处理与生物信息处理工具也迅速涌现,碱基识别和模型仿真是其中两个较为关键的研究方向。首先介绍纳米孔测序基本原理与信号处理流程,探讨其目前面临的挑战,归纳近年来在碱基识别与纳米孔模型仿真两个方面的主要进展与发展趋势,并用实测数据比较了不同碱基识别方法的性能。继而搭建了纳米孔测序集成仿真平台,为信号处理算法的评估提供支撑。进一步,随着全球数据量的爆发式增长,DNA数据存储正成为未来非常有潜力的海量数据存储方式,采用纳米孔测序读出是一种非常有效的方法。总结了纳米孔测序技术在DNA数据存储中的应用进展,分析了其可行性。分析了基于纳米孔测序实现的人工染色体数据存储的快速读出方法,探讨了与实际测序数据结合的纳米孔测序读段仿真在DNA数据存储中的应用,为开发适合DNA数据存储的方案提供参考。     

Optimal pooling for genome re-sequencing with ultra-high-throughput short-read technologies

New generation sequencing technologies offer unique opportunities and challenges for re-sequencing studies. In this article, we focus on re-sequencing experiments using the Solexa technology, based on bacterial artificial chromosome (BAC) clones, and address an experimental design problem. In these specific experiments, approximate coordinates of the BACs on a reference genome are known, and fine-scale differences between the BAC sequences and the reference are of interest. The high-throughput characteristics of the sequencing technology makes it possible to multiplex BAC sequencing experiments by pooling BACs for a cost-effective operation. However, the way BACs are pooled in such re-sequencing experiments has an effect on the downstream analysis of the generated data, mostly due to subsequences common to multiple BACs. The experimental design strategy we develop in this article offers combinatorial solutions based on approximation algorithms for the well-known max n-cut problem and the related max n-section problem on hypergraphs. Our algorithms, when applied to a number of sample cases give more than a 2-fold performance improvement over random partitioning.     

Next-generation sequencing technologies for environmental DNA research

Since 2005, advances in next-generation sequencing technologies have revolutionized biological science. The analysis of environmental DNA through the use of specific gene markers such as species-specific DNA barcodes has been a key application of next-generation sequencing technologies in ecological and environmental research. Access to parallel, massive amounts of sequencing data, as well as subsequent improvements in read length and throughput of different sequencing platforms, is leading to a better representation of sample diversity at a reasonable cost. New technologies are being developed rapidly and have the potential to dramatically accelerate ecological and environmental research. The fast pace of development and improvements in next-generation sequencing technologies can reflect on broader and more robust applications in environmental DNA research. Here, we review the advantages and limitations of current next-generation sequencing technologies in regard to their application for environmental DNA analysis.     

Reconstruction of Microbial Haplotypes by Integration of Statistical and Physical Linkage in Scaffolding

DNA sequencing technologies provide unprecedented opportunities to analyze within-host evolution of microorganism populations. Often, within-host populations are analyzed via pooled sequencing of the population, which contains multiple individuals or “haplotypes.” However, current next-generation sequencing instruments, in conjunction with single-molecule barcoded linked-reads, cannot distinguish long haplotypes directly. Computational reconstruction of haplotypes from pooled sequencing has been attempted in virology, bacterial genomics, metagenomics, and human genetics, using algorithms based on either cross-host genetic sharing or within-host genomic reads. Here, we describe PoolHapX, a flexible computational approach that integrates information from both genetic sharing and genomic sequencing. We demonstrated that PoolHapX outperforms state-of-the-art tools tailored to specific organismal systems, and is robust to within-host evolution. Importantly, together with barcoded linked-reads, PoolHapX can infer whole-chromosome-scale haplotypes from 50 pools each containing 12 different haplotypes. By analyzing real data, we uncovered dynamic variations in the evolutionary processes of within-patient HIV populations previously unobserved in single position-based analysis.     

Toward next-generation sequencing of mitochondrial genomes — Focus on parasitic worms of animals and biotechnological implications

Helminths (worms) include parasitic nematodes (roundworms) and platyhelminths (flatworms). These worms are abundant, and many of them are of agricultural, aquacultural, veterinary and medical importance and cause substantial socioeconomic losses worldwide. The genetic characterization of parasitic nematodes using advanced molecular tools is central to the diagnosis of infections and the control of parasitism. The accurate analysis of genetic variation also underpins studies of their taxonomy, epidemiology and evolutionary history. Although the nuclear genome contains suitable genetic markers (e.g., in ribosomal DNA) for the identification of many species, the large size and high variability of the mt genome consistently provides a rich source of such markers for informative systematic and epidemiological studies both within and among species. There is significant value in establishing a practical platform for the rapid sequencing, annotation and analysis of mt genomic datasets to underpin such fundamental and applied studies of parasitic worms (= helminths). In the last decade, there have been some important advances in the mt genomics of helminths, but next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation. In this article, we provide a background on mt genomics, cover technological challenges and recent advances, and provide a perspective on future mt genome research of parasitic helminths and its fundamental scientific and biotechnological implications.     

Optical mapping of DNA: single-molecule-based methods for mapping genomes

The technologies associated with DNA sequencing are rapidly evolving. Indeed, single-molecule DNA sequencing strategies are cheaper and faster than ever before. Despite this progress, every sequencing platform to date relies on reading the genome in small, abstract fragments, typically of less than 1000 bases in length. The overarching aim of the optical map is to complement the information derived from DNA sequencing by providing long-range context on which these short sequence reads can be built. This is typically done using an enzyme to target and modify at short DNA sequences of, say, six bases in length throughout the genome. By accurately placing these short pieces of sequence on long genomic DNA fragments, up to several millions of bases in length, a scaffold for sequence assembly can be obtained. This review focuses on three enzymatic approaches to optical mapping. Optical mapping was first developed using restriction enzymes to sequence-specifically cleave DNA that is immobilized on a surface. More recently, nicking enzymes have found application in the sequence-specific fluorescent labeling of DNA for optical mapping. Such covalent modification allows the DNA to be imaged in solution, and this, in combination with developing nanofluidic technologies, is enabling new high-throughput approaches to mapping. And, finally, this review will discuss the recent development of mapping with subdiffraction-limit precision using methyltransferase enzymes to label the DNA with an ultrahigh density.     

Nanopore sequencing technology: research trends and applications

Nanopore sequencing is one of the most promising technologies being developed as a cheap and fast alternative to the conventional Sanger sequencing method. Protein or synthetic nanopores have been used to detect DNA or RNA molecules. Although none of the technologies to date has shown single-base resolution for de novo DNA sequencing, there have been several reports of alpha-hemolysin protein nanopores being used for basic DNA analyses, and various synthetic nanopores have been fabricated. This review will examine current nanopore sequencing technologies, including recent developments of new applications.     

新一代测序技术的研究进展

大规模DNA测序技术是揭秘人类和其它生物遗传密码的重要技术,在分子生物学和基础医学领域有广泛应用。第二代测序技术的出现使DNA测序的通量大幅提高,测序的成本大幅下降,原来只有在大型测序中心才能完成的测序任务现在已经可以在更多的实验室展开。但是,早期的第二代测序技术仍然存在诸如文库构建过程复杂、测序成本依然较高等缺点。为了克服上述缺点,近三年发展了几种新的第二代和第三代测序技术,这些技术不仅继承了早期第二代测序技术通量高的优点,而且在文库构建等方面取得了重要突破,进一步简化了测序操作,降低了测序成本,缩短了测序时间。本文就几种最新的大规模测序技术的原理、特点与发展趋势进行简要介绍。     

高通量测序技术及其在表观遗传学上的应用

DNA测序技术是现代生命科学研究的重要工具之一,而高通量测序技术在全基因组的研究中发挥着越来越重要的作用。简要回溯DNA测序技术的产生与发展,着重从PCR扩增测序和单分子测序两个方面全面描述了高通量测序中众多代表性的技术及直接测序技术,并从DNA甲基化、组蛋白修饰、非编码RNA调控等方面阐述了高通量测序技术在表观遗传学上的运用。