单细胞全基因组扩增技术与应用

同一组织中的细胞往往具有类似的结构和功能,然而通过对单个细胞进行测序分析后,发现每个细胞都具有一定异质性.单细胞全基因组扩增技术是进行单细胞测序的前提,该技术可用于揭示单细胞基因组结构差异,同时在肿瘤研究、发育生物学、微生物学等研究中发挥重要作用,并成为生命科学研究技术的热点之一.单细胞全基因组扩增技术的难点在于单细胞的分离和全基因组的扩增.本文介绍了单细胞全基因组扩增技术中常用的单细胞分离技术和单细胞全基因组扩增技术,并对各技术间的优缺点进行比较,同时着重讨论该技术在肿瘤研究、发育生物学和微生物学研究中的应用.     

未培养微生物研究:方法、机遇与挑战

自然界中绝大部分的微生物仍是未培养的,称之为未培养微生物或微生物"暗物质"。对其进行研究不仅有助于认识微生物多样性及其代谢特征,加深对环境中微生物参与的生态学过程的理解,还有利于重构生命之树,揭示微生物的进化历程,具有重要的科学意义。同时未培养微生物是发现新基因资源和新活性物质的巨大宝库。随着现代分子生物学研究方法和培养技术的成熟和完善,从环境中直接破译未培养微生物的遗传信息,并实现培养逐渐成为可能。本文主要介绍了基于宏基因组技术和单细胞基因组技术或两者结合运用,研究环境中未培养微生物的主要方法和挑战,总结分析了目前已经解析的未培养微生物的主要类群,并对未来研究的机遇进行了展望。     

环境与工业微生物基因组学研究进展

环境微生物是维持生态圈中能量和物质循环的重要因素之一,在各种污染物和有害物质的降解等方面发挥着重要作用,在进行能源生产和可再生利用等研究领域具有潜在应用价值．在超过150种被破译的非致病微生物基因组中,绝大多数是环境和工业细菌．随着新一代测序技术的投入使用和元基因组学方法的出现,国际上的微生物基因组方面的工作进入了高通量和高产出的阶段．我国已经完成全基因组测序的7株环境细菌中涵盖了嗜高温、嗜酸、耐高压、耐低压等各种极端环境微生物,研究人员从中发现了众多与环境和工业应用密切相关的代谢途径、遗传功能和生物酶,目前,国内多家单位相继启动了元基因组计划,必将使我国在环境和工业微生物基因组研究领域取得一大批原创成果．     

单细胞基因组学分析的技术前沿

基因组学已经深刻地改变了生命科学的诸多领域的面貌。目前它的主要内容是新的全基因组碱基序列的测定和在全基因组范围内鉴定那些在不同水平上影响生命活动的基因群的功能和相互作用。为达此要求,近年出现的第二代测序(深度测序)技术和基因芯片技术发挥了关键作用,但是两者都需要足够的高质量的核酸样品。所以,在只有或只能用单细胞或极少量细胞的情况下,如果没有特殊手段,上述分析往往不能常规、方便地进行。文章以DNA扩增为主线,综合阐述了目前在单细胞(特别是微生物)全基因组测序和大基因组的靶向重测序,以及对单细胞或微量细胞进行的基于深度测序或芯片杂交的功能基因组分析,如转录组、ChIP和DNA的CpG甲基化分析等的最新策略和技术,评价了单细胞基因组测序和功能基因组学各技术的特点并对发展前景进行了展望。     

微生物单细胞分离方法研究进展

自然界中大多数微生物处于未培养状态,被称为“微生物暗物质”。随着微生物单细胞分离方法的不断更新,利用新技术、新方法应对微生物纯培养的挑战获得了重要进展,这些新的分离及培养策略对推动微生物资源学的发展具有重要意义。尽管宏基因组学和基因组学数据相关成果日益增多,但微生物单细胞的分离与培养对于系统研究微生物的生态功能、遗传进化等仍至关重要。本文主要概述了目前使用的或正在研发的膜扩散培养法、微流控分选、荧光激活细胞分选、单细胞拉曼分选、光镊技术、显微操作技术等单细胞分离技术的原理与应用,及其在微生物单细胞分离和培养方面的优点与不足,同时展望了这些单细胞分离技术未来的发展和应用前景。     

环境微生物不依赖于培养的基因组学研究及应用

微生物在生物圈中分布广泛,并且在地球物质循环中占有重要地位,但是约99﹪的微生物目前还不能通过传统的培养方法得到纯培养物（即未培养微生物）,给这些未培养微生物的研究带来很大的困难。随着分子生物学的快速发展及其在微生物研究中的广泛运用,促进了以环境中未培养微生物为研究对象的新兴学科--环境基因组学的产生和发展。在不进行相关微生物培养分离的情况下,通过从环境样品中直接提取获得所有微小生物的全部遗传物质,并构建环境基因组文库;进一步利用功能基因组学研究策略,从文库中寻找编码产生新的有生物活性产物的基因;通过对系统发育相关锚定位点基因序列分析,从而确定特定生态环境体系中未培养微生物的种类结构组成及进化地位,并最终重建该体系中微生物群体的基本物质循环模式。此外,环境基因组学也可以在对未培养微生物生理生化特性深入了解的基础上,建立发展合适的培养体系,最终获得某些特定微生物的纯培养物。本文对环境基因组的构建及相关分析研究策略的进展进行了综述;同时介绍了其在微生物分类及生态学研究的应用。     

不同单细胞全基因组扩增方法的比较及MALBAC在辅助生殖中的应用

单细胞全基因组扩增(whole genome amplification, WGA)是指在单细胞水平对全基因组进行扩增的新技术,其原理是将分离的单个细胞的微量全基因组DNA进行扩增,获得高覆盖率的完整的基因组后进行高通量测序,用于揭示细胞异质性。目前,WGA方法主要包括引物延伸预扩增(primer extension preamplification PCR, PEP-PCR)、简并寡核苷酸引物PCR (degenerate oligonucleotide primed PCR, DOP-PCR)、多重置换扩增(multiple displacement amplification, MDA)、多次退火环状循环扩增(multiple annealing and looping-based amplification cycles, MALBAC)等。本文对不同的单细胞WGA方法的原理及应用情况分别进行了阐述,并对其扩增效率进行评价和比较,包括基因组覆盖度、均一性、重现性、SNV (single-nucleotide variants)和CNV (copy number variants)检测力等。综合对比不同单细胞WGA方法后发现,MALBAC的扩增均一性最高、等位基因脱扣率最低、重现性最好,且对于CNV和SNV的检测效果最好。本文还阐述了MALBAC技术在人类单精子减数重组、非整倍体分析以及人类卵细胞基因组研究中的应用。     

动物胃肠道微生物元基因组学研究进展

动物胃肠道中寄居着庞大复杂的微生物,它们对宿主营养、健康和生产有着重要的作用.随着分子生物学的发展,未培养微生物的研究越来越被重视,宏基因组学方法研究胃肠道微生物不仅能了解未培养微生物多样性,还能获得微生物的遗传、代谢和生理等方面的信息.探讨了元基因组文库的构建和分析方法,并重点介绍了元基因组学在动物胃肠道尤其是反刍动物瘤胃微生物研究中的应用.     

环境微生物基因组技术研究

绝大多数微生物难以有效地进行人工培养,而且实验室工程菌几乎没有野生型功能,因此限制了基于自然微生物多样性的生物技术的应用.为了把对生物群落的结构、功能和细菌在自然环境中进化的认识应用到微生物工艺学中、微生物学家们正致力于把基因组学和相关的高通量技术应用到微生物培养体系和环境样品中,而这必将增添对生态系统及其生物学功能的新见解并带动生物技术的发展.从微生物鉴别及其基因功能的分析、确证和评价等方面综述了基因组技术在环境样品中的应用,提出了现今面临的问题,同时也对环境基因组技术做了展望.     

元基因组学方法在环境微生物中的研究进展

新一代测序技术的快速发展,使得元基因组学研究方法成为了理解环境微生物群落结构和相互作用的重要手段之一。元基因组学方法不需要将环境样本中的微生物单独分离培养,而是作为整体进行研究,因而可以回避传统研究时分离培养微生物的困难。基于这一优势,人体、海洋和土壤等环境有关的各项环境微生物测序计划相继启动,并取得了一系列重要的研究进展。探讨了元基因组测序数据分析中所经常采用的方法,以及有关流程的优势和局限性,并进一步讨论了这些方法在各种环境微生物研究中的应用和成果。     

Single-cell metagenomics: challenges and applications

With the development of high throughput sequencing and single-cell genomics technologies, many uncultured bacterial communities have been dissected by combining these two techniques. Especially, by simultaneously leveraging of single-cell genomics and metagenomics, researchers can greatly improve the efficiency and accuracy of obtaining whole genome information from complex microbial communities, which not only allow us to identify microbes but also link function to species, identify subspecies variations, study host-virus interactions and etc. Here, we review recent developments and the challenges need to be addressed in single-cell metagenomics, including potential contamination, uneven sequence coverage, sequence chimera, genome assembly and annotation. With the development of sequencing and computational methods, single-cell metagenomics will undoubtedly broaden its application in various microbiome studies.     

From bacterial genomics to metagenomics: concept,tools and recent advances

In the last 20 years, the applications of genomics tools have completely transformed the field of microbial research. This has primarily happened due to revolution in sequencing technologies that have become available today. This review therefore, first describes the discoveries, upgradation and automation of sequencing techniques in a chronological order, followed by a brief discussion on microbial genomics. Some of the recently sequenced bacterial genomes are described to explain how complete genome data is now being used to derive interesting findings. Apart from the genomics of individual microbes, the study of unculturable microbiota from different environments is increasingly gaining importance. The second section is thus dedicated to the concept of metagenomics describing environmental DNA isolation, metagenomic library construction and screening methods to look for novel and potentially important genes, enzymes and biomolecules. It also deals with the pioneering studies in the area of metagenomics that are offering new insights into the previously unappreciated microbial world. The authors have contributed equally to the work     

Single virus genomics: a new tool for virus discovery

Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called 'Single Virus Genomics', which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.     

Improved Multiple Displacement Amplification (iMDA) and Ultraclean Reagents

Background

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA).

Results

A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome.

Conclusions

The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.     

Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis

A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively.     

New trends in fluorescence in situ hybridization for identification and functional analyses of microbes

Fluorescence in situ hybridization (FISH) has become an indispensable tool for rapid and direct single-cell identification of microbes by detecting signature regions in their rRNA molecules. Recent advances in this field include new web-based tools for assisting probe design and optimization of experimental conditions, easy-to-implement signal amplification strategies, innovative multiplexing approaches, and the combination of FISH with transmission electron microscopy or extracellular staining techniques. Further emerging developments focus on sorting FISH-identified cells for subsequent single-cell genomics and on the direct detection of specific genes within single microbial cells by advanced FISH techniques employing various strategies for massive signal amplification.