高通量测序和DGGE分析土壤微生物群落的技术评价

【目的】比较新一代高通量测序与传统的变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis,DGGE)指纹图谱技术,评价两种技术研究土壤微生物群落结构的优缺点。【方法】针对新西兰典型草地和森林土壤,以16S rRNA基因为标靶,通过高通量测序和DGGE技术分析土壤微生物群落的组成、丰度和多样性,比较两种方法在土壤微生物研究中的适用性。【结果】在不同的微生物分类水平,高通量测序草地土壤检测到22门,54纲,60目,131科,350属;而DGGE仅检测到6门,9纲,8目,10科,10属,表明DGGE显著低估了土壤微生物的群落组成。森林土壤也得到了类似规律,高通量测序的检测灵敏度是DGGE的3.8、6.7、6.4、19.2及39.4倍。进一步分析土壤中主要微生物类群的相对丰度,发现分类水平越低,高通量测序与DGGE的结果差异越大,尤其在科和属的水平上差异最大。以高通量测序结果为标准,DGGE明显高估了土壤中大多数微生物类群的相对丰度,最高可达2000倍。两种方法都表明草地土壤的多样性指数高于森林土壤,但DGGE多样性指数的绝对值远低于高通量测序结果。【结论】高通量测序能够较为全面和准确的反映土壤微生物群落结构,而DGGE仅能够反映有限的优势微生物类群,在很大程度上极可能低估土壤微生物的物种组成并高估其丰度。     

基于高通量测序技术大亚湾肥胖软箭虫(Flaccisagitta enflata)的食性分析

采用Illuminate高通量测序技术分析了大亚湾肥胖软箭虫(Flaccisagitta enflata)的食性。在夏季(2017年8月)和冬季(2018年1月)对大亚湾内不同站位拖网采获的四组样品中, 分别挑取毛颚类优势种肥胖软箭虫进行18S rDNA V4区扩增; 通过高通量测序得到四组样品的序列, 经过处理每组样品得到约30,000条高质量序列。分析结果表明, 大亚湾肥胖软箭虫的食物来源于16个门类生物, 主要的优势类群分别来自于3个门类的浮游动物(刺胞动物门46.16%、节肢动物门的桡足类19.16%和栉水母动物门14.22%), 真菌类的2个门类(子囊菌门14.04%和担子菌亚门4.48%), 以及少量浮游植物类群的2个门 (褐藻门0.06%和隐藻门0.03%)。另外, 还检测出少量肥胖软箭虫可能摄食的纤毛虫、线虫、住囊虫、海葵等。夏季湾内肥胖软箭虫食物中子囊菌门贡献最高 (43.3%), 这与大亚湾海水养殖等人类活动影响有关; 夏季湾口外海水带来丰富的暖水性栉水母, 占肥胖软箭虫总食物丰度的55.25%。冬季湾内肥胖软箭虫摄食哲水蚤比例达49.94%, 而湾口肉质介穗水母占比高达85.3%。分析结果可见, 肥胖软箭虫的摄食具有偏好性, 且明显存在季节和区域差异。研究探讨了大亚湾不同环境下肥胖软箭虫食性转变及其食物来源的季节和区域差异, 为深入揭示浮游动物在海湾生态系统物质与能量传递过程的作用机制提供了重要基础资料。     

一种结合单张芯片序列捕获和高通量测序技术测序外显子组的方法

随着高通量测序技术的发展,全外显子测序已经成为一种研究人类疾病的重要方法.本文展示了一种通过Nimblegen2.1M芯片进行外显子DNA序列捕获和高通量测序的方法,包括两步法文库制备.测序的平均覆盖深度达33倍时,95.6%的34M目标区域得到均衡覆盖,特异性达到80%.对比全基因组鸟枪法测序的结果,此方法在检测SNP时的假阳性率为0.97%,假阴性率为6.27%.本方法对于全基因组扩增的DNA也适用.结果显示,全外显子测序技术可以在大规模的群体研究和医学研究中起到重要作用.     

应用高通量测序技术对蚊携带RNA病毒的检测研究

目的:基于高通量测序技术,分析我国不同边境地区蚊虫携带病原的多样性,为快速筛查蚊媒病原提供参考依据。方法:将采自不同边境地区的田间样品随机分为6组,提取病毒核酸,然后使用Ion S5 XL测序仪进行高通量测序,并进行生物信息学分析。结果:在6个蚊类混合样本中,有4个混合样本发现2种及2种以上的可疑病毒。结论:高通量测序检测体系有效地检测出蚊媒病毒的存在,初步揭示了蚊类所携带的病原体种类的多样性。     

基于新一代测序技术的mtDNA突变检测方法学的建立

目的:大量研究证实线粒体DNA(mtDNA)突变与肿瘤发生及进展密切相关,但使用传统测序方法难以高通量、高精确度的检测mtDNA突变,为此本研究建立了基于新一代测序技术的mtDNA突变检测方法.方法:提取肝癌患者癌、癌旁组织以及外周血细胞总DNA,利用PCR技术对线粒体基因组进行富集并对PCR产物进行平末端、粘性末端连接或对PCR引物进行氨基修饰,构建mtDNA测序文库.经Illumina HiSeq 2000平台测序后利用生物信息学方法与人类mtDNA参考序列进行比对,并进行测序数据分析.结果:通过对不同质量基因组DNA进行评估后,发现三对引物法适用于大部分DNA样本的mtDNA富集.进一步我们发现PCR引物的氨基修饰可显著提高测序数据覆盖均一性,降低测序成本.结论:本研究利用新一代测序技术通过对线粒体DNA富集方法以及测序覆盖度均一性进行优化,建立了一套灵敏、特异、高通量的mtDNA突变检测策略,为mtDNA突变与疾病研究提供了新方法.     

黄海中部高眼鲽的摄食及随体长的变化

对2004年1月-2005年10月在黄海中部采集的622尾高眼鲽样品的胃含物进行分析.结果表明：高眼鲽摄食的食物种类包括11类38种,其中甲壳类和瓣鳃类为优势饵料类群,共占食物相对重要性指数百分比的99%;食物中的优势饵料种类为太平洋磷虾、脊腹褐虾和樱蛤.与20世纪80年代相比,黄海高眼鲽的摄食强度下降,食物组成发生了较大的变化;鱼类不再是优势饵料类群,鳀鱼在食物中所占的比例明显下降.通过聚类分析和列联表检验等方法对高眼鲽摄食随体长的变化进行研究,结果表明：体长在51～370 mm范围内的高眼鲽的摄食强度没有显著的体长变化,但不同体长组摄食的食物种类有一定差异.＜119 mm的主要以磷虾类和底层虾类为食;体长在120～199 mm的除以磷虾类和底层虾类为主要食物外,还摄食相当比例的瓣鳃类和鱼类;＞200 mm的则主要摄食底层虾类和鱼类,磷虾类所占比例减小.     

基于高通量测序对呼伦贝尔陶顺阿尔山细菌群落多样性的研究

对内蒙古呼伦贝尔陶顺阿尔山水体与沉积物的细菌群落结构和多样性，进行16S rRNA基因(V3～V5区)高通量测序。运用Illumina MiSeq高通量测序平台，共获得594 226条优化序列。结果显示，水体中最主要的细菌类群为变形菌门(Proteobacteria),而沉积物样品中的主要优势类群为盐厌氧菌门(Haloanaerobium);细菌在属分类水平上，水体中优势菌群为嗜盐单胞菌属(Halomonas)、冷弯菌属(Psychroflexus)、盐厌氧菌属(Haloanaerobium);沉积物样品中优势菌群为盐厌氧菌属、盐单胞菌属、未分类菌(no-rank-f-unclassified),其中存在大量的未知物种。陶顺阿尔山水体与沉积物中存在着丰富的微生物群落，且差异性大，未知菌种居多。本研究首次阐明了陶顺阿尔山细菌群落的多样性，为今后开发和利用陶顺阿尔山的功能菌群提供参考。     

一种基于多重PCR的人类线粒体基因组高通量测序方法

人类线粒体基因组DNA(mtDNA)是一个16569 bp的双链闭合环状DNA分子,具有母系遗传、多拷贝、高异质性及高变异率等特点,是研究人类遗传和进化上广泛使用的分子标记.近几年,高通量测序技术的出现,使得在短时间内准确测定mtDNA序列成为可能;但目前常用的高通量测序建库方法操作复杂、研究费用相对较高.基于多重PCR扩增的测序方法具有高效率、高灵敏度、低成本的特点,因而适用于大规模线粒体基因组的变异检测分析.利用73个相互重叠的扩增子通过多重PCR方法来扩增中国人的线粒体全基因组,在扩增片段两端连接特定的接头序列,然后在IlluminaHiSeq X Ten平台上进行高通量测序.对获得的测序数据分析发现,mtDNA每个位点的测序深度均达到2000×以上;当测序深度为100×时,所有样本的序列覆盖度都达到100%;数据质量适用于后续的变异检测分析.利用本研究建立的基于多重PCR的二代测序方法无需片段化即可直接上机测序,在复杂遗传病的研究中有着广泛的应用前景.     

基于高通量测序研究青藏高原茶卡盐湖微生物多样性

【目的】茶卡盐湖(Chaka Salt Lake,CSL)是青藏高原有名的天然结晶盐湖,具有独特的石盐盐湖矿床,盛产青盐。盐湖卤水环境中存在丰富的嗜盐菌资源和潜在的新种,细菌和古菌的群落结构特征和物种多样性尚不明确。【方法】采用Illumina高通量测序平台对茶卡盐湖水样和底泥混合物中的细菌和古菌群落进行16S r RNA基因(V3-V5区)高通量测序,检测4个样本的群落结构差异和微生物多样性。【结果】获得细菌和古菌总有效序列分别为117 192和110 571条。结果分析表明细菌和古菌的物种注释(Operational taxonomic unit,OTU)数目分别为421和317,获得分类地位明确的细菌种类为14门28纲170属,古菌为5门4纲34属。细菌的优势类群是厚壁菌门(Firmicutes),所占比例为68.37%,其次为变形菌门Proteobacteria(20.49%);优势种属依次为芽孢杆菌属Bacillus(41.94%)、海洋芽孢杆菌属Oceanobacillus(8.03%)、假单胞菌属Pseudomonas(7.67%)、盐厌氧菌属Halanaerobium(7.42%)和乳球菌属Lactococcus(7.38%);古菌的优势类群以广古菌门(Euryarchaeota)盐杆菌纲(Halobacteria)为主,优势菌是Halonotius(17.21%)和盐红菌属Halorubrum(16.23%)。【结论】揭示了茶卡盐湖中细菌和古菌的群落结构及物种多样性,为嗜盐菌的开发及后续微生物资源的挖掘提供了理论依据。     

肠道微生物与疾病关系的研究新进展- 运用454 测序技术分析

由于传统研究方法成本和速度的限制,远远满足不了对微生物群落大规模的研究,以454测序为代表的新一代高通量测序技术凭借低成本、高通量、流动自动化的优势为研究微生物的多样性和组成提供了新的技术平台。本文就近年来454测序技术在研究人体肠道微生物与疾病关系的应用进行了综述。     

High-throughput sequencing offers insight into mechanisms of resource partitioning in cryptic bat species

Sympatric cryptic species, characterized by low morphological differentiation, pose a challenge to understanding the role of interspecific competition in structuring ecological communities. We used traditional (morphological) and novel molecular methods of diet analysis to study the diet of two cryptic bat species that are sympatric in southern England (Plecotus austriacus and P. auritus) (Fig. 1). Using Roche FLX 454 (Roche, Basel, CH) high-throughput sequencing (HTS) and uniquely tagged generic arthropod primers, we identified 142 prey Molecular Operational Taxonomic Units (MOTUs) in the diet of the cryptic bats, 60% of which were assigned to a likely species or genus. The findings from the molecular study supported the results of microscopic analyses in showing that the diets of both species were dominated by lepidopterans. However, HTS provided a sufficiently high resolution of prey identification to determine fine-scale differences in resource use. Although both bat species appeared to have a generalist diet, eared-moths from the family Noctuidae were the main prey consumed. Interspecific niche overlap was greater than expected by chance (O(jk) = 0.72, P < 0.001) due to overlap in the consumption of the more common prey species. Yet, habitat associations of nongeneralist prey species found in the diets corresponded to those of their respective bat predator (grasslands for P. austriacus, and woodland for P. auritus). Overlap in common dietary resource use combined with differential specialist prey habitat associations suggests that habitat partitioning is the primary mechanism of coexistence. The performance of HTS is discussed in relation to previous methods of molecular and morphological diet analysis. By enabling species-level identification of dietary components, the application of DNA sequencing to diet analysis allows a more comprehensive comparison of the diet of sympatric cryptic species, and therefore can be an important tool for determining fine-scale mechanisms of coexistence.     

Unexpected prey of juvenile spotted scat (Scatophagus argus) near a wharf: The prevalence of fouling organisms in stomach contents

A knowledge of fish diets can contribute to revealing the trophic role and ecological function of species in aquatic ecosystems. At present, however, there are no efficient or comprehensive methods for analyzing fish diets. In this study, we investigated the diets of juvenile Scatophagus argus collected near a wharf in Daya Bay, China, by dissection and high‐throughput sequencing (HTS) using the 18S rDNA V4 region. Microscopy disclosed large amounts of bryozoans and unrecognizable detritus. In contrast, HTS analysis indicated that the fish diets were considerably more diverse than visual inspection suggested. After eliminating fish sequences, approximately 17,000 sequences from taxa in nine phyla (Ciliophora, Bryozoa, Annelida, Bacillariophyta, Chlorophyta, Arthropoda, Dinoflagellata, Tunicata, and Phaeophyta) were identified from the analysis of stomach contents. Twenty‐one food categories were identified, most of which (95.2%) were benthic fouling organisms that could easily be collected around wharfs. These consisted of bryozoans (31.9%), ciliates (45.7%), polychaetes (14.6%), and green algae (3.0%). Therefore, to adapt to anthropogenic habitat modification, the fish had probably shifted from planktonic to benthic feeding. The prevalence of fouling organisms in the stomachs of juvenile S. argus indicates that the fish have responded to habitat changes by widening their food spectrum. This adaptation may have increased their chances of survival. The fouling organisms that inhabit highly perturbed coastal ecosystems could represent a food source for animals at higher trophic levels. Our results accordingly suggest that human activity might significantly influence fish feeding behavior and material transfer along the food chain.     

Seasonal and inter‐island variation in the foraging strategy of the critically endangered Red‐headed Wood Pigeon Columba janthina nitens in disturbed island habitats derived from high‐throughput sequencing

We studied the feeding ecology of the critically endangered Red‐headed Wood Pigeon Columba janthina nitens, a subspecies endemic to a very remote and highly disturbed oceanic island chain, the Ogasawara Islands. An analysis based on high‐throughput sequencing (HTS) was undertaken on 627 faecal samples collected over 2 years from two island habitats, and food availability and the nutrient composition of the major fruits were also estimated. The HTS diet analysis detected 122 food plant taxa and showed clear seasonal and inter‐island variation in the diet of the Pigeons. The results indicated a preference for lipid‐rich fruits, but the diet changed according to the availability of food resources, perhaps reflecting the foraging strategy of the Pigeons in isolated island habitats with poor food resources. Pigeons also frequently consumed introduced plants at certain times of year, perhaps compensating for the lack of preferred native food resources. However, the degree of dependence on introduced plants appeared to differ between the two island habitats, so the different impacts of introduced plant eradication on the foraging conditions for the Pigeons on each island should be considered. HTS diet analysis combined with field data may be useful for monitoring the foraging conditions of endangered species and may also inform an appropriate conservation strategy in oceanic island ecosystems with complicated food webs that include both native and introduced species.     

Food habits of fishes in azostera marina bed at aburatsubo, central japan

To clarify the feeding habits of seagrass fishes, the gut contents of 31 fish species collected in aZostera marina bed at Aburatsubo central Japan, were examined. Ontogenetic changes in food preference were recognized in 12 species. In general, juveniles of the latter preyed mainly on small crustaceans (e.g., gammaridean amphipods) or planktonic animals (e.g., calanoid and cyclopoid copepods), subsequently changing to other prey items (e.g., hard-shelled animals) with growth. Cluster analysis based on dietary overlaps showed that the seagrass fish assemblage comprised seven feeding guilds (small-crustacean, planktonic-animal, large-crustacean, polychaete, fish, hard-shelled mollusc and detritus feeders). Of these, small-crustacean feeders and planktonic-animal feeders were the most abundantly represented, including juveniles of several species, which, when adult, transferred to other feeding guilds. On the other hand detritivores were represented by a single species.     

Feeding ecology of pelagic fish species in the Gulf of Riga (Baltic Sea): the importance of changes in the zooplankton community

The feeding ecology of four pelagic fish species was studied in relation to their prey availability in the Gulf of Riga (Baltic Sea) during the summer 1999-2006. The zooplankton community was dominated by the cladoceran Bosmina longispina, rotifers Keratella cochlearis and K. quadrata and the copepod Eurytemora affinis, with the highest interannual variability in abundance recorded for B. longispina. The last influenced the diet of adult sprat Sprattus sprattus, juvenile smelt Osmerus eperlanus and three-spined stickleback Gasterosteus aculeatus as these were strongly selecting for B. longispina. The fish feeding activity did not match the abundance dynamics of their preferred prey, suggesting that fishes may switch to consume other prey in case the preferred diet was limited. A considerable dietary overlap indicated high potential competition between pelagic fish species. While herring Clupea harengus membras and G. aculeatus were relying on very different food, the diets of young O. eperlanus and G. aculeatus were very similar. Interannual variability in zooplankton composition and abundance significantly affected the diet composition of fishes, but those changes were insufficient to exert a consistent influence upon fish feeding activity and total amounts of zooplankton consumed.     

太湖新银鱼移植对(鳖)早期摄食和生长的影响

研究选择长江中游西洞庭湖水系太湖新银鱼移植水体(黄石水库)和未移植水体(蒙泉水库),研究太湖新银鱼(Neosalanx taihuensis Chen)移植对浮游动物食性鱼类(Hemiculter leucisculus Basilewsky)早期生长和摄食的影响。2009年7月下旬和8月中旬共采集稚鱼157尾,其中7月下旬采集稚鱼在14—23日龄之间,两水体间生长差异不显著;8月中旬采集稚鱼在20—49日龄之间,黄石水库生长率显著小于蒙泉水库。对样品耳石日轮分析发现25日龄之前两水体稚鱼生长率相似,之后黄石水库稚鱼较蒙泉水库生长慢。食性分析发现25日龄前两水体稚鱼食物组成相似,主要摄食轮虫、小型枝角类和桡足类;25日龄后黄石水库稚鱼食性没有显著变化,而蒙泉水库稚鱼则转食大型枝角类、昆虫及鱼卵和仔鱼。两水体气候条件、营养状况、鱼类区系组成上基本相同,是否有太湖新银鱼移植是两水体间的主要差别。太湖新银鱼春群在1—5月间繁殖,而的繁殖在6月下旬之后。因此在早期生活史阶段与太湖新银鱼的食物竞争会主要发生在转食大型浮游动物之后。太湖新银鱼摄食使黄石水库大型浮游动物饵料资源短缺,稚鱼在25日龄后不能转食,是导致黄石水库幼鱼在25日龄后生长减慢的重要因素。     

长江口中华鲟幼鱼的食物组成及摄食习性

分析了2004年5-9月长江口水域中华鲟幼鱼的食物组成及摄食的月份变化.结果表明:长江口中华鲟幼鱼以底栖小型鱼类、多毛类和端足类为主要食物,兼食虾类、蟹类及瓣鳃类等小型底栖动物.幼鱼的摄食率和摄食强度较高,且摄食强度存在着月份变化.主要饵料生物组成有明显的月变化,且食物个体有逐渐增大的趋势,其中5月主食多毛类和蟹类,6月主食鱼类和端足类,7月为鱼类和多毛类,8月样本较少且为空胃,9月为鱼类和虾类.与历史资料进行比较发现,曾是幼鱼重要饵料的鲬类所占比例极小,其质量百分比仅占1.81%;而以前未曾记录的虾虎鱼类在食物中所占比例较高, 其质量百分比达50.54%;等足类以前也未曾记录,其出现频率达10.07%.     

高通量测序技术在野生动物食性分析中的应用

食性研究是动物生态学颇受关注的一个重要内容,而食性分析方法由于受到技术和适用范围的限制,也在不断改进和更新。随着高通量测序技术的发展,该技术逐渐扩展到野生动物的食性分析,使食性分析的效率得到极大提升,并拓宽了食性分析的应用范围。尽管高通量测序应用于食性分析在数据量、灵敏度和分辨率方面的优势较为明显,但由于涉及到的步骤较多,受到的影响因素较为复杂,目前高通量测序应用于食性分析还属于研究比较薄弱的领域。概述了高通量测序技术应用于食性分析的基本流程,总结了该技术在食物组成分析、种内和种间食性关系、食物与栖息地、行为关系方面的研究动态,分析了PCR、污染和定量分析对该技术应用性的影响,提出了相应的解决对策和建议,并对其应用前景进行了展望。     

Why do juvenile fish utilise mangrove habitats?

Three hypotheses to discern the strong positive association between juvenile fish and mangrove habitat were tested with field and laboratory experiments. Artificial mangrove structure in the field attracted slightly more juvenile fish than areas without structure. Artificial structure left to accumulate fouling algae attracted four-times the total number of juvenile fish than areas without structure or areas with clean structure. Community composition of fish attracted to structure with fouling algae was different when compared with areas with no structure or clean structure; five species were attracted by structure with fouling algae whilst two species were associated with structure regardless of fouling algae. Algae were linked to increased food availability and it is suggested that this is an important selection criteria for some species. Other species were apparently attracted to structure for different reasons, and provision of shelter appears to be important. Predation pressure influenced habitat choice in small juvenile fish in laboratory experiments. In the absence of predators, small juveniles of four out of five species avoided shelter but when predators were introduced all species actively sought shelter. Large fish were apparently less vulnerable to predators and did not seek shelter when predators were added to their tank. Feeding rate was increased in the mangrove habitat for small and medium-sized fish compared with seagrass beds and mudflats indicating increased food availability or foraging efficiency within this habitat. Larger fish fed more effectively on the mudflats with an increased feeding rate in this habitat compared with adjacent habitats. The most important aspect of the mangrove habitat for small juvenile fish is the complex structure that provides maximum food availability and minimises the incidence of predation. As fish grow a shift in habitat from mangroves to mudflat is a response to changes in diet, foraging efficiency and vulnerability to predators.     

Contributions of juvenile and adult diet to the lifetime reproductive success and lifespan of a spider

Food availability can vary widely for animals in nature and can have large effects on growth, reproduction and survival. While the consequences of food limitation for animals have been extensively studied, significant questions still remain including how ontogenetic variation in food availability contributes to lifetime reproductive success. We tested the effects of juvenile and adult food limitation on the lifetime reproductive success and lifespan of bridge spiders, Larinioides sclopetarius. Food availability was manipulated (low or high) over the entire juvenile and adult stage in a full‐factorial design and reproductive output and lifespan were measured. Juvenile and adult food limitation both reduced lifetime egg and hatchling production with effect sizes that were not significantly different from each other. Unlike some other arthropods, where juvenile food limitation reduces fecundity by reducing adult body size, body size was not affected by juvenile diet in bridge spiders. Clutch size was also significantly reduced by both juvenile and adult food limitation. The effect of adult diet on clutch size was stronger than that of juvenile diet. Juvenile and adult food limitation both extended total lifespan, and adult food limitation extended adult longevity (i.e. time from maturation to death). However, juvenile food limitation decreased adult longevity, in contrast to what would be predicted by dietary or caloric restriction. Compensatory feeding and growth are widely recognized mechanisms through which animals can ameliorate some of the negative effects of periods of food limitation. Yet our results combined with studies of a range of other species suggest that there may be lasting consequences of juvenile food limitation on lifetime reproductive success that cannot be compensated for by adult feeding in some species.