高通量测序分析DNA提取引起的对虾肠道菌群结构偏差

【目的】通过高通量测序技术,评价不同DNA试剂盒提取引起的对虾肠道菌群结构偏差,了解健康凡纳滨对虾肠道菌群结构特征。【方法】分别以细菌、粪便和组织DNA试剂盒3次重复提取凡纳滨对虾肠道总DNA(分别编号为SIB,SIS和SIT),检测DNA含量、纯度及其16S r DNA V4区可扩增性,进一步采用Illumina Mi Seq高通量测序比较SIB和SIS样品菌群组成和多样性。【结果】细菌试剂盒提取的虾肠总DNA效果最好,粪便试剂盒次之,而组织试剂盒所提DNA含量低且难以被扩增。从SIB和SIS样品分别获得52151±5085和55296±5147条有效序列,同一(46800条)测序深度下,SIS样品OTU(operational taxonomic unit)数量和Shannon多样性指数均显著高于SIB的,而SIB样品间OTU重复性则优于SIS样品间的。从SIB和SIS样品鉴定的优势门一致,均包括变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、浮霉菌门(Planctomycetes)、放线菌门(Actinobacteria)和蓝细菌门(Cyanobacteria),但不同分类水平上绝大多数优势菌群丰度在两种样品间差异明显。【结论】高通量测序分析表明对虾肠道菌群结构因DNA提取方法不同而呈现显著偏差;本研究健康凡纳滨对虾肠道核心菌群主要由发光杆菌属(Photobacterium),乳球菌属(Lactococcus),弧菌属(Vibrio),Aliivibrio和3个分类未定属构成。     

Investigation of the intestinal microbiota in preterm infants using different methods

Modifications in microbial colonization of the human gut are believed to affect intestinal homeostasis and increase the risk of gastrointestinal diseases. The present study examined different methods for investigating the dynamic characterization of the intestinal microbiota in preterm infants. Fecal samples were collected weekly from ten preterm infants during their stay in a neonatal intensive care unit. The infants had a mean gestational age of 29 weeks (range: 28–32 weeks) and a mean birth weight of 1233 g (range: 935–1450 g). Bacterial colonization was assessed using conventional culture techniques and molecular biological methods. More specifically, the recently developed denaturing high performance liquid chromatography (dHPLC) technique was compared to established methods such as temporal temperature gradient gel electrophoresis (TTGE) and rRNA gene library sequencing. Our results indicate that the gastrointestinal tract of preterm infants, born at a gestational age of less than 33 weeks, has a low biodiversity of mainly, culturable bacteria. Finally, dHPLC was evaluated in terms of speed, labor and sensitivity for its use as a tool to analyze microbial colonization in preterm infants. We found that this technique provided major improvements over gel-based fingerprinting methods, such as TTGE, that are commonly used for studying microbial ecology. As such, it may become a common analytical tool for this purpose.     

采用高通量测序技术研究抗生素对小鼠肠道菌群的影响

目的 探讨不同浓度抗生素对小鼠肠道菌群多样性和结构的影响,并预测相关功能变化。方法 15只SPF级ICR小鼠随机分为正常组、低浓度抗生素组和高浓度抗生素组,连续灌胃5 d后,采集小鼠新鲜粪便样本。利用Illumina MiSeq测序平台,对细菌的16S rRNA V3‒V4区进行高通量测序,并对测序结果进行生物信息学分析。结果 高、低浓度抗生素组小鼠肠道菌群组成与正常组存在明显差异。与正常组相比,高剂量组小鼠肠道肠球菌属、志贺埃希菌属相对丰度显著升高（t=‒2.71,P=0.026;t=‒2.30,P<0.05）;分节丝状菌属、拟普雷沃菌属相对丰度显著降低（t=2.88,P=0.020;t=2.49,P=0.037）,理研菌属极显著降低（t=3.79,P=0.005）。低剂量组小鼠肠道菌群变形菌纲成为优势菌,芽胞杆菌属、粪球菌_2、苏黎世杆菌属、普雷沃菌属_2、普雷沃菌属_7、志贺埃希菌属、沙雷菌属和放线菌属等相对丰度显著升高（均P<0.05）;梭杆菌属、泛菌属极显著升高（t=‒3.19,P=0.013;t=‒3.50,P=0.008）;分节丝状菌属、理研菌属相对丰度显著降低（t=2.69,P=0.028;t=2.33,P=0.048）。PICRUSt功能预测分析显示,抗生素组显著增加人类疾病、细胞过程和环境信息处理功能层的基因拷贝数,显著降低有机系统、遗传信息处理和代谢功能层的基因拷贝数。结论 广谱抗生素能破坏小鼠肠道的微生态平衡,有必要深入研究抗生素对心血管、免疫性、感染性及神经退行性疾病发展的潜在作用。     

基于ITS2高通量测序 研究强直性脊柱炎患者肠道真菌特征

目的探究强直性脊柱炎(ankylosing spondylitis,AS)患者肠道真菌菌群的多样性特征,分析健康人群与AS患者肠道真菌的结构差异。方法收集17例健康人群新鲜粪便样本和24例AS患者新鲜粪便样本,分别称为HC组和AS组,提取两组粪便样本总DNA;根据真菌ITS2区设计引物进行扩增,利用Illumina HiSeq PE250平台进行ITS2高通量测序;测序结果经过Reads拼接,OTUs(operational taxonomic units)聚类,Alpha和主成分分析,物种组成统计,显著性差异分析,最终得到样本物种信息。结果对肠道真菌菌群进行Alpha分析,各多样性指标中,shannon和observed_species指数差异有统计学意义(P0.05),其余指数差异均无统计学意义,故不能明确两组间多样性的差异。主成分分析提示差异显著。对肠道真菌结构进行分析,门的水平分析显示,担子菌门(Basidiomycota)在HC组和AS组中差异显著,接合菌门(Zygomycota)在两组间差异极显著;纲和属的水平分析显示,Ascomycota_unidentified纲在两组中差异显著,伞菌纲(Agaricomycetes)、Incertae_sedis_10纲、Orbiliomycetes纲差异极显著。Agaricomycetes_unidentified_1属、Amphinema属、Geoglossales_unidentified_1属、腔块菌属(Hydnotrya)差异显著,鹅膏菌属(Amanita)和锁瑚菌属(Clavulina)差异极显著。结论本实验证实了在AS患者中存在肠道真菌菌群失调,其特征是生物多样性和结构的改变,揭示肠道真菌也可能在AS发病中发挥作用。     

454焦磷酸测序分析几种哺乳动物粪便细菌多样性

摘要:【目的】分析比较几种哺乳动物粪便细菌群落多样性,了解粪便细菌多样性与动物进化和食物的关系。【方法】采集6 种哺乳动物粪便样品;提取总DNA;PCR扩增,获得16S rDNA V3标签片段;用454焦磷酸测序技术进行高通量测序;主要基于QIIME平台分析比较粪便细菌多样性。【结果】分析发现,厚壁菌门和拟杆菌门广泛存在于各样品中,并且占绝对优势。α多样性分析发现,杂食性的长臂猿、黑猩猩和川金丝猴的多样性最高,其次是肉食性的东北虎,来自熊科杂食性的亚洲黑熊和大熊猫的多样性最低。β 多样性分析发现,白眉长臂猿、黑猩猩、川金丝猴几种灵长目动物粪便的菌群相似,而大熊猫菌、黑熊、孟加拉虎几种食肉目动物粪便细菌群相似但食肉动物孟加拉虎主要又因含梭杆菌门而区别于其他动物。【结论】动物粪便细菌优势类群明显;同种动物重复样本的相似性最高,各种动物多样性存在差异,但几种灵长类动物粪便细菌多样性更丰富;动物粪便细菌的组成和动物的进化及食物相关。本研究为哺乳动物粪便菌资源及后续研究提供了借鉴。     

Lactobacilli in the intestinal microbiota of Swedish infants

Lactobacillus colonisation was examined in 112 Swedish infants. Faecal samples obtained at 1, 2, 4 and 8 weeks and at 6, 12 and 18 months of age were cultivated quantitatively on Rogosa agar. Lactobacilli were speciated by PCR and typed to the strain level by randomly amplified polymorphic DNA (RAPD). Lactobacilli reached a peak at 6 months when 45% of the infants were colonised. L. rhamnosus and L. gasseri were the most common species in this period. Colonisation by lactobacilli in general (P < 0.01) and L. rhamnosus in particular (P < 0.05) was more common in breast-fed than in weaned infants at 6 months of age. Lactobacillus isolation reached a nadir of 17% by 12 months (P < 0.0001), but increased to 31% by 18 months of age P < 0.05). The food-related species L. paracasei, L. plantarum, L. acidophilus and L. delbrueckii dominated in this second phase. A single strain persisted for at least 3 weeks in 17% of the infants during the first 6 months, most commonly L. rhamnosus. Lactobacillus population counts in colonised infants increased from 10(6.4) cfu/g at 1 week to 10(8.8) cfu/g at 6 months, and then dropped to 10(5.4) cfu/g faeces at 12 months of age. Lactobacillus colonisation was not significantly related to delivery mode, or to presence of siblings or pets in the household. Our results suggest that certain Lactobacillus species, especially L. rhamnosus, thrive in the intestinal flora of breast-fed infants. After weaning they are replaced by other Lactobacillus species of types found in food.     

Diversity of Entamoeba spp. in African great apes and humans: an insight from Illumina MiSeq high-throughput sequencing

Understanding the complex Entamoeba communities in the mammalian intestine has been, to date, complicated by the lack of a suitable approach for molecular detection of multiple variants co-occurring in mixed infections. Here, we report on the application of a high throughput sequencing approach based on partial 18S rDNA using the Illumina MiSeq platform. We describe, to our knowledge, for the first time, the Entamoeba communities in humans, free-ranging western lowland gorillas and central chimpanzees living in the Dja Faunal Reserve in Cameroon. We detected 36 Entamoeba haplotypes belonging to six haplotype clusters, containing haplotypes possessing high and low host specificity. Most of the detected haplotypes belonged to commensal Entamoeba, however, the pathogenic species (Entamoeba histolytica and Entamoeba nuttalli) were also detected. We observed that some Entamoeba haplotypes are shared between humans and other hosts, indicating their zoonotic potential. The findings are important not only for understanding the epidemiology of amoebiasis in humans in rural African localities, but also in the context of wild great ape conservation.     

应用Illumina MiSeq高通量测序技术分析河西走廊地区盐碱土壤微生物多样性

【目的】以甘肃省河西走廊地区的9个盐碱土壤样品(原生盐碱土、次生盐碱土、农田土)为材料,研究该地区盐碱土壤中微生物群落的多样性。【方法】提取土壤微生物总DNA,应用Illumina Mi Seq高通量测序技术进行分析。【结果】从分布在河西走廊3个流域的9个盐碱土样品中共获得325 089条微生物的16S r RNA基因序列。冗余分析和热图分析表明,原生盐碱土与次生盐碱土、原生盐碱土与农田土微生物群落构成差异较大,次生盐碱土与农田土微生物群落差异较小。土壤p H对微生物群落组成的影响最显著。多样性指数和稀释性曲线分析得出,在9个土壤样品中,S6号Shannon指数最大,S1号Shannon指数最小,S1号Simpson指数最大,S6号Simpson指数最小,说明原生盐碱土的微生物群落多样性最低,次生盐碱土的微生物群落多样性最高。盐碱土壤中主要的微生物群落包括9个门,其中变形菌门占主导地位,其余依次是放线菌门、拟杆菌门、酸杆菌门、浮霉菌门、绿弯菌门、芽单胞菌门、厚壁菌门和疣微菌门。原生盐碱土和农田土中占优势的微生物群落是变形菌门,次生盐碱土中占优势的微生物群落是放线菌门。【结论】河西走廊地区盐碱土壤中微生物多样性非常丰富,存在大量的微生物类群,尤其是在次生盐碱土壤中。     

Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems

Background  

The generation and analysis of high-throughput sequencing data are becoming a major component of many studies in molecular biology and medical research. Illumina's Genome Analyzer (GA) and HiSeq instruments are currently the most widely used sequencing devices. Here, we comprehensively evaluate properties of genomic HiSeq and GAIIx data derived from two plant genomes and one virus, with read lengths of 95 to 150 bases.     

Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems

Background

The generation and analysis of high-throughput sequencing data are becoming a major component of many studies in molecular biology and medical research. Illumina's Genome Analyzer (GA) and HiSeq instruments are currently the most widely used sequencing devices. Here, we comprehensively evaluate properties of genomic HiSeq and GAIIx data derived from two plant genomes and one virus, with read lengths of 95 to 150 bases.

Results

We provide quantifications and evidence for GC bias, error rates, error sequence context, effects of quality filtering, and the reliability of quality values. By combining different filtering criteria we reduced error rates 7-fold at the expense of discarding 12.5% of alignable bases. While overall error rates are low in HiSeq data we observed regions of accumulated wrong base calls. Only 3% of all error positions accounted for 24.7% of all substitution errors. Analyzing the forward and reverse strands separately revealed error rates of up to 18.7%. Insertions and deletions occurred at very low rates on average but increased to up to 2% in homopolymers. A positive correlation between read coverage and GC content was found depending on the GC content range.

Conclusions

The errors and biases we report have implications for the use and the interpretation of Illumina sequencing data. GAIIx and HiSeq data sets show slightly different error profiles. Quality filtering is essential to minimize downstream analysis artifacts. Supporting previous recommendations, the strand-specificity provides a criterion to distinguish sequencing errors from low abundance polymorphisms.     

基于16S rRNA基因高通量测序研究狞猫肠道微生物多样性

[目的]研究狞猫肠道微生物多样性特征。[方法]对7只健康成年野生狞猫（2只雄性,5只雌性;2只来自济南野生动物园,5只来自威海野生动物园）粪便微生物16S rRNA基因V3-V4区进行高通量测序,对狞猫肠道微生物多样性进行研究。[结果] 7只狞猫共获得肠道微生物16S rRNA基因V3-V4区有效序列1458741条,平均208392条,序列平均长度433 bp。通过以97%的序列相似性进行分类,获得操作分类单元（OTU）平均 233个。经序列比对和分类鉴定,这些OTU都属于细菌域,包括13个门,26个纲,43个目,75个科,119个属。其中,丰度最高的细菌门是厚壁菌门（Firmicutes,平均占61.7%）、放线菌门（Actinobacteria,12.42%）、拟杆菌门（Bacteroidetes,7.79%）、梭杆菌门（Fusobacteroidetes,7.79%）和变形菌门（Proteobacteria,7.53%）。丰度最高的科依次是消化链球菌科（Peptostreptococcaceae,平均占16.15%）,梭菌科I（Clostridiaceae_I,14.78%）,毛螺菌科（Lachnospiraceae,13.13%）,红蝽杆菌科（Coriobacteriaceae,12.31%）等。丰度最高的属是柯林斯氏菌属（Collinsella,11.44%）,艰难梭菌属（Peptoclostridium,10.91%）,狭窄梭菌属1（Clostridium_sensu_stricto_1,10.3%）,拟杆菌属（Bacteroides,7.41%）,消化链球菌属（Peptostreptococcus,5.21%）等。7只狞猫的肠道微生物中有平均15.35%的OTU没有归类到属。样本间聚类分析结果表明,来自同一动物园的样本聚为一支。[结论]本文通过高通量测序技术研究了狞猫肠道微生物的组成和多样性特征,为狞猫的救护饲养和消化生理学研究提供基础数据。     

基于Illumina MiSeq高通量测序技术分析硇洲岛海星共附生微生物多样性

【背景】海星作为海洋生物中的一类比较高级的棘皮类动物,其体内蕴藏着丰富且具有生物活性的共附生微生物资源。【目的】分析湛江硇洲岛海星中共附生微生物的多样性。【方法】采用IlluminaMiSeq高通量测序技术分别对硇洲岛海星进行共附生细菌16SrRNA基因V3-V4区和共附生真菌18S rRNA基因ITS1-ITS2区的测序,并根据测序结果进行OTU聚类分析、α多样性分析及物种分类分析等。【结果】高通量测序获得细菌和真菌Filtered的数目分别为61992和71196个,OTU数目分别为2384和529个。经物种分类分析,共附生细菌主要为变形菌门(Proteobacteria),其平均相对含量高达77.37%;其次是厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)和梭杆菌门(Fusobacteria);其中优势细菌属为嗜冷杆菌属(Psychrobacter)和乳球菌属(Lactococcus)。共附生真菌主要为子囊菌门(Ascomycota),其相对含量高达92.33%;其次是霉菌门(Fungi)、担子菌门(Basidi...     

结直肠癌患者肠道黏膜菌群多样性变化研究

目的应用PCR-DGGE技术对结直肠癌患者肠道黏膜局部菌群多样性进行研究,为结直肠病变的防治提供微生态调节思路。方法收集正常对照组、结直肠息肉组及结直肠癌组患者各30例,采集结直肠黏膜局部肛拭子,提取细菌基因组DNA,采用PCR-DGGE对肠道黏膜局部菌群进行指纹图谱分析。结果正常对照组、结直肠息肉组和结直肠癌组患者PCR-DGGE指纹图谱分析显示3组肠道黏膜局部菌群多样性发生了显著的变化,3组肠道黏膜局部菌群发生了显著的菌群变迁。结论 PCR-DGGE分析结直肠癌患者肠道黏膜局部菌群多样性变化对监测肠道局部微生态变化在结直肠癌的发生过程中的作用具有重要价值。     

高通量测序分析小头裸裂尻鱼皮肤和肠道的微生物多样性

【目的】解析小头裸裂尻鱼不同部位的微生物群落结构、物种组成、多样性特征以及菌群功能差异。【方法】通过Illumina MiSeq扩增子高通量测序,分析小头裸裂尻鱼皮肤黏膜、肠道黏膜和肠道内容物3个部位微生物菌群组成差异,并通过Tax4Fun预测菌群潜在功能。【结果】皮肤黏膜微生物α多样性最高,其Shannon指数显著高于肠道黏膜(P<0.05)和肠道内容物(P<0.001)。主坐标分析表明,皮肤黏膜微生物显著区别于其他2个部位。在门水平小头裸裂尻鱼3个部位相对丰度前五的微生物类群均为放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)和蓝藻门(Cyanobacteria),其中肠道内容物中放线菌门相对丰度(46.53%)显著高于肠道黏膜(29.23%,P<0.05)和皮肤黏膜(25.83%,P<0.01);肠道黏膜中变形菌门的相对丰度(40.33%)显著高于肠道内容物(26.10%,P<0.05)。对各部位相对丰度前10的菌群进行分析发现,小头裸裂尻鱼皮肤黏...     

早期灌喂母源粪菌对新生仔猪肠道菌群发育的影响

【目的】粪菌移植(fecal microbiota transplantation,FMT)作为一种治疗手段,已在人类肠道疾病治疗中有较多应用,但在干预新生仔猪肠道菌群上的研究未见报道。本文旨在研究早期母源粪菌移植对新生仔猪肠道菌群发育的影响。【方法】选取一窝12头杜长大新生仔猪,随机分为粪菌处理组(feces treatment,FT)和对照组(control,CO)。FT组仔猪出生后1–5 d每日灌注母源粪菌接种液,CO组灌注等量生理盐水。于1、3、5、7、10、14、18和22日龄采集仔猪粪样,Miseq高通量测序分析仔猪粪便菌群。【结果】灌喂母源粪菌有增加仔猪肠道菌群丰富度的趋势;主坐标分析显示,两组仔猪粪样菌群结构簇并未分开,并在18和22日龄时靠近母猪粪样菌群结构簇;随日龄增加,两组仔猪肠道中的变形菌门丰度均显著降低,而厚壁菌门的丰度显著增加,且从10日龄起拟杆菌门和厚壁菌门之和约为90%;与对照组相比,灌喂母源粪菌增加了10日龄时Escherichia-Shigella的丰度,而降低了18日龄时该菌属的丰富度,18日龄时肠球菌属和普氏菌属的丰度则显著增加。【结论】1–3日龄口服灌喂母源粪菌液并不能影响仔猪肠道菌群的定殖,这一阶段主要受母体微生物结构的影响;灌喂粪菌液对仔猪肠道菌群定殖的影响最多持续10–14 d;而且仔猪在22 d左右,肠道菌群结构逐渐趋同于母猪肠道菌群。     

高通量测序分析一只临床死亡阿拉伯狒狒的胃肠道菌群

运用高通量测序分析一只临床死亡的阿拉伯狒狒胃肠道菌群的组成及多样性,结合其临床症状和检测结果进一步分析该动物的死亡原因,为野生动物的饲养管理和物种健康提供基础资料。

以2017年12月20日成都动物园一只死亡的幼龄雌性阿拉伯狒狒为研究对象,收集其胃、十二指肠、空肠、回肠、盲肠和结肠的内容物及粪便,利用高通量测序技术对细菌16S rRNA基因的V4区进行测序,分析细菌菌群的组成和多样性差异。

十二指肠的菌群有效序列数较高,而盲肠和粪便菌群的alpha多样性较高;胃肠道菌群丰度不同,但都主要来自变形菌门、厚壁菌门和拟杆菌门,而大肠具有较多的梭杆菌门;海尔曼螺杆菌和大肠埃希菌分别在胃和小肠富集,嗜肺巴氏杆菌和具核梭杆菌在结肠富集,并且结肠具有较多来自变形菌门的特有细菌;菌群的功能预测提示胃和小肠具有腹泻和胃肠炎风险,结肠具有菌群失调风险。

阿拉伯狒狒的胃肠道细菌菌群的高通量测序分析和临床诊断及器官组织病理检测结果表明该动物患有的间质性肺炎与小肠富集的大肠埃希菌关系密切,其直接死亡可能与嗜肺巴氏杆菌有关,这些症状进一步导致其结肠菌群失调而加剧了死亡,提示饲养管理中应加强对动物园阿拉伯狒狒等野生动物肠道大肠埃希菌和嗜肺巴氏杆菌的监测,提高野生动物的饲养管理和物种健康。

     

基于高通量测序的不同年龄恒河猴肠道菌群结构差异分析

目的观察不同年龄恒河猴肠道菌群结构和丰度的变化。方法 50只恒河猴,分为成年(5～10岁)、老年(10岁以上)2组,其中成年组33只,老年组17只。采集动物的新鲜直肠粪便,提取DNA后使用Illumina高通量测序平台对样本中细菌16S rDNA-V3区进行测序,定量分析肠道菌群的结构和丰度。结果老年组和成年组获得的优化序列数差异无显著性(P> 0.05)。α-多样性分析,老年组恒河猴肠道菌群的Chao1指数(P=0.0174)、Simpson指数(P=0.0258)、ACE指数(P=0.0121)与成年组比较降低,Shannon指数与成年组比较升高(P=0.0132)。老年组与成年组相比,在门水平,拟杆菌门(Bacteroidetes)相对丰度升高(P=0.0013),厚壁菌门(Firmicutes)、螺旋体门(Spirochaetes)、疣微菌门(Verrucomicrobia)、粘胶球形菌门(Lentisphaerae)相对丰度降低(P=0.0283,P=0.0002,P=0.0482,P=0.0242)。在科水平,Prevotellaceae相对丰度升高(P=0.0001),Ruminococcaceae、Clostridiales、Spirochaetaceae、Christensenellaceae相对丰度降低(P=0.0039,P=0.0080,P=0.0002,P=0.0021)。在属水平,老年组unidentified_Prevotellaceae相对丰度升高(P=0.0001),乳杆菌属(Lactobacillus)、八迭球菌(Sarcina)、unidentified_Spirochaetaceae相对丰度降低(P=0.0114,P=0.0227,P=0.0028)。β-多样性分析,老年组肠道菌群与成年组分布在不同区域,差异有显著性(P=0.003)。LEfSe分析,在成年组,链球菌属(Streptococcus)、布赫纳氏菌属(buchnera)、乳杆菌属(lactobacillus)是具有统计学意义的生物标记物。结论恒河猴肠道菌群的结构随年龄增长而改变。丰度降低,多样性增高。