基于Illumina HiSeq 2000测序技术对当归根的转录组特性研究

目的 识别当归Angelica sinensis根药用活性物质的基因序列,阐释当归次生代谢产物生物合成的遗传基础.方法 从当归头、当归尾提取总RNA,质量鉴定合格后构建测序文库.测序文库检测合格后在Illumina HiSeq 2000测序仪上完成双端测序.采用相关生物信息学方法分析当归根的转录组特性.结果 采用Illumina HiSeq 2000高通量测序获得当归根转录组的66 431 540个原始短读序.用生物信息学方法从头组装并注释了30 432个功能基因序列(unigene).应用Uniprot蛋白数据库进行序列相似性比对,当归功能基因序列主要分布于目前研究较深入的7种重要经济作物,如葡萄、蓖麻、毛果杨、大豆、苜蓿、拟南芥和烟草.研究发现,当归根表达的127、69、70、94个unigene分别映射到类苯基丙烷生物合成、N-聚糖生物合成、黄酮类化合物生物合成和叶酸生物合成等路径,可能参与药用活性物质阿魏酸、当归多糖、当归总黄酮和叶酸的生物合成.结论 参与当归根重要药效物质(阿魏酸、当归多糖、当归总黄酮等)生物合成的功能基因序列可能是当归补血、活血功效的分子生物学基础.     

冬病夏治方穴位贴敷对哮喘豚鼠气道炎症及肠道菌群的影响

目的:探究冬病夏治方穴位贴敷给药对哮喘豚鼠的治疗作用及其对肠道菌群的影响。方法:将75只豚鼠随机分成空白组、哮喘模型组、阳性(地塞米松磷酸钠)组、穴位给药组、非穴位给药组(n=15),采用卵蛋白注射及雾化吸入的方法建立豚鼠哮喘模型,通过肺组织病理苏木素-伊红(HE),马松(Masson)染色观察冬病夏治方对豚鼠哮喘的治疗效果。提取豚鼠粪便总基因组DNA,对16 S rRNA V4可变区基因进行PCR扩增及Illumina Hi Seq测序,将基因序列相似度高于97%的序列进行聚类归并,并进行生物信息学分析。结果:与哮喘模型组比较,各给药组豚鼠肺组织炎症细胞减少,支气管收缩、气道上皮增生症状改善明显,恢复良好,胶原纤维沉积均有不同程度减少,且地塞米松磷酸钠组与穴位给药组的减少程度明显,穴位给药组的治疗效果优于非穴位给药组。通过操作分类单元(OTU)及其丰度分析发现,冬病夏治方穴位贴敷给药后豚鼠的肠道菌群多样性升高,普氏菌属显著增多,拟杆菌门减少,变形菌增多;哮喘模型组厚壁菌数目较空白组及各给药组减少。结论:冬病夏治方穴位贴敷能有效治疗哮喘,改善哮喘症状,调控哮喘豚鼠肠道菌群的构成。提示该方可能通过调控肠道菌群从而影响机体免疫,达到治疗哮喘的效果,为研究哮喘的治疗机制提供一定的理论基础。     

A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

PRRSV is a positive-sense RNA virus with a high degree of genetic variability among isolates. For diagnostic sensitivity and vaccine design it is essential to monitor PRRSV genetic diversity. However, to date only a few full genome sequences of PRRSV isolates have been made publicly available. In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally.     

SPF鸡不同生长阶段粪便菌群组成及多样性研究

目的 提供SPF鸡不同生长阶段肠道菌群的组成及多样性数据.方法 采集10日龄、12周龄、23周龄和52周龄SPF鸡粪便,利用Illumina HiSeq 2500高通量测序方法,对样品16S rRNA的两个高变区(V3-V4)进-测序分析,并进行了α-多样性指数、β-多样性指数和粪便群落特征分析.结果 四个生长阶段粪便样品菌群具有相似的较高的丰富度和良好的均匀度,物种多样性无显著差异(P＞0.05);厚壁菌门(Firmicutes)、变形菌门(Proteobacte ria)和拟杆菌门(Bacte roidetes丰度占95％以上;乳杆菌属(Lactobacillus)、链球菌属(Streptococcus)、拟杆菌属(Bacteroides)、肠球菌属(Enterococcus)为优势菌属,不同时期丰度差异达显著水平(P＜0.05).结论 四个生长阶段菌群组成多样,且优势菌群主要为有益菌.该实验结果能够为SPF鸡品质评定、健康监测和安全性评价提供依据.     

Analysis of hepatitis B virus genotyping and drug resistance gene mutations based on massively parallel sequencing

Drug resistance to nucleoside analogs is a serious problem worldwide. Both drug resistance gene mutation detection and HBV genotyping are helpful for guiding clinical treatment. Total HBV DNA from 395 patients who were treated with single or multiple drugs including Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir and Emtricitabine were sequenced using the HiSeq 2000 sequencing system and validated using the 3730 sequencing system. In addition, a mixed sample of HBV plasmid DNA was used to determine the cutoff value for HiSeq-sequencing, and 52 of the 395 samples were sequenced three times to evaluate the repeatability and stability of this technology. Of the 395 samples sequenced using both HiSeq and 3730 sequencing, the results from 346 were consistent, and the results from 49 were inconsistent. Among the 49 inconsistent results, 13 samples were detected as drug-resistance-positive using HiSeq but negative using 3730, and the other 36 samples showed a higher number of drug-resistance-positive gene mutations using HiSeq 2000 than using 3730. Gene mutations had an apparent frequency of 1% as assessed by the plasmid testing. Therefore, a 1% cutoff value was adopted. Furthermore, the experiment was repeated three times, and the same results were obtained in 49/52 samples using the HiSeq sequencing system. HiSeq sequencing can be used to analyze HBV gene mutations with high sensitivity, high fidelity, high throughput and automation and is a potential method for hepatitis B virus gene mutation detection and genotyping.     

基于高通量测序分析连栀矾溶液发酵炮制过程中真菌菌群多样性变化

目的探究传统连栀矾溶液发酵炮制过程中真菌菌群多样性和优势菌群,为科学发酵提供理论依据。方法采用Illumina HiSeq高通量测序技术测定不同发酵时间上下层连栀矾溶液中真菌ITS2区序列,应用QIIME、Mothur和R等软件整理和统计样品序列数目和操作分类单元(OTU)数量,分析样品中真菌菌群的组成、丰度、分布、alpha多样性、beta多样性,阐明炮制过程中真菌菌群结构、多样性及丰富度的动态变化。结果 4个不同的发酵时期共获得用于分析的有效序列数为207 068,稀疏曲线表明测序深度充分,OTUs的数量接近于饱和,能充分展示连栀矾溶液的真菌群落结构,其主要由子囊菌门(Ascomycota,74.21%)、担子菌门(Basidiomycota,7.31%)、罗兹菌门(Rozellomycota,2.62%)、接合菌门(Zygomycota,0.66%)和球囊菌门(Glomeromycota,0.01%)组成,优势菌门为子囊菌门。通过分析OTUs数量、chao1指数,ACE指数、Shannon指数,表明连栀矾溶液真菌菌群有较高的丰富度和多样性,并且在炮制过程中上下层总丰富度和多样性随发酵时间的增加逐渐增加,其中上层样品的丰富度、多样性逐渐增加,下层样品的丰富度、多样性先降低后增加,且上层样品的丰富度、多样性明显高于下层样品。结论以高通量测序技术分析连栀矾溶液发酵炮制过程中真菌菌群结构、多样性及丰富度的动态变化可为指导其科学发酵提供参考。     

基于转录组测序的银杏类黄酮生物合成关键基因表达分析

目的对银杏进行转录组测序,分析银杏生长发育不同时期类黄酮生物合成关键基因的表达。方法以银杏幼年树和成年树不同时期叶片作为供试材料,利用Illumina HiSeq 2000进行转录组测序,对Unigene进行功能注释,分析银杏类黄酮生物合成关键基因的表达特征。结果转录组测序共获得43 073条Unigene,其中35 179条被注释,基因差异表达(DEG)筛选得到5 117个基因。通过对类黄酮合成相关KEGG途径进行分析,筛选出50条候选基因。分析50条候选基因的表达规律,发现类黄酮合成关键基因均在银杏幼叶中高表达,但在成年树与幼年树同时期叶片之间表达差异不显著。分析与类黄酮合成关系密切的13个基因发现,其中肉桂酸4-羟化酶(C4H)、查耳酮合成酶(CHS)、花青素合成酶(ANS)、花青素还原酶(ANR)和类黄酮O-甲基转移酶(FOMT)基因的表达量较高,类黄酮3′-羟化酶(F3′H)、类黄酮3′,5′-羟化酶(F3′5′H)和黄酮醇合成酶(FLS)基因的表达量则相对较低。结论通过转录组高通量测序,筛选分析了银杏类黄酮生物合成的关键基因及其表达特征,为提高银杏类黄酮产量提供了分子生药学理论基础。