High-throughput Sequencing Technology and Its Application

Gene sequencing is a great way to interpret life, and high-throughput sequencing technology is a revolutionary technological innovation in gene sequencing researches. This technology is characterized by low cost and high-throughput data. Currently, high-throughput sequencing technology has been widely applied in multi-level researches on genomics, transcriptomics and epigenomics. And it has fundamentally changed the way we approach problems in basic and translational researches and created many new possibilities. This paper presented a general description of high-throughput sequencing technology and a comprehensive review of its application with plain, concisely and precisely. In order to help researchers finish their work faster and better, promote science amateurs and understand it easier and better.

Comparison of rumen archaeal diversity in adult and elderly yaks(Bos grunniens)using 16S rRNA gene high-throughput sequencing

This study was conducted to investigate the phylogenetic diversity of archaea in the rumen of adult and elderly yaks. Six domesticated female yaks, 3 adult yaks （（5.3±0.6） years old）, and 3 elderly yaks （（10.7±0.6） years old）, were used for the rumen contents collection. Illumina MiSeq high-throughput sequencing technology was applied to examine the archaeal composition of rumen contents. A total of 92 901 high-quality archaeal sequences were analyzed, and these were assigned to 2 033 operational taxonomic units （OTUs）. Among these, 974 OTUs were unique to adult yaks while 846 OTUs were unique to elderly yaks; 213 OTUs were shared by both groups. At the phylum level, more than 99% of the obtained OTUs belonged to the Euryarchaeota phylum. At the genus level, the archaea could be divided into 7 archaeal genera. The 7 genera （i.e., Methanobrevibacter, Methanobacterium, Methanosphaera, Thermogymnomonas, Methanomicrobiu, Meth- animicrococcus and the unclassified genus） were shared by all yaks, and their total abundance accounted for 99% of the rumen archaea. The most abundant archaea in elderly and adult yaks were Methanobrevibacterand Thermogymnomonas, respectively. The abundance of Methanobacteria （class）, Methanobacteriales （order）, Methanobacteriaceae （family）, and Methanobrevibacter （genus） in elderly yaks was significantly higher than in adult yaks. In contrast, the abundance of Ther-mogymnomonas in elderly yaks was 34% lower than in adult yaks, though the difference was not statistically significant. The difference in abundance of other archaea was not significant between the two groups. These results suggested that the structure of archaea in the rumen of yaks changed with age. This is the first study to compare the phytogenetic differences of rumen archaeal structure and composition using the yak model.

Genome-wide identification of RNA editing in seven porcine tissues by matched DNA and RNA high-throughput sequencing

Background: RNA editing is a co/posttranscriptional modification mechanism that increases the diversity of transcripts, with potential functional consequences. The advent of next-generation sequencing technologies has enabled the identification of RNA edits at unprecedented throughput and resolution. However, our knowledge of RNA editing in swine is still limited.Results: Here, we utilized RES-Scanner to identify RNA editing sites in the brain, subcutaneous fat, heart, liver,muscle, lung and ovary in three 180-day-old Large White gilts based on matched strand-specific RNA sequencing and whole-genome resequencing datasets. In total, we identified 74863 editing sites, and 92.1% of these sites caused adenosine-to-guanosine(A-to-G) conversion. Most A-to-G sites were located in noncoding regions and generally had low editing levels. In total, 151 A-to-G sites were detected in coding regions(CDS), including 94 sites that could lead to nonsynonymous amino acid changes. We provide further evidence supporting a previous observation that pig transcriptomes are highly editable at PRE-1 elements. The number of A-to-G editing sites ranged from 4155(muscle) to 25001(brain) across the seven tissues. The expression levels of the ADAR enzymes could explain some but not all of this variation across tissues. The functional analysis of the genes with tissuespecific editing sites in each tissue revealed that RNA editing might play important roles in tissue function.Specifically, more pathways showed significant enrichment in the fat and liver than in other tissues, while no pathway was enriched in the muscle.Conclusions: This study identified a total of 74863 nonredundant RNA editing sites in seven tissues and revealed the potential importance of RNA editing in tissue function. Our findings largely extend the porcine editome and enhance our understanding of RNA editing in swine.

Microbial diversity in Huguangyan Maar Lake of China revealed by high-throughput sequencing

Huguangyan Maar Lake is a typical maar lake in the southeast of China. It is well preserved and not disturbed by anthropogenic activities. In this study, microbial community structures in sediment and water samples from Huguangyan Maar Lake were investigated using a high-throughput sequencing method. We found significant differences between the microbial community compositions of the water and the sediment. The sediment samples contained more diverse Bacteria and Archaea than did the water samples. Actinobacteria, Betaproteobacteria, Cyanobacteria, and Deltaproteobacteria predominated in the water samples while Deltaproteobacteria, Anaerolineae, Nitrospira, and Dehalococcoidia were the major bacterial groups in the sediment. As for Archaea, Woesearchaeota (DHVEG-6), unclassified Archaea, and Deep Sea Euryarchaeotic Group were detected at higher abundances in the water, whereas the Miscellaneous Crenarchaeotic Group, Thermoplasmata, and Methanomicrobia were significantly more abundant in the sediment. Interactions between Bacteria and Archaea were common in both the water column and the sediment. The concentrations of major nutrients (NO^3-, PO4^3-, SiO3^2- and NH4^+) shaped the microbial population structures in the water. At the higher phylogenetic levels including phylum and class, many of the dominant groups were those that were also abundant in other lakes;however, novel microbial populations (unclassified) were often seen at the lower phylogenetic levels. Our study lays a foundation for examining microbial biogeochemical cycling in sequestered lakes or reservoirs.

Differential mRNA expression profiling of oral squamous cell carcinoma by high-throughput RNA sequencing

Differentially expressed genes are thought to regulate the development and progression of oral squamous cell carcinomas （OSCC）. The purpose of this study was to screen differentially expressed mRNAs in OSCC and matched paraneoplastic normal tissues, and to explore the intrinsic mechanism of OSCC development and progres- sion. We obtained the differentially expressed mRNA expression profiles in 10 pairs of fresh-frozen OSCC tissue specimens and matched paraneoplastic normal tissue specimens by high-throughput RNA sequencing. By using Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analyses, the functional significance of the differentially expressed genes were analyzed. We identified 1,120 sig- nificantly up-regulated mRNAs and 178 significantly down-regulated mRNAs in OSCC, compared to normal tissue. The differentially expressed mRNAs were involved in 20 biological processes and 68 signal pathways. Compared to adjacent normal tissue, the expression of MAGEAll was up-regulated; TCHH was down-regulated. These find- ings were verified by real-time PCR. These differentially expressed mRNAs may function as oncogenes or tumor suppressors in the development and progression of OSCC. This study provides novel insights into OSCC. However, further work is needed to determine if these differentially expressed mRNAs have potential roles as diagnostic bio- markers and candidate therapeutic targets for OSCC.

Characterization of Bacterial Community Associated with Four Organs of the Yesso Scallop (Patinopecten yessoensis) by High-Throughput Sequencing

We used Illumina high-throughput sequencing of PCR-amplified V3-V4 16 S rRNA gene regions to characterize bacterial communities associated with the adductor muscles, gills, gonads and intestines of the Yesso scallop(Patinopecten yessoensis) from waters around Zhangzidao, Dalian, China. Overall, 421,276 optimized reads were classified as 25 described bacterial phyla and 308 genera. Firmicutes, Proteobacteria, Tenericutes, Bacteroidetes, Chlamydiae and Spirochaetae accounted for > 97% of the total reads in the four organs. The bacterial 16 S rDNA sequences assigned to Firmicutes and Proteobacteria were abundant in the adductor muscles, gills and gonads; while reads from Tenericutes were dominant in the intestines, followed by those from Firmicutes, Chlamydiae, Proteobacteria and Bacteroidetes. At the genus level, the dominant genera in the adductor muscles, gills and gonads appeared to be Bacillus, Enterococcus and Lactococcus, whereas Mycoplasma was dominant in the intestines. The relative abundances of Bacillus, Enterococcus, Lactococcus, Alkaliphilus, Raoultella, Paenibacillus and Oceanobacillus were significantly lower in the intestine than in the other three organs. Cluster analysis and principal coordinates analysis of the operational taxonomy units profile revealed significant differences in the bacterial community structure between the intestine and the other three organs. Taken together, these results suggest that scallops have intestine-specific bacterial communities and the adductor muscles, gills and gonads harbor similar communities. The difference in the bacterial community between organs may relate to unique habitats, surroundings, diet and their respective physiological functions.

Gelatin filter capture-based high-throughput sequencing analysis of microbial diversity in haze particulate matter

Airborne particulate matter(PM),especially PM2.5,can be easily adsorbed by human respiratory system.Their roles in carrying pathogens for spreading epidemic diseases has attracted great concern.Herein,we developed a novel gelatin filter-based and culture-independent method for investigation of the microbial diversity in PM samples during a haze episode in Tianjin,China.This method involves particle capture by gelatin filters,filter dissolution for DNA extraction,and high-throughput sequencing for analysis of the microbial diversity.A total of 584 operational taxonomic units(OTUs)of bacteria and 370 OTUs of fungi at the genus level were identified during hazy days.The results showed that both bacterial and fungal diversities could be evaluated by this method.This study provides a convenient strategy for investigation of microbial biodiversity in haze,facilitating accurate evaluation of airborne epidemic diseases.

High-throughput sequencing analysis of differentially expressed mi RNAs and target genes in ischemia/reperfusion injury and apelin-13 neuroprotection

miRNAs regulate a variety of biological processes through pairing-based regulation of gene expression at the 3＇ end of the noncoding region of the target miRNA, miRNAs were found to be abnormally expressed in ischemia/reperfusion injury models. High-throughput sequencing is a recently developed method for sequencing miRNAs and has been widely used in the analysis of miRNAs. In this study, ischemia/reperfusion injury models were intracerebroventricularly injected with 50 pg/kg apelin-13. High-throughput sequencing showed that 357 known miRNAs were differentially expressed among rat models, among which 78 changed to 〉 2-fold or 〈 0.5-fold. Quantita- tive real-time polymerase chain reaction was selected to confirm the expression levels of four miRNAs that were differentially expressed, the results of which were consistent with the results of high-throughput sequencing. Gene Ontology analysis revealed that the predicted targets of the different miRNAs are particularly associated with cellular process, metabolic process, single-organism process, cell, and binding. Kyoto Encyclopedia of Gene and Genome analysis showed that the target genes are involved in metabolic pathways, mitogen-ac- tivated protein kinase signaling pathway, calcium signaling pathway, and nuclear factor-KB signaling pathway. Our findings suggest that differentially expressed miRNAs and their target genes play an important role in ischemia/reperfusion injury and neuroprotection by apelin-13.

High throughput RNA sequencing utility for diagnosis and prognosis in colon diseases

RNA sequencing is the use of hight hroughput next generation sequencing technology to survey, characterize, and quantify the transcriptome of a genome. RNA sequencing has been used to analyze the pathogenesis of several malignancies such melanoma, lung cancer, and colorectal cancer. RNA sequencing can identify differential expression of genes(DEG's), mutated genes, fusion genes, and gene isoforms in disease states. RNA sequencing has been used in the investigation of several colorectal diseases such as colorectal cancer, inflammatory bowel disease(ulcerative colitis and Crohn's disease), and irritable bowel syndrome.

High Throughput Sequencing of circRNAs in Tomato Leaves Responding to Multiple Stresses of Drought and Heat

Our aim is to study the roles of a new emerging group of non-coding RNAs, circRNAs, in tomato(Solanum lycopersicum L.) plants grown at the combination of drought and heat, two of the most usual stress conditions known to frequently happen in field. Tomato seedlings from cultivar‘Jinling Meiyu’ were treated without stresses(control), at water shortage, high temperature and subjected the multiple stresses. In total, 467 circRNAs were identified with 87.82% from exon using high throughput sequencing technology. Among the circRNAs, 70 were from chr1 with the range from 23 to 49 from the other chromosomes. In detail, 156 circRNAs were shared in the four libraries, while 21, 17 and 36 circRNAs were only shown in drought, heat and multiple stresses libraries, respectively. Through a differential expression analysis, four, seven and nine circRNAs were differentially regulated in tomato at drought, heat and multiple stresses as compared with control. These circRNAs played roles on photosynthesis, starch and sucrose metabolism, RNA transport, RNA degradation, spliceosome, ribosome, etc. Our study underlined the potential role of circRNAs involved in the abiotic stress response in tomato, which might pave the way for studying biological roles of circRNAs responding to multiple stresses in plants.

Microbial Diversity in Rhizosphere Soil of Cotinus coggygria Based on High Throughput Sequencing

In order to reveal the influence of different plant configurations on the microbial community structure and diversity in rhizosphere soil of Cotinus coggygria in Fragrant Hills park,the ITS+5.8S rDNA gene and 16S rDNA gene V3-V4 region sequencing analysis for fungi and bacteria,respectively,were conducted by high throughput sequencing(Illumina MiSeq).The results showed that the fungal diversity in the rhizosphere soil samples of C.coggygria in Fragrant Hills park in 2018 was significantly higher than that in 2016,and it was higher in the rhizosphere soil of healthy C.coggygria in Xunlupo than that in diseased ones in 2018.Verticillium dahliae,which is the causal agent of C.coggygria wilt,was detected in five soil samples.In 2018,the bacterial diversity in the rhizosphere soil of diseased C.coggygria in Xunlupo was the lowest,while it was the highest in the rhizosphere soil of healthy C.coggygria under Platycladus orientalis in Langfengting.

基于高通量测序的眼-鼻区系微生物菌群研究进展

近年来,微生物在眼部的作用日益受到重视。传统的眼部微生物鉴定主要集中于涂片、细菌培养等检查手段,但受到培养条件等各种因素限制导致阳性率低。随着人类宏基因组计划的启动实施,高通量测序技术因其通量高、灵敏度及特异性高、信息量丰富、成本低的特点可将微生物群落定义到属、种水平,有助于更好的理解眼部疾病的病因、诊断、治疗及预防,推动眼科实现精准化医疗进程。由于眼部与鼻部在解剖结构上相通,本文旨在探讨眼部和鼻部微生物菌群策略的研究进展及眼-鼻区系微生物可能的传播途径,为眼鼻相关疾病的研究提供基础。

网箱养殖对喀斯特深水型湖泊水体细菌群落结构的影响--以万峰湖为例

微生物群落结构的变化可反映养殖环境的变化,研究水体微生物群落结构组成及功能对管理和维护湖泊生态环境具有重要意义。2019年6月,在贵州省万峰湖(104.5~105.1°E,24.7~24.9°N)网箱上游1000 m处(对照点)、网箱内、网箱下游200、400、600、800 m处设置采样点,每个采样点均采集表层、中层(25 m)、底层(50 m)水体水样。利用高通量测序技术分析水样中细菌群落多样性,结果显示:18个水样中共鉴定出细菌38门、699属,其中Flavobacterium(黄杆菌属)和Pseudomonas(假单胞菌属)是万峰湖水体中优势菌属,网箱底层水体中分布有Lactobacillus(乳杆菌属)、Barnesiella、Shewanella(希瓦氏菌属)、Sphingomonas(鞘氨醇单胞菌属)等特异性优势菌属。对不同采样点细菌多样性分析表明:万峰湖水体与网箱的水平距离是影响细菌群落结构的关键,影响范围在800 m以内。网箱养殖造成了底层水体大量特异性细菌种属的产生。pH、总磷和化学需氧量是影响网箱水体细菌群落的关键因子。研究结果可为湖泊渔业养殖可持续性发展和湖泊生态环境保护提供参考。

肝癌细胞株-乙型肝炎病毒DNA整合事件的高通量靶向捕获测序分析

目的 采用探针捕获和高通量测序技术研究HepG2.2.15与HepAD38 2个乙型肝炎病毒(hepatitis B virus, HBV)肝癌细胞系中病毒DNA在宿主基因组中的整合位点特征。方法 提取HepG2.2.15与HepAD38细胞基因组DNA,采用探针捕获和高通量测序策略对2个肝癌细胞株中整合的HBV DNA进行捕获测序,使用Trimmomatic、BBAP、BWA(Burrows-Wheeler Aligner)等生物信息学软件进行数据分析;最后通过聚合酶链反应(polymerase chain reaction, PCR)及克隆测序对病毒DNA整合特征进行验证。结果 利用探针捕获和高通量测序技术分别在HepG2.2.15与HepAD38细胞系中检测出12、7个HBV DNA整合事件,随机分布在chr1、chr2、chr7、chr9、chr16、chr17、chr19、chrX染色体上,整合位点上下游基因出现频率较高的有DPP7、TRIM56、GPC3等。结论 成功建立基于探针捕获和高通量测序技术检测HBV DNA在宿主基因组上的整合片段的方法,HepG2.2.15与HepAD38细胞系中HBV整合事件在染色体上随机发生,大部分整合位点发生在HBV基因组上的X基因区域。

荒漠-绿洲过渡带不同生境芦苇根际土壤真菌多样性及群落结构分析

以沙漠-绿洲边缘4种不同生境(湿地生境、盐渍生境、盐渍-沙地过渡生境和沙地生境)芦苇群落为研究对象,基于高通量测序法探究芦苇根际土壤真菌多样性及群落结构。结果表明,4种生境芦苇根际真菌共检测出3门18纲51目98科128属145种。在门分类水平,子囊菌门(Ascomycota)、担子菌门(Basidiomycota)分别为盐渍-沙地过渡生境、湿地生境主要优势菌门,而毛霉门(Mucoromycota)在盐渍生境中相对丰度最高。在属分类水平,湿地生境、盐渍生境、盐渍-沙地过渡生境和沙地生境的优势物种分别为棉革菌属(Tomentella)、古根菌属(Archaeorhizomyces)、梨孢属(Pyricularia)和小脆柄菇属(Psathyrella)。4种生境间真菌多样性和物种组成存在显著差异,有益菌与有害菌共存。湿地生境真菌物种丰富度和多样性最高,沙地生境和盐渍-沙地过渡生境主要优势物种更多,沙地生境和盐渍-沙地过渡生境的菌群组成相似,但真菌群落与环境因子关系尚不明确。上述研究结果对于认识河西走廊不同生境芦苇根际微环境具有一定参考价值。

基于RNA-seq测序的弓獭蛤(Lutraria rhynchaena)不同组织转录组差异研究

弓獭蛤(Lutraria rhynchaena)是我国南方沿海重要的海水养殖贝类,为探讨弓獭蛤不同组织的基因表达差异,本研究采用RNA-seq测序技术对北部湾海域弓獭蛤7种组织(肌肉、外套膜、鳃、肝胰腺、虹吸管、雌性性腺和雄性性腺)进行转录组测序及生物信息学分析。结果显示,21个样本共获得939 197 518条质控后序列,各组织质控后的碱基数为15.39—29.26 G。测序结果经过Denovo组装和ORF查找共获得56 773个Unigenes。在5个公共数据库NR (Non-Redundant protein sequence database)、GO (Gene Ontology)、eggNOG(evolutionary genealogy of genes:Non-supervised Orthologous Groups)、KEGG (Kyoto Encyclopedia of Genes and Genomes)和SWISS-Prot对比各注释分别得到24 557、13 094、17 524、10 352和13 857条有效注释信息。本研究获得了大量弓獭蛤组织间的差异表达基因,丰富了弓獭蛤的基因数据库,为更深入地了解弓獭蛤各组织功能,进一步挖掘和开发利用弓獭蛤功能基因提供基础数据。

稻-红螯螯虾共作模式下浮游生物的群落变化特征及其与虾生长之间的关系

为探究稻渔综合种养模式下浮游生物的群落变化特征及其与养殖对象之间的关系,2022年6月-9月采用18S rRNA基因高通量测序技术调查了浙江省海宁市6处稻红螯螯虾(Cherax quadricarinatus)养殖水体中浮游生物的群落组成及多样性的变化,并结合Spearman相关性热图分析了优势种类和多样性指数与生长性状之间的关系。结果显示:红螯螯虾的体长、头胸甲宽、体重、增重率、特定生长率和肥满度在不同稻田间无显著差异,但体长、头胸甲宽和体重随着养殖时间的延长明显增加,特定生长率则显著下降。虾引入稻田后会相对改善水体中浮游生物的群落多样性,然而,随着养殖时间的延长,群落稳定性逐渐降低。隐藻(Cryptomonas)、衣藻(Chlamydomonas)、小球藻(Chlorella)和中剑水蚤(Mesocyclops)是共作期间常见的优势种类。浮游植物的优势种或主要种类(浮游生物中排名前50的种类)与浮游动物的变化趋势不同,前者的相对丰度逐渐增加,后者则逐渐降低。分析发现,季节变化是驱动稻虾共作模式下浮游生物群落演替的主要原因。此外,虾的摄食作用也会对群落变化产生一定影响。相关性分析显示,肥满度与小球藻生物量显著负相关,表明养殖水体的水质对虾的生长和健康状况具有重要影响,因此需平衡好水稻种植区和水产养殖区对营养水平需求不同的矛盾。

Characterization of root-associated bacterial community structures in soybean and corn using locked nucleic acid(LNA) oligonucleotide-PCR clamping and 454 pyrosequencing

supported in part by grants from the Strategic Priority Research Program of Chinese Academy of Sciences （XDB15010103）;the National Natural Science Foundation of China （41201247）

浓香型白酒大曲微生物群落α-多样性分析

本文采用高通量测序技术,对泸州某制曲厂的浓香型白酒大曲进行微生物群落α-多样性动态分析,共获得细菌有效序列23505条和真菌有效序列533410条。研究结果表明,浓香型白酒大曲中的微生物α-多样性指数在发酵过程中呈现动态变化。在发酵初期,大曲中生物群落的α-多样性均逐步增加,到达峰值后会受到生物抑制作用而逐渐降低;在发酵中期,微生物群落的α-多样性指数达到最低;在发酵后期,微生物的α-多样性指数逐渐升高并形成种群稳定的群落结构。由此可见,发酵后期对大曲的微生物群落结构及浓香型白酒品质的形成具有重要作用。

叠氮丙啶-实时荧光聚合酶链式反应-高通量测序技术鉴定冷链食品中多种病原菌

目的 应用叠氮丙啶-实时荧光聚合酶链式反应-高通量测序技术鉴定冷链食品中多种病原菌,构建病原菌分子进化树。方法 以冷链食品中的沙门氏菌、金黄色葡萄球菌、蜡样芽胞杆菌、副溶血性弧菌、单核细胞增生李斯特氏菌5种病原菌作为研究对象,应用叠氮丙啶-实时荧光聚合酶链式反应技术作为冷链食品中病原菌检测初筛手段,应用微生物培养法以及生化鉴定仪器法进行方法比对与结果验证,运用高通量测序以及分子进化树构建作为冷链食品中所分离病原菌的种属地位确证方法。结果 叠氮丙啶-实时荧光聚合酶链式反应技术成功扩增了冷链食品中生活状态病原菌的特征性核酸片段,排除了死亡细菌以及阴性对照菌的干扰,病原菌检出限可达到1×10^(3)CFU/mL,一次反应可检测42份试样,可以在18 h内完成检测工作。在冷链食品中病原菌抽样检测调查中,随机采集的751份冷链食品,共检出62株病原菌,病原菌总体检出率为8.3%(62/751)。通过后续的16S rRNA测序以及葡萄球菌属、弧菌属以及李斯特菌属分子进化树的构建,成功溯源了金黄色葡萄球菌的污染来源并完成病原菌种属定位。结论 本方法特异性好、灵敏度高、检测通量高,为冷链食品及相关食品中病原菌的精确检测与溯源分析提供新的思路与方法。