High-throughput Sequencing Technology and Its Application
ngs高通量测序流程
ngs高通量测序流程英文回答：NGS (Next-Generation Sequencing) is a high-throughput sequencing technology that has revolutionized the field of genomics. It allows for the rapid and cost-effective sequencing of large amounts of DNA or RNA samples. The NGS workflow involves several steps, including sample preparation, library construction, sequencing, and data analysis.The first step in the NGS workflow is sample preparation. This involves extracting DNA or RNA from the biological sample of interest. The quality and quantity of the extracted nucleic acids are crucial for the success of downstream steps. Various extraction methods and kits are available depending on the sample type.Once the nucleic acids are extracted, the next step is library construction. In this step, the DNA or RNA isfragmented into smaller pieces, and adapters are added to the ends of the fragments. These adapters contain sequences that are necessary for the attachment of the fragments to the sequencing platform. The size selection of the fragments is also performed to ensure that only the desired fragment lengths are sequenced.After library construction, the samples are ready for sequencing. There are different sequencing platforms available, such as Illumina, Ion Torrent, and PacBio. Each platform has its own advantages and limitations in terms of read length, throughput, and error rate. The choice of platform depends on the specific research needs and budget.During the sequencing process, the DNA or RNA fragments in the library are amplified and attached to a solid surface, such as a flow cell in the case of Illumina sequencing. The fragments are then sequenced using fluorescently labeled nucleotides, and the emitted signals are detected and converted into digital data.Once the sequencing is complete, the raw data undergoesquality control and data analysis. Quality control involves checking the sequencing quality scores, read length distribution, and other metrics to ensure the reliability of the data. Data analysis involves aligning the reads to a reference genome or transcriptome, identifying genetic variations, and quantifying gene expression levels.NGS has a wide range of applications in genomics research, including genome sequencing, transcriptome analysis, epigenetics, metagenomics, and personalized medicine. It has enabled researchers to study complex biological processes at an unprecedented level of detail and has led to significant advancements in our understanding of genetics and disease.中文回答：NGS（Next-Generation Sequencing）是一种高通量测序技术，已经彻底改变了基因组学领域。
高通量测序技术在动植物研究领域中的应用
高通量测序技术在动植物研究领域中的应用一、本文概述随着生物技术的飞速发展，高通量测序技术（High-throughput sequencing technology）已成为动植物研究领域的重要工具。
该技术以其快速、准确、高效的特点，极大地推动了动植物基因组学、转录组学、表观遗传学等多个研究领域的进步。
本文旨在全面综述高通量测序技术在动植物研究领域的应用，包括动植物基因组测序、基因表达分析、基因功能研究、种质资源鉴定、遗传育种以及生态保护等方面。
通过深入剖析这些应用案例，旨在为读者提供一个清晰、全面的高通量测序技术应用全景，以期推动该技术在动植物研究领域的进一步发展和应用。
二、高通量测序技术的基本原理与方法高通量测序技术，又称为下一代测序技术（Next Generation Sequencing，NGS），是近年来生物信息学领域的一项革命性技术。
其基本原理是通过将待测样本的DNA或RNA片段化，然后利用高通量测序平台对这些片段进行大规模并行测序。
这种方法大大提高了测序速度和效率，降低了成本，使得研究者可以对基因组、转录组甚至单细胞进行全面的深入研究。
高通量测序的方法主要包括样本准备、文库构建、测序及数据分析等步骤。
在样本准备阶段，研究者需要从动植物组织中提取高质量的DNA或RNA，并通过特定的酶处理将其片段化。
文库构建则是将这些片段与测序引物连接，形成适合测序的文库。
测序阶段则通过高通量测序仪器对文库进行大规模的并行测序，得到原始的测序数据。
在数据分析阶段，研究者需要使用生物信息学工具对原始数据进行处理、组装和注释，最终得到基因组的序列信息、基因结构、表达水平等关键信息。
通过这些信息，研究者可以对动植物的基因组结构、功能、进化等方面进行深入的研究。
高通量测序技术在动植物研究领域的应用广泛，包括但不限于基因组测序、转录组测序、表观遗传学研究、单细胞测序等。
这些应用不仅有助于我们更深入地理解动植物的生物学特性，也为动植物育种、疾病防治、生态保护等领域提供了新的思路和方法。
全转录组测序 辐射损伤生物标志物 英语
全转录组测序辐射损伤生物标志物英语Whole transcriptome sequencing (WTS) is a high-throughput sequencing technology that captures the entire transcriptome, including both coding and non-coding RNA species. It provides comprehensive information about gene expression, alternative splicing, transcript variants, and novel transcripts within a biological sample.Radiation injury biomarkers are molecules or indicators that can be used to detect and monitor the effects of radiation exposure on biological systems. These biomarkers can provide information about the extent and severity of radiation-induced damage, as well as potential responses and recovery processes.In the context of radiation injury, whole transcriptome sequencing can be employed to identify potential biomarkers. By analyzing the transcriptome changes in response to radiation exposure, researchers can identify genes, transcripts, or pathways that are differentially expressed or modulated. These differentially expressed genes or transcripts may serve as candidate biomarkers for radiation injury.Whole transcriptome sequencing offers several advantages in biomarker discovery. It provides a global view of the transcriptome, capturing changes in both known and unknown genes and transcripts. This comprehensive approach increases the chances of identifying novel and potentially overlooked biomarkers.Furthermore, whole transcriptome sequencing allows for the analysis of transcriptional regulation and epigenetic modifications, which can provide insights into the underlying mechanisms of radiation-induced biological responses. By integrating transcriptome data with other omics data, such as genomics and proteomics, a more comprehensive understanding of radiation injury and biomarker discovery can be achieved.In summary, whole transcriptome sequencing is a powerful tool for identifyingradiation injury biomarkers. By analyzing the global transcriptome changes, researchers can uncover candidate biomarkers that may aid in the early detection, monitoring, and therapeutic intervention of radiation-induced injuries. The application of whole transcriptome sequencing in radiation biology holds promise for improving our understanding of radiation toxicity and developing personalized radiation therapy strategies.。
生物知识汇总英文名词缩写
1 HTS高通量测序技术（High-throughput sequencing，HTS）2 WGS全基因组测序3 Chip染色质免疫共沉淀（ChromatinImmunoprecipitation，ChIP）4 CHIRP( Chromatin Isolation by RNA Purification )是一种检测与RNA绑定的DNA和蛋白的高通量测序方法。
方法是通过设计生物素或链霉亲和素探针，把目标RNA拉下来以后，与其共同作用的DNA染色体片段就会附在到磁珠上，最后把染色体片段做高通量测序，这样会得到该RNA能够结合到在基因组的哪些区域，但由于蛋白测序技术不够成熟，无法知道与该RNA结合的蛋白。
5 RIPRNA Immunoprecipitation是研究细胞内RNA与蛋白结合情况的技术，是了解转录后调控网络动态过程的有力工具，能帮助我们发现miRNA的调节靶点。
这种技术运用针对目标蛋白的抗体把相应的RNA-蛋白复合物沉淀下来，然后经过分离纯化就可以对结合在复合物上的RNA进行测序分析。
6 CLIP-seqCLIP-seq,又称为HITS-CLIP，即紫外交联免疫沉淀结合高通量测序(crosslinking-immunprecipitation and high-throughput sequencing)7 SNP单核苷酸多态性singlenucleotide polymorphism，SNP8 SNV单核苷酸位点变异。
单核苷酸变异是一种体细胞突变（somatic mutation），称做SNV9 INDEL (基因组小片段插入）基因组上小片段（>50bp）的插入或缺失，形同SNP/SNV。
10 CNV(copy number variation)基因组拷贝数变异11SV (structure variation)基因组结构变异12 RPKMRPKM,Reads Per Kilobase of exon model per Million mapped reads, is defined in thisway [Mortazavi etal., 2008]:每1百万个map上的reads中map到外显子的每1K个碱基上的reads个数。
高通量测序技术
的玻璃表面(即Flow cell)，这些DNA片段经过延伸和桥式扩增后，在Flow cell上形成了数以亿计Cluster，每个Cluster是具有数千份相同模板的单分子簇。
然后利用带荧光基团的四种特殊脱氧核糖核苷酸，通过可逆性终止的SBS(边合成边测序)技术对待测的模板DNA进行测序。
ABI SOLiD连接法测序(sequence by ligation)技术应用测序技术推进科学研究的发展。
随着第二代测序技术的迅猛发展，科学界也开始越来越多地应用第二代测序技术来解决生物学问题。
比如在基因组水平上对还没有参考序列的物种进行从头测序(de novo sequencing)，获得该物种的参考序列，为后续研究和分子育种奠定基础;对有参考序列的物种，进行全基因组重测序(resequencing)，在全基因组水平上扫描并检测突变位点，发现个体差异的分子基础。
在转录组水平上进行全转录组测序(whole transcriptome resequencing)，从而开展可变剪接、编码序列单核苷酸多态性(cSNP)等研究;或者进行小分子RNA测序(small RNA sequencing)，通过分离特定大小的RNA分子进行测序，从而发现新的microRNA分子。
在转录组水平上，与染色质免疫共沉淀(ChIP)和甲基化DNA免疫共沉淀(MeDIP)技术相结合，从而检测出与特定转录因子结合的DNA区域和基因组上的甲基化位点。
这边需要特别指出的是第二代测序结合微阵列技术而衍生出来的应用--目标序列捕获测序技术(Targeted Resequencing)。
这项技术首先利用微阵列技术合成大量寡核苷酸探针，这些寡核苷酸探针能够与基因组上的特定区域互补结合，从而富集到特定区段，然后用第二代测序技术对这些区段进行测序。
目前提供序列捕获的厂家有Agilent和Nimblegen ，应用最多的是人全外显子组捕获测序。
科学家们目前认为外显子组测序比全基因组重测序更有优势，不仅仅是费用较低，更是因为外显子组测序的数据分析计算量较小，与生物学表型结合更为直接。
高通量测序技术的数据分析方法教程
高通量测序技术的数据分析方法教程随着生物技术的发展，高通量测序技术（high-throughput sequencing technology）已成为生物学、医学和生物信息学研究中的重要工具。
高通量测序技术可以快速而准确地测定DNA或RNA序列，透过大量的数据来揭示生物体的基因组、转录组以及其他生物学过程中的变化。
然而，正确且高效地分析测序数据是高通量测序技术应用的关键一步。
本文将介绍高通量测序技术的数据分析方法教程。
首先，分析高通量测序数据前，我们需要了解常见的测序平台和数据格式。
当前常用的高通量测序平台包括Illumina、ABI SOLiD、Ion Torrent等，而测序数据通常以FASTQ、SAM/BAM和VCF等格式存储。
FASTQ格式用于存储原始测序数据，其中包含了每个测序读段的序列信息及其对应的质量分数。
而SAM/BAM格式则是将测序读段比对到参考基因组之后的结果，其中SAM是比对结果的文本格式，而BAM则是对应的二进制格式。
VCF（Variant Call Format）格式则用于存储基因型变异信息。
接下来，我们将介绍高通量测序数据的基本分析流程。
通常，测序数据分析可以分为质控、比对、变异检测和功能注释几个主要步骤。
在质控步骤中，我们需要对测序数据进行质量评估和过滤。
质量评估可以通过查看测序数据的质量分数、GC含量、碱基分布和测序错误率等指标来判断测序数据的质量。
使用质量评估工具如FastQC和NGS QC Toolkit可以帮助我们快速准确地评估测序数据的质量，并进行相应的过滤工作，去除低质量的测序读段。
接下来，我们需要将测序读段比对到参考基因组上。
比对工作可以通过软件如Bowtie、BWA和HISAT等进行。
比对结果通常以SAM格式存储，然后可以进行排序、去重和索引等处理，生成最终的BAM格式文件。
在变异检测步骤中，我们需要从比对后的BAM文件中检测样本中存在的变异信息。
变异检测可以通过多种工具来实现，如GATK、Samtools和VarScan等。
NuGEN建库产品介绍资料
按照应用类型分类，NGS市场被分为肿瘤、遗传筛查、传染病、药物和生物标志 的发现、分子流行病学、农业等。在所有的这些应用领域中，肿瘤检测在收益和 样本容量上占据最大的市场份额(超过35%)。
背景介绍
背景介绍
测序市场分布
3% 10%
16%
71%
Illumina Life Roche PacBio
Illumina 测 序 仪 升 级 Hiseq2500 ， 单 条 测 序 长 度增长，通量增加，更 受欢迎。
Life 的 Ion Torrent 相 对 通量高，测序时间短， 受到一些客户的喜好。
Roche在2013年10月份 关闭454平台。
PacBio作为新的技术， 通量高，侧许长但稳定序原理
cBot cluster 产生
测序
数据分析
测序技术
核酸提纯
DNA
Total成
Library Construction
Ovation Ultralow Library Systems v2
1 ng DNA input
Ovation® Rapid DR Multiplex System
100 ng DNA input
Ovation Ultralow Methyl-Seq Library Systems
背景介绍
测序技术的发展已经经历了很长的时间，而下一代测序彻底改变了人类基因组测 序的方式。有关预算，未来的几年内，人类基因组测序的费用将在1000美元，12天即可出结果。自实现第一个人类基因组测序以来(花了十年时间，30亿美元)， 测序所需的费用和时间大幅度降低。到2020年，全球NGS市场将达48.92亿美元， 复合年增长率为20.7%。 NGS技术成本的降低和分析速度的提升大大提高了它在临床领域的运用，这样良 好的增长趋势也要源于个性化医疗的增长，医疗开支的增加以及从微阵列到NGS 的转变。
High-throughput sequencing and Platforms--高通量测序以及平台介绍
姓名：刘传宇High-throughput sequencing and PlatformsAbstractHigh-throughput sequencing or called "Next-generation" sequencing technology can sequence hundreds of thousands to millions of DNA molecules in one time and its read is shorter than previous sequencing technique,yet these advantages are symbol of sequencing ,which is the same case of former techniques.However，high-throughput sequencing canenable the transcriptome and genome of a species the panorama of a detailed analysis，so it was also called deepsequencing. Keywordshigh-throughput sequencing deepsequencing next-generation sequencing application platforms third generation sequencing technologyIntroductionsDevelopment Situation of sequencing technique:According to the history of development andinfluence of sequencing principle and technique,it has the following kinds: Massively Parallel Signature Sequencing (MPSS), Polony Sequencing1, 454 pyrosequencing2, illumina (Solexa) sequencing3, ABI SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing and so on,scientific community has been widely used in second generation sequencing technology to solve biological problems. Such as sequencing species which have no referential sequences at the genome level4(de novo sequencing), obtaine the referential sequences of that species.This work lays a foundation for subsequent research and molecular breeding. However,some species which have referential sequence, process whole genome resequencing, scanning and detecting mutations at the whole genome level to find that the molecular basis of individual differences,and do the whole transcriptome sequencing at the transcriptome level , thus to research alternative splicing and coding sequence of single nucleotide polymorphisms (cSNP) or small molecular RNA sequencing5, sequencing by separateing specific size RNA to discover new microRNA.At the transcriptome level, high-throughput sequencing works with the help of Chromatin Immunoprecipitation (ChIP) and Methylated DNA immunoprecipitation technology (MeDIP) to detect DNA areas which binding with specific transcription factors and methylated sites on the genome .Particularly, targeted Resequencing is a great application in high-throughput sequencing and microarray technology. Firstly,this technology uses microarray to synthesise oligonucleotide probeswhich could combined with certain parts of the genome, so that it assemble es specific segment and then use the second generation sequencing to sequencing these sections.Currently, manufacturers which provide sequence capture are Agilent andNimblegen ,but exome sequencing is used much more frequently.Scientists now think that exome sequencing is superiorer to whole genome sequencing not only because of cost lower , but also because that exome sequencing has a smaller amount of data analysis , and it also combines with biological phenotypes more straightforwardly. At present, high throughput sequencing are used widely in searching for candidate genes of disease.Summary of the PlatformsThe first generation of sequencing technology can sequence long fragments and has high accuracy, but it cost too much and it is not able to practise the sequencing of microscale DNA samples.454 was the 1st commercial NGS platform, it was acquired by Roche, but is still known as by the name 454. This sequencer uses pyrosequencing technology. Instead of using dideoxynucleotides to terminate the chain amplification, pyrosequencing technology relies on the detection of pyrophosphate released during nucleotide incorporation. It 454 uses beads that start with a single template molecule which is amplified via emPCR. Millions of beads are loaded onto a picotitre plate designed so that each well can hold only a single bead. All beads are then sequenced in parallel by flowing pyrosequencing reagents across the plate.SOLiD was purchased by Applied Biosystems in 2006. SOLiD was the 3rd commercial NGS platform. The sequencer adopts the technology of two-base sequencing based on ligation sequencing. On a SOLiD flowcell, the libraries can be sequenced by 8 base-probe ligation. The fluorescent signal will be recorded during the probes complementary to the templa te strand and vanished by the cleavage of probes’ last 3 bases. And the sequence of the fragment can be deduced after 5 round of sequencing using ladder primer sets.Solexa was subsequently acquired by Illumina and is now known by the name Illumina. Solexa developed the 2nd commercial NGS platform. Illumina uses a solid glass surface 6(similar to a microscope slide) to capture individual molecules and bridge PCR to amplify DNA into small clusters of identical molecules. These clusters are then sequenced with a strategy that is similar to Sanger sequencing, except only dye-labelled terminators are added, the sequence at that position is determined for all clusters, then the dye is cleaved and another round of dye-labelled terminators are added.Helicos7developed the HeliScope, which was the first commercial single-molecule sequencer. Unfortunately, the high cost of the instruments and short read lengths limited adoption of this platform. Helicos no longer sells instruments, but conducts sequencing via a service centre model.ConclusionThe second generation sequencing technology makes an indelible contribution to biology, although there are still some technical defects. Now, third generation Sequencing technology has emerged, such as Single Molecule Real Time (SMRT ™) DNA Sequencing8, but the second generation Sequencing is still used for large-scalesequencing. Therefore, before the fully maturity of the third generation sequencing technology, the second generation sequencing technology will not exit from sequencing projects.Recently, the third generation sequencing technology, which is able to determine the base composition of single DNA molecules, has joined the race.It is based on single molecular nanopore sequencing9, and a lot of companies, such as Pacific Biosciences10, Oxford Nanpore, Genia, NABsys, NobloGen, and so on. Among these gene chip technology are being used. Its advantages include read longer, faster, more accueate and it does not need to prepare samples.References1 Porreca, G. J., Shendure, J. & Church, G. M. Polony DNA sequencing. Current protocols inmolecular biology / edited by Frederick M. Ausubel ... [et al.]Chapter 7, Unit 7 8, doi:10.1002/0471142727.mb0708s76 (2006).2 Serkebaeva, Y. M., Kim, Y., Liesack, W. & Dedysh, S. N. Pyrosequencing-based assessmentof the bacteria diversity in surface and subsurface peat layers of a northern wetland, with focus on poorly studied phyla and candidate divisions. PloS one8, e63994, doi:10.1371/journal.pone.0063994 (2013).3 Castoe, T. A.et al.Rapid microsatellite identification from Illumina paired-end genomicsequencing in two birds and a snake. PloS one7, e30953, doi:10.1371/journal.pone.0030953 (2012).4 Sanders, S. J.et al.De novo mutations revealed by whole-exome sequencing are stronglyassociated with autism. Nature485, 237-241, doi:10.1038/nature10945 (2012).5 Gausson, V. & Saleh, M. C. Viral small RNA cloning and sequencing. Methods Mol Biol721,107-122, doi:10.1007/978-1-61779-037-9_6 (2011).6 Hofs, B., Brzozowska, A., de Keizer, A., Norde, W. & Cohen Stuart, M. A. Reduction ofprotein adsorption to a solid surface by a coating composed of polymeric micelles with a glass-like core. Journal of colloid and interface science325, 309-315, doi:10.1016/j.jcis.2008.06.006 (2008).7 Kapranov, P., Ozsolak, F. & Milos, P. M. Profiling of short RNAs using Helicossingle-molecule sequencing. Methods Mol Biol822, 219-232, doi:10.1007/978-1-61779-427-8_15 (2012).8 Liu, Y. & Wu, B. Q. [Third-generation DNA sequencing: single molecule real-time DNAsequencing]. Zhonghua bing li xue za zhi Chinese journal of pathology40, 718-720 (2011).9 Astier, Y., Braha, O. & Bayley, H. Toward single molecule DNA sequencing: directidentification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter. Journal of the American Chemical Society128, 1705-1710, doi:10.1021/ja057123+ (2006).10 Quail, M. A. et al. A tale of three next generation sequencing platforms: comparison of IonTorrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC genomics13, 341, doi:10.1186/1471-2164-13-341 (2012).。