生物信息學(xué)-第4次理論轉(zhuǎn)錄組學(xué)

RNA測序技術(shù)原理及應(yīng)用遺傳的中心法則基因組—轉(zhuǎn)錄組—表觀遺傳組—蛋白組層次什么是轉(zhuǎn)錄組？All

transcripts

All

mRNAsRNA是解讀基因組的關(guān)鍵RNAProteinPhenotypeGenotype

DNA測序技術(shù)RNA-SeqSmallRNA測序降解組測序Non-codingRNA測序研究對象mRNASmallRNAmRNANon-codingRNA鑒定新分子OOOO表達(dá)譜研究OOOO基因結(jié)構(gòu)分析O/XXXOEST測序O/XXXX

篩選分子標(biāo)記

OOOX轉(zhuǎn)錄融合基因表達(dá)O/XXXXRNA測序技術(shù)WorkflowofRNA-Seq樣品檢測文庫制備ClusterStationIlluminaSequencing生物信息分析ApplicationRNA-Seq(單端測序---Quantification)RNA-Seq

(雙端測序---Transcriptome)Expression-profiling√√AlternativeSplicing－√FusionGene－√SNPdetection－√HiSeq2500ApplicationsofRNA-Seq7RNA-Seq(Transcriptome)Characterizethetranscriptomeinunparalleleddetail8RNA-Seq(Denovotranscriptomeassembly)RNA-Seq(Transcriptomeresequencing)Transcriptomeresequencing分析流程應(yīng)用---優(yōu)化轉(zhuǎn)錄組注釋信息基因結(jié)構(gòu)優(yōu)化可變剪接鑒定基因融合鑒定RNA水平SNP分析GenomicintergenicregionReadsclusterPairedReadsdistribution優(yōu)化基因結(jié)構(gòu)

鑒定新的轉(zhuǎn)錄本Paired-End(PE)ReadsReads比對到參考序列基因間區(qū)域鑒定可變剪接（AlternativeSplicing）exon1exon2exon3exon1exon2exon3exon1exon3commonreadsjunctionreadsmRNA鑒定基因融合PairedReadsSingleReadsGeneAGeneB分析RNA水平SNP轉(zhuǎn)錄組重測序比對軟件：SOAPDenovo轉(zhuǎn)錄組測序:組裝軟件：SoapDenovo比對軟件：SoapSNP15轉(zhuǎn)錄組研究技術(shù)橫向比較TechnologyTilingArraycDNAorESTsequesingTranscriptomeSequencingPrincipleHybridizationSangersequencingNext-GensequencingResolutionFromseveralto100SinglebaseSinglebaseThroughputHighLowHighRelianceongenomicsequenceYesNoInsomecasesBackgroundnoiseHighLowLowApplicationSimutaneouslymaptranscribedregionsandgeneexpressionYesLimitedforexpressionYesDynamicrangetoquantifygeneexpressionlevelUptoafew-hundredfoldNotpractical>8,000-foldAbilitytodistinguishdifferentisoformsandallelicexpressionLimitedYesYes轉(zhuǎn)錄組測序的優(yōu)勢RNA測序與芯片檢測基因數(shù)比較RNA測序與芯片檢測基因的表達(dá)量分布Technique1RNA-Seq與基因芯片優(yōu)缺點比較技術(shù)優(yōu)點缺點RNA-seq1）檢測基因數(shù)比基因芯片多25%2）定量準(zhǔn)，可重復(fù)性高（重復(fù)相關(guān)系數(shù)≥0.99）3）數(shù)字化信號，無背景噪音，無交叉雜交4）高、低豐度基因均可檢測5）不受研究物種限制，模式生物和非模式生物均可檢測6）數(shù)據(jù)可與時俱進(jìn)，即隨數(shù)據(jù)庫更新而更新7）具有較好的分析兼容性，數(shù)據(jù)格式與芯片相同，可與芯片的分析軟件兼容1）樣本要求量比基因芯片多2）數(shù)據(jù)量大，需要具備一定的生物信息分析基礎(chǔ)，才能更好的挖掘數(shù)據(jù)蘊含的豐富信息基因芯片1）平臺應(yīng)用較早2）信息分析軟件較多3）有些平臺要求樣品量少1）檢測靈敏度較低、重復(fù)性差、檢測閾值較狹窄2）有背景噪音，假陽性率＞1%3）受物種限制，只能檢測部分模式生物4）受基因拷貝數(shù)限制，無法檢測出低豐度基因5）只能檢測已知轉(zhuǎn)錄本，無法檢測出新轉(zhuǎn)錄本6）受數(shù)據(jù)庫數(shù)據(jù)所限，因探針依靠現(xiàn)有數(shù)據(jù)庫或比較舊的版本的數(shù)據(jù)庫來設(shè)計的，可能出現(xiàn)注釋不準(zhǔn)確的情況RNA-Seq

應(yīng)用領(lǐng)域2023/2/718RNA-Seq面臨的挑戰(zhàn)龐大的數(shù)據(jù)量所帶來的信息學(xué)難題；如何針對更