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Genomesequencingandassembly刘志发
OUTLINEDNAsequencingGenomeAssembly
2012PublishedGenomes
WhatisDNAsequencing?DNAsequencingistodeterminetheorderofthenucleotidebasesinamoleculeofDNA.Firstgenerationsequencer:Chain-terminationmethodsNextgenerationsequencer(Illumina/Roche):High-throughputsequencingThirdgenerationsequencer(PacBio):Singlemolecule
Chain-terminationmethods根本原理:聚丙烯酰胺凝胶电泳可以区分长度只差一个核苷酸的DNA分子;利用DNA聚合酶不能够区分dNTP和ddNTP的特性,使ddNTP渗入到寡核苷酸链的3’-末端。因为ddNTP3’不是-OH,不能与下一个核苷酸聚合延伸,从而终止DNA链的增长。
Chain-terminationmethodsDr.FredSanger
Illuminasequencing
454sequencing
Whatisgenomeassembly?SequenceassemblyreferstoaligningandmergingfragmentstoamuchlongerDNAsequenceinordertoreconstructtheoriginalsequence.Usingthereadfilestogetlongersequence.
Whydoassemble?sanger测序:500~1000bp?Illumina测序:200-500bp?pacific:10kbp?人的基因组大小为3Gbp
Howtoassemble?很长的毕业照是怎么拍的呢??寻找reads之间的overlapATACCTTGCTAGCGTGCTAGCGTAGGTCTGA=ATACCTTGCTAGCGTAGGTCTGA
genomeassemblyInshotgunsequencing,DNAisbrokenuprandomlyintonumeroussmallsegments.MultipleoverlappingreadsforthetargetDNAareobtainedbyperformingseveralroundsofthisfragmentationandsequencing.Computerprogramsthenusetheoverlappingendsofdifferentreadstoassemblethemintoacontinuoussequence.ABCABCABCABC小片段测序计算机拼装
ABC小片段测序计算机拼装ProblemCAATGCATTA……GCAGCCAATGCGAP错装
Twostrategies
AdvantageanddisadvantageHierarchicalshotgunComplexandslowonexperimentstepHighcost,andhighqualityUsedonlybythehumangenomeprojectWhole-genomeshotgunEasyandfastonexperimentstepMuchlowercost,andalittlelowerqualityUsedwidelybymanyothergenomeprojects
SequenceassemblySequenceassemblyreferstoaligningandmergingfragmentstoamuchlongerDNAsequenceinordertoreconstructtheoriginalsequence. Overlap: contig Ge+en+no+om+mi+ic+cs?Geno
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