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Next generation sequencing
Next generation sequencing techniques allow for high-throughput DNA sequencing at a lower cost compared to Sanger sequencing. The document focuses on Illumina sequencing and 454 pyrosequencing. In Illumina sequencing, DNA fragments are attached to a flow cell and undergo bridge amplification and sequencing by synthesis using fluorescently labeled nucleotides. 454 pyrosequencing involves emulsion PCR to amplify DNA fragments attached to beads, followed by sequencing using DNA polymerase and a bioluminescent detection of incorporated nucleotides. Both techniques allow for massively parallel sequencing of millions of DNA fragments.
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Next generation sequencing
- 1. Shaheen Alam NEXT GENERATIONSEQUENCING DNA sequencing - the process of determining the precise order of nucleotides within a DNA molecule or includes any method or technology that is used to determine the order of the four bases -Adenine(A), Guanine(G), Cytosine(C),and Thymine(T) in a strand of DNA. Generally there are three generation for Nucleic acid sequencing. These are – 1st generationsequencing technique Sanger sequencing- The main method for sequencing DNA for the past thirty years! Maxam Gilbert Sequencing Method. 2nd generationsequencing techniques (next generationsequencing) Differ from Sanger sequencing in their basic chemistry There are two types of Next generation sequencing Sequencing by synthesis (SBS) 1. 454/Pyrosequencing 2. Illumina/Solexa 3. Helicos 4. Pacbio 5. Iron torrent sequencing Sequencing by hybridization or ligation 1. SOLiD (Support Oligonucleotide LigationDetection) 3rd generationsequencing technique 1. SMRT (Single Molecule Real Time Sequencing) 2. Oxford Nanopore, etc Here, I make to focus only on NEXT GENERATION SEQUENCING. Basic characteristic of Next-generationsequencing The principle behind Next Generation Sequencing (NGS) is similar to that of Sanger sequencing which relies on capillary electrophoresis. The genomic strand is fragmented, and the bases in each fragment are identified by emitted signals when the fragments are ligated against a template strand.
- 2. Shaheen Alam The Sangermethod required separate steps for sequencing, separation (by electrophoresis) and detection, which made it difficult to automate the sample preparation and it was limited in throughput, scalability and resolution. The NGS method uses array-based sequencing which combines the techniques developed in Sanger sequencing to process millions of reactions in parallel, resulting in very high speed and throughput at a reduced cost. The genome sequencing projects that took many years with Sanger methods can now be completed in hours with NGS, although with shorter read lengths (the number of bases that are sequenced at a time) and less accuracy. Basic steps of all Next generation methodsof DNA sequencing are- 1. Librarypreparation:librariesare createdusingrandomfragmentationof DNA,followedby ligationwithcustomlinkers 2. Amplification:the libraryisamplifiedusingclonal amplificationmethodsandPCR 3. Sequencing:DNA issequencedusingone of several differentapproaches I will discuss detail only on four following method . These are- 1) Illumina/Solexasequencing 2) 454/Pyrosequencing 3) Iron torrentsequencingand 4) SOLiD(Support OligonucleotideLigationDetection) Illumina/Solexa sequencing Principle : Illumina sequencing was initially developed by Solexa Inc. and introduced in 2006; shortly after its introduction, Solexa was purchased by Illumina Inc. This technique uses a flow cell surface that allows for the detection of over 1 million individual reads. This flow surface has eight channels on it, so eight separate sequencing experiments can be run per cell. The process uses the four nucleotides labeled with four different fluorescent mole cules and involves the following steps
- 3. Shaheen Alam Step-1:Prepare GenomicDNASample A strand cannotbe sequencedif itistolarge or if it is double stranded.Therefore the DNA isfragmentedinto between300 and800 bps longthrough– Sonication Nebulization Enzyme digestion Adaptersare neededtobe ligatedonthe endsof the fragmentsinorderto getthe sequence toanneal to where the DNA sequence canbe determinedbythe sequencingmachine. Denature dsDNA intossDNA byheatingto95° C Step-2-AttachDNA to Surface In bridge amplification,the adapterconstructsaddedto the DNA sequence of interesthave flowcell binding sites,P5and P7, whichallow the P5 and P7 regionsof the single-strandedlibraryfragmentstoanneal totheir complimentaryoligosonthe flow cell surface.This meansif one of the DNA fragmentsP5 annealstothe flow cell,itwill anneal byattachingtoa P7 oligosthatis attachedto the flow cell,andvise versa. Several samplescanbe loadedone the 8 lane flowcell for simultaneousanalysis Step-3-Bridge Amplification In thisstepthe sequence makesakindof bridge shape whenitis beingcopied. Unlabelednucleotidesandpolymeraseenzymeare addedto initiate the solid phase bridge amplification
- 4. Shaheen Alam Step-4-FragmentsBecome DoubleStranded The reagentsneededtosequenceare addedsuchas primerstostart the sequencing,nucleotidestoformthe new sequence,polymerase toactuallysequence the nucleotidestogether,and buffertokeepthe pHat an optimal levelforthe enzymaticreaction.The flow cell oligosact as primersanda strand complimentarytothe libraryfragmentissynthesized. Step-5-Denature the Double-StandedMolecules The original strandiswashedaway,leavingbehind fragmentcopiesthatare covalentlybondedtothe flow cell surface ina mixture of orientations. Step-6-Complete Amplification The steps5 through7 of the additionof the sequencing reagents,creationof double strandedDNA,and the denaturationare repeatedmultiple timestocreate thousandsof identical copiesof the same sequence in each cluster.Inotherwordseachclusteris made of up thousandsof the identical sequence andeachclusterisa differentfragmentof the original largersequenceof interest.
- 5. Shaheen Alam Step-7-Determine FirstBase TheP5 regioniscleaved,resultinginclusterscontainingonly fragmentswhichare attachedby the P7 region.Thisensuresthat all copiesare sequencedinthe same direction.The sequencing primerannealstothe P5 endof the fragment,andbeginsthe sequencingbysynthesisprocess. The reagentsneededtosequenceare addedsuchas primersto start the sequencing,fluorescentlylabelednucleotidestoform and detectthe base addedto eachcluster,polymerasetoactually add the nucleotidestothe formingstrand,andbuffertokeepthe pH at an optimal level forthe enzymaticreaction. Step-8-Image FirstBase The unincorporatedbases,whichare the basesthatwere not attachedby a polymerase toone of newlyformingsequencesin the clusters,are washedaway.Thenthe machine can clearly detectthe fluorescentsignalsthatare leftandrecordthe base that wasaddedto each of the clusters. Afterthat the blockon the newlyaddedbase’s3prime partof the sugar isremovedsothat anotherbase will be able toaddedtoit by polymerase.The fluorescentisalsoremovedsothatonlythe new base’sfluorescentisdetectedinthe new cycle.
- 6. Shaheen Alam Step-9-Determine SecondBase •Addsequencingreagents • Primers • Polymerase • Fluorescentlylabelednucleotides • Buffer • Secondbase incorporated Step-10-Image SecondChemistryCycle Againthe image isdetectedbyfirstwashingawaythe unincorporatedbases.Thenthe flow cellispreparedforthe next cycle by removingthe fluorescentanddeblockingthe newly addedbase so that polymerase canaddanotherbase toit. Step-11-SequencingOverMultiple ChemistryCycles The sequencingcyclesare repeatedtodeterminethe sequence of basesina fragment,one base ata time.
- 7. Shaheen Alam Step-12-AlignData • Afterthesequencingisfinishedtheyare aligned • Each was once one largersequence that had beenfragmented • Needstobe realignedto findthe original sequence of the largersequence . Bioinformaticstoolsare usedtodothisusinga reference sequence.A reference sequenceisa digital nucleicacidsequence database,assembled by scientistsas arepresentativeexampleof a species'setof genes. SNPsare alsocall by the alignmenttoolsaswell. SNPsstandsfor Single Nucleotide Polymorphism . Usingthis variationsinthe new sequence canbe found populationinwhichaSingle Nucleotide — A,T, C or G — inthe genome differsbetween membersof a biological speciesorpaired chromosomes Applications of illumine sequencing:- 1) DNA sequencing 2) Gene RegulationAnalysis 3) Sequencing-basedTranscriptomeAnaysis 4) SNPsand SVsdiscovery 5) CytogeneticAnalysis 6) ChIP-sequencing 7) Small RNA discoveryanalysis 454/Pyrosequencing Principle: Pyrosequencing is non-electrophoretic, bioluminescencemethod that measures the release of inorganic pyrophosphateby proportionally converting it into visible light using a series of enzymatic reaction. 454 sequencing was the first commercially available advanced sequencing technique. Itwas introduced in 2005 by the 454 Corporation.
- 8. Shaheen Alam Step-1 DNA isfirst denatured into single strands ,joined to adapters at either end of the fragmented DNA and attached to microscopic beads. Step-2 The DNA on the beads is amplified by an emulsion PCR. PCR amplified allowing up to 1 million identical fragments around one bead Spet-3 each bead is then placed in a well of a Pico Titer tube, which is put into a flow cell whereit is incubated with DNA polymerase,ATP sulfurylase, luciferase, and apyrasealong with the substrates luciferin and adenosine 5’- phosphosulfate (ASP).
- 9. Shaheen Alam Step-4 The firstof four deoxyribonucleotide triphosphates (dNTP) is added to the reaction. DNA polymerasecatalyzes the incorporation of the deoxyribon ucleotide triphosphateinto the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosp hate(PPi) in a quantity equimolar to the amount of incorporated nucleotide Step-5 ATP sulfurylasequantitatively converts PPito ATP in the presence of adenosine5-phosphosulfate(APS). This ATP drives the luciferase mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportionalto the amount of ATP. The light produced in the luciferase- catalyzed reaction is detected by a chargecoupled device (CCD) camera and seen as a peak in a PyrogramT”4. The height of each peak (light signal) is proportionalto the number of nucleotides incorporated Step-6 Apyrase, a nucleotide degrading enzyme, continuously degrades ATP and unincorporated dNTPs. This switches off the light and regenerates the reaction solution. The next dNTP is then added.
- 10. Shaheen Alam Step-7 Addition ofdNTPs is performed one at a time. It should be noted that deoxyadenosine alfa-thio triphosphate (dATPalfaS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the Pyrogram. Applicationof 454/Pyrosequencing 1) Whole genome sequencing 2) Targetedresequencing 3) Sequencing-basedTranscriptome Analysis 4) Metagenomics Ion Torrent system Principle: The Ion Torrent system( uses a "sequencing by synthesis) is unique among NGS technologies in that the detection for sequencing is not based upon fluorescent dyes but rather measuring the pH change as the result of the release of a H-ion upon nucleotide incorporationa using semiconductor technology. Methods Following PCR colonies formation using emulsion PCR, the DNA library fragment is flooded sequentially with each nucleoside triphosphate (dNTP), as in pyrosequencing. The dNTP is then incorporated into the new strand if complementary to the nucleotide on the target strand. Each time a nucleotide is successfully added, a hydrogen ion is released, and it detected by the sequencer's pH sensor. As in the pyrosequencing method, if more than one of the same nucleotide is added, the change in pH/signal intensity is correspondingly larger.
- 11. Shaheen Alam Figure -IonTorrent semiconductor sequencing Ion torrent sequencing is the first commercial technique not to use fluorescence and camera scanning; it is therefore faster and cheaper than many of the other methods. Unfortunately, it can be difficult to enumerate the number of identical bases added consecutively. For example, it may be difficult to differentiate the pH change for a homorepeat of length 9 to one of length 10, making it difficult to decode repetitive sequences. SOLiD (Support Oligonucleotide Ligation Detection ) Methods: Life Technologies/Applied Biosystems has created the Support Oligonucleotide Ligation Detection (SOLiD) platform that utilizes sequencing by ligation to determine DNA sequence composition. There are four main steps of SOLiD sequencing process-Detection) is comprised of 4 parts. 1) LibraryPreparation 2) EmulsionPCRandBead EnrichmentBeads 3) BeadDeposition 4) SequencingbyLigationandDataAnalysis
- 12. Shaheen Alam Fragment Library Mate-PairLibrary Steps-1 The SOLiD systemsupportssample preparationforfragmentormate- pairedlibrarieswithinsertsizesranging from60-110bp and 600-6kbp, respectively..Eitherforafragmentor Mate-pairedlibrary,atemplate (RNA, DNA,cDNA,or PCR prodcut) issheared by sonication(CovarisS2) orphysical disruption(HydroShear). The shearedtemplate isend-repaired, purifiedandligatedtoadaptorsto obtaina fragmentlibrary.These adaptors,P1 and P2, are usedto amplifycopynumberof the genomic target(s).Afterthe libraryisamplified,a size-selectionisperformedinAgarose or PAGE gelsinorderto obtainthe desiredfragmentsize containingthe template ligate to adaptors. A few additional stepsare performed aftershearingandend-repairingthe template toobtaina mate-paired library.A mate-pairedlibraryconsistsof a pair of DNA fragmentsthatare “mates”because theyoriginatedfrom the two endsof the same genomicDNA fragment.The shearedend-repaired template ismethylatedandcapedwith EcoP15I CAPadapters.EcoP15I CAP adaptersconnectthe DNA mate-pairs togetherthrougha biotinylatedinternal adapterresultinginDNA circularization. EcoP15I cleaves25-27 bp awayfrom the unmethylatedenzymerecognition sitesinthe CAPlinker,yieldingmate- pairedgenomicDNA attachedtoboth sidesof the internal adapter.P1and P2 are thenligatedtothe endsof the mate-pairedlibraryforsubsequent amplificationbyPCRandenrichmentof amplifiedtemplate.
- 13. Shaheen Alam Steps-2 Emulsion PCR(ePCR) ePCRtakes place inoil inwater microreactorscontainingP1-coupled beads,templates,primers,andall requiredPCRreactioncomponents. Templates(eitherafragmentor mate-pairedlibrary) are amplified approximately30,000 timesinthese 10-µm microreactors.In thisprocess, a polymerase extendsfromthe P1 adapterafterthe template anneals to the P1-coupledbeads.A complementarysequence is extendedoff beadsurface followed by template dissociation
- 14. Shaheen Alam Enrichment ofTemplated Beads AfterePCR,the templates are denaturedanda beadenrichment stepisperformedina glycerol gradienttoseparate beadswith extendedtemplatesfromthe unwantednon-templatedbeads.P2- coatedPolystyrene beadsare usedto collectP2-Positive templatedbeads, recoveringapproximately200 Million beads,or 80% of the total P2-Positive beads. Steps-3 3'-end Modification The templatedbeadsare 3’ modified inorder to facilitate acovalent linkage tothe slide. Bead Deposition After3' modification,the beads depositedonaglass slide ina randomarray. Differentdeposition chamberscan segmentaslide into one,fouror eightchambers,and consequentlydifferentsamplescan be loadedinthe same sequence run. In general,the one,fourandeight chambersslide canhold165 million, 30 million(perindividual well)and14 million(perindividual well) Millionof P2-PositiveBeads,respectively.
- 15. Shaheen Alam Properties ofprobe Probe structure Steps-4 A 8-mer probes which have Template basesare the correspondingnucleotidesthatwill matchtothe next-readpositionsonthe sequencedtemplate strand. Degenerate bases are correspondingnucleotides that will matchto the UNREAD positionsadjacentto the next-readpositions.These nucleotidesmust match the relevantbasesonthe template strandfor the probe to anneal,however,theiridentityisnot important.The identityof the degenerate basesis neitherknownnorusedbythe sequencingprocess. Thisdegenerate base runof three nucleotides requires3^4 versionsof eachof the fourgreen fluorescentprobes(CA,AC,TGor GT) to ensure bindingabilitybythe probe forall possible reads encounteredonthe sequencedtemplate strand. Universal basescan bindtoany of the four nucleotides. The firsttwo “template”basesmustmatchthe next positionsonthe template strandbeforeDNA ligase will jointhe probe tothe extending(working) complimentarystrand:
- 16. Shaheen Alam Once beaddeposition has occurred, a primer of length N is hybridized to the adapter, then the beads are exposed to a library of 8-mer probes which have different fluorescent dye at the 5' end and a hydroxyl group at the 3' end. DNA ligase is then uses to join the 8-mer probe to the primer. A phosphorothioate linkage between bases 5 and 6 allows the fluorescent dye to be cleaved from the fragment using silver ions. This cleavage allows fluorescence to be measured (four different fluorescent dyes are used, all of which have different emission spectra) and also generates a 5’-phosphate group which can undergo further ligation. Once the first round of sequencing is completed, the extension product is melted off and then a second round of sequencing is perfomed with a primer of length N−1. Many rounds of sequencing using shorter primers each time (i.e. N−2, N−3 etc) and measuring the fluorescence ensures that the target is sequenced. .
- 17. Shaheen Alam Comparisonof next-generationsequencing platforms platform library Sequencing principle Advantage Disadvantage Roche 454 Fragment /emultionPCR pyrosequencing Longerreads improve mapping inrepetitive region, Fast run time Highreagent cost Higherror ratesin homopolymer repeates Illumina Fragment/PCR colonies Sequencingby synthesis Potential forhigh sequence yield. Low multiplexing capabilityof samples. Equipment can be very expensive Iron Torrent Fragment/PCR colonies Sequencingby synthesis faster and cheaper than many of the other methods it may be difficult to differentiate the pH change for a homorepeat of length 9 to one of length 10, making it difficult to decode repetitive sequences. SOLiD Fragment /emultionPCR Sequencingby ligation Due to the two- base sequencing method the SOLiD technique is highly accurate (at 99.999% with a sixth primer, it is the most accurate of the second generation platforms) inexpensive read lengths are short, making it unsuitable for many applications longrun time
- 18. Shaheen Alam Reference 1) http://www.authorstream.com/Presentation/vasu2891-1737182-next-generation- sequencing/ 2)http://www.atdbio.com/content/58/Next-generation-sequencing 3) www.illumina.com/content/dam/illumina.../illumina_sequencing_introduction.pdf 4) Next Generation Sequencing for DUMMIES 5) http://grf.lshtm.ac.uk/sequencing.htm 6) https://gtc.soe.ucsc.edu/content/solid-technology-overview