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Next generation sequencing
1. Shaheen Alam
NEXT GENERATION SEQUENCING
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 Sanger method 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 GenomicDNA Sample
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 Double Stranded
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
The P5 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
• Afterthe sequencingisfinishedtheyare
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 is first
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 first of 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 of dNTPs 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 -Ion Torrent 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-Pair Library
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 of Templated
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 of probe
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 bead deposition 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-generation sequencing 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
