Biologia molecolare applicata alle biotecnologie, Biologia molecolare, inglese

25.01.2017! Sequencing cycle 2 Sequencing cycle 3

Sequencing cycle 1 Sequencing cycle 2 Sequencing cycle 4

Introduction*to*NGS*approaches* record

intensities

record

record intensities

intensities

add dye-labelled bases

In!1973!the!first!sequence!was!sequenced!by!Gilbert!and!Maxam,!it!was!long!24bp.! .!

.! .!

.! .!

remove block .!

.! .!

.! .!

.!

.! .!

.! .!

In!1977!was!developed!the!Sanger'method:!it!is!also!used!today!and!it!is!based!on!the!use!of! A!

A! A!

A! A! Fire laser

C!

C! C!

C! C!

dideoxy!nucleotides!that!lack!hydroxyl!group!in!the!position!3!so!when!they!are!incorporated! Fire laser

T!

T! T!

T! T!

Fire laser

G!

G! G!

G! G!

they!block!polymerization.! A!

A! A!

A! A!

A!

A! A!

A! A!

Using!a!3’!labelled!primer!and!a!small!amount!of!ddnt,!they!introduce!a!stop!when!they!are! .!

.! .!

.! .!

.!

.! .!

.! .!

incorporated! so! the! result! is! fragmentation! of! the! DNA! and! it! is! possible! to! separate! these! .!

.! .!

.! .!

fragments!by!poliacrilammide!gels!and!obtain!the!picture!of!the!length!of!the!fragments.! !

! The!trick!of!this!system!is!the!removal!of!the!blocks,!these!are!transient!blocker!that!can!be!

The!next!step!was!the!use!of!ddnt'conjugated'with'fluorophores!so!that!they!can!be!added!all! removed.!!

together!in!the!reaction.! !

Coupling!this!with!capillary!electrophoresis!this!was!the!first!sequencer.!Using!this!approach! Using!this!technique!we!can!read!sequences!of!300nt,!but!usually!sequences*of*100=150*nt.!

we!can!sequence!600bp!but!is!quite!a!long!process!and!expensive.! !

! The! efficiency! of! this! technique! is! high! but! it! is! not! 100%,! so! after! 100! polymerizations! we!

Human*genome*project* start! have! problems! in! the! removing! of! the! block! and! one! cluster! begins! to! be! a! mixed!

They!were!able!to!complete!the!first!tract!of!our!genome.!In!the!2000!a!new!technique!NGS! population! of! asynchronous! strands! (for! example! there! is! a! small! %! of! nt! that! are! not!

emerged;!we!can!divide!it!into!two!different!techniques:! deblocked);!so!we!can!read'only'short'sequences,!and!this!is!one!of!the!two!biggest!issue!of!this!

S Clonally'amplified'DNA!→!the!technique!we!use!today!with!Illumina!machines! technology.! The! second! is! the! fact! that! in! order! to! obtain! a! sequence! you! have! to! perform!

S Single'molecule'DNA!(NSNGS)!→! different!PCR!steps,!so!you!are!not'sequencing'the'initial'molecule'of'DNA.! Single read vs. paired-end

! !

! We!can!sequence!in!two!ways:!

Illumina*technology* Single*read*

•

The!first!part!of!the!technology!is!the!preparation'of'the'genomic'leaning;!in!this!case!we!work! Paired=end! we! flip! the! molecule! and! perform! a! new! step! of!

• →!

on!cDNA!(we!are!interested!to!study!the!RNA):!! PCR!and!remove!the!other!primer.!In!this!way!you!are!flipping!the!

1. Fragmentation'of'the'cDNA!→!to!obtain!a!population!of!fragments!ranging!between!to! molecule!and!restart!the!sequencing!on!the!other!side.!!

300?600bp! So! we! obtain! a! pairedSend! sequence:! so! if' we' have' a' fragment' of' Se

2. Addition'of'adapters!→!universal!molecules!(oligodt)!ligated!to!DNA!and!fundamental! 250nt' we' can' read' 125nt' from' one' side' and' 125nt' from' the' other'

for!the!capture!of!DNA.!One!line!usually!contains!8!lanes:!the!plex!contains!a!lot!of!ssS side.' And! this! is! fundamental,! because! we! know! roughly! the! REV

FW

FW

oligont!complementary!to!the!adapters!and!covalently!bind!to!the!glass.! dimension!of!our!fragments,!and!when!we!map!the!sequences!on!

3. Loading' of' the' DNA! when! you! load! DNA! you! also! perform! a! denaturation' and' the!reference!genome,!you!know!exactly!that!these!two!sequences!

→!

renaturation'step:!these!oligont!can!work!as!primers!because!they!are!complementary! are!physically!linked!and!they!are!roughly!500b!of!distance.! Single!Read! Paired1End!

to!the!adapters,!so!you!can!obtain!a!copy!of!your!DNA!of!interest!in!both!the!strands! And! this! is! fundamental! especially! for! the! RNAseq! and! the!

Single read vs. paired-end

(depends!on!which!the!adapter!is!bound)! definition!of!different!isoforms!of!splicing.!

!

4. Denaturation' and' wash! the! only! material! that! remains! is! the! ssDNA! covalently!

→! !

bound!to!the!glass! !

5. Bridge?PCR! we! have! millions! of! DNA! molecules,! so! we! can! perform! a! bridgeSPCR:!

→! !

these!single!molecules!contain!another!copy!of!adapter!at!the!end,!so!we!can!perform!a! !

cycle! of! PCR! without! adding! other! primers! (simply! with! poly! and! nt),! these! ssDNA! Sequencing “read”

!

molecules!can!bind!the!different!adapters!that!are!covalently!bound!in!proximity!and! 35-300 nt

Sequencing*depth'is!the!number!of!sequencing!reads!generated!by!a!sequencing!run.!'

form!a!cluster'of'1000'molecules!after!different!cycles!of!PCR.!! REV

FW

FW

The!higher'the'number'of'the'reads,'the'higher'the'coverage.!

These! clusters' are' all' DNA' molecules' that' are' identical,! so! you! have! cluster! of! DNA! !

molecules!that!are!the!same!copy!of!the!original!one.! Single!Read! Paired1End!

!

We!have!1!million!of!clusters/mm3!which!can!be!visualized:!if!we!have!another!primer! !

that!drives!the!polymerization!with!fluorescent!nt!that!are!blocked!temporally!so!they! !

can!only!add!on!nt!each!time,!we!have!the!addition!of!the!first!nt!after!the!primer.!! !

This! is! enough! to! be! detected! so! it’s! possible! to! detect! fluorescence! of! the! first! !

nucleotide!added.!Repeating!the!scanning!is!possible!to!obtain!the!sequencing.! !

6. Sequencing! !

*for!each!cycle!of!PCR!just!one!nt!can!be!added!!!

! 1! ! 2!

NGS: different type of experiments

Pacific Biosciences: Real Time Sequencing Technology

Third*generation*Sequencing* NGS:*types*of*experiments*

The! idea! is! to! perform! a! real' time' sequencing,! in! which! you! are! able! to! detect! the!

incorporation! of! each! nucleotide! without! stopping! the! reaction! and! recording! the!

incorporation!of!the!nt.!! !

We!can!have!an!amazing!amount!of!techniques!based!on!NGS:!

Whole'genome'sequencing'

• Whole?exome'(1%)'sequencing'→!capture!of!the!exon!parte!of!the!human!genome;!we!

• can!perform!fragmentation!of!genomic!DNA!and!then!an!enrichment!of!the!exon!part!

! using! an! array! of! tailed! oligont! that! can! capture! the! exon! part;! then! is! possible! to!

The!idea!is!to!use!nucleotides!in!which!fluorophore!is!linked!to!the!terminal!phosphate!of!the!

Zero-Mode Waveguides (ZMW)

nt,!so!the!fluorophore!is!removed!by!the!nt!after!incorporation!during!polymerization.! perform!a!sequencing!of!a!very!shot!part!of!our!genome!(1.5%).!!

With! this! approach! we! lose! all! the! information! of! ncRNA,! so! it! is! not! a! very! useful!

Using!this!type!of!approach!we!can!detect!the!incorporation!of!nt!in!real!time.!!

DNA polymerase is affixed to the bottom

Only the bottom portion of the hole (70 approach.!

We! have! grid! with! tiny! holes! (50S70! nm):! when! illuminated! with! a! light! which! has! a!

of a tiny hole (~70nm). Incorporation of

nm) is illuminated by the attenuated light

wavelength!similar!to!the!dimension!of!the!hole,!the!light!is!not!able!to!pass!through!the!grid! PCR' amplification! we! can! perform! a! targeted! sequencing! using! amplicons,! this! is!

• →!

dye-labeled nucleotide is detected as a

of the excitation beam defining a 20

but!we!have!a!refraction'of'the'light!that!is!present!only!on!a!small!volume!on!the!base!of!the! useful!if!we!have!to!sequence!thousand!of!different!samples!just!focusing!on!a!defined!

-21 flash of bright light

zeptoliter (10 ) detection volume

hole.!So!the!light!is!stopped!by!the!grid!but!because!of!the!refraction,!a!small!amount!of!light!is! region!of!DNA!

present!on!the!bottom!of!the!hole.!! Trancriptome'RNA!sequencing!

•

This!gives!the!possibility!to!create!a!reactor!in!which!only!in!a!small!portion!the'presence'of' Exon'capture'transcriptome'

•

light'is'able'to'excite'the'fluorophore.!! !

Pacific Biosciences: Real Time Sequencing Technology

On! the! bottom! of! each! hole! we! have! only! one' molecule' of' DNA' polymerase! which! is! able! to! DNA'sequencing'applications'

induce! the! polymerization! DNA;! the! different! nt! that! enter! in! the! hole! There!are!other!techniques!that!are!used!for!the!definition!of!epigenomic!data:!

are!excited!from!the!light,!so!only!nucleotides!that!are!entering!the!hole! ChIP* seq! fundamental! for! the! definition! of! interaction! of! different! proteins! or!

• →!

are!excited.!! Library Preparation modification!of!DNA!

• DNA template is circularized by the use

In!this!way!we'can'avoid'stopping'each'cycle'of'polymerization!so!we!can! Chromatin*packaging*and*accessibility*

of “bell” shaped adapters. •

• As long as the polymerase is stable this

obtain' very' long' reads' in' a' very' fast' way;! the! problem! is! that! the! !

allows for continuous sequencing of both

frequency'of'mistakes'is'quite'high.!! Another! application! of! DNA! sequencing! is! the! identification' of' SNPs:! the! diploid! genome! is!

strands.

! more! than! 3.2! billion! of! base! pairs,! and! we! have! 3/4! millions! of! variants! present! in! our!

The!trick!is!to!prepare!libraries!with!circular'adapters:!in!this!way!you! genome,! they! can! be! quite! common! or! rare! variants.! With! whole! genome! sequencing! is!

can! have! a! circular! sequence! of! DNA! and! when! you! can! use! a! polymerase! that! displays! the! possible!to!analyse!these!variants.!!

DNA! that! is! produced,! you! can! roll! trough! the! DNA! several! times,! so! you! are! sequencing! The!vast!majority!of!SNPs!are!usually!located!in!nonScoding!regions!!

always!the!same!portion!of!DNA!hundred!of!times:!so!we!can!obtain!a!consensus!sequence.! !

! Identification'of'DNA'mutations'in'cancer'

Advantages:! The!most!important!application!is!sequencing!of!different!types!of!cancer:!cancer!in!general!is!

! No!amplification!required!→!we!can!start!from!our!DNA! characterized! by! a! strong! heterogeneity! between! the! population! of! cancer! cells,! and! it’s! not!

! Extremely!long!read!lengths! easy! to! define! which! is! the! most! important! cell! and! which! are! the! driver' mutations! for! the!

! Average!2500bp!(longest!15000bp)! tumor! and! distinguish! them! from! the! passenger'mutations! that! contribute! for! the! growth! of!

Disadvantages! the!cancer!but!are!not!relevant!for!the!cancer!phenotype.!

! High!error!rates!(but!we!can!use!a!trick)! Now! with! the! possibility! to! sequence! at! single! cell! level! the! genome! of! the! different! cancer!

For! the! moment! Biosciences! sequencing! is! used! only! in! few! peculiar! needs,! for! example! for! cells,!we!are!trying!to!create!a!different!type!of!analysis!in!which!we!have!hierarchical!tree!of!

the! sequencing! of! prokaryotes! when! you! don’t! have! a! reference! genome,! or! if! you! are! the!different!mutations!in!order!to!understand!which!are!the!most!important!ones.!!

interested!in!splicing!isosomes.!! !

! !

! !

* !

! 3! ! 4!

Assay for Transposase Accessible-Chromatin seq: a

method for mapping chromatin accessibility

ChIP-Seq: histone modifications

ChIP=Seq:*histone*modifications* incubate! chromatin! with! it,! the! enzyme! binds'

preferentially'the'accessible'part'of'the'chromatin'

• H3K4me3, promoter regions; and' cut' this' part! of! the! DNA! and! it! is! able! to!

• H3K4me, enhancer regions; catalyse!the!entry'of'two'adapters.!!

• H3K36me3, transcribed regions; In!this!way!is!possible!to!use!the!two!adapters!to!

• H3K27me3, Polycomb repression; expand! by! a! PCR! and! then! sequence! the!

• H3K9me3, heterocromatin regions fragments! and! to! have! a! view' of' the' accessible'

• H3K27ac and H3K9ac, increased activation of chromatin.!

enhancer and promoter regions

!!!!!!!!!!!!!!!!!!!!!!! ! It!is!fundamental!for!the!definition!of!promoters,!

! ChIP-Seq: histone modifications enhancers! and! sites! of! binding! for! the!

• H3K4me3, promoter regions;

ChIPSSeq! allows! to! define! the! interaction! of! the! portions' of' DNA' that' are' in' contact' with' transcription!factors.!!

• H3K4me, enhancer regions;

histones! (in! particular! modified! histones)! or! binding'proteins,! like! transcription! factors,! that!

• H3K36me3, transcribed regions; !

interact!with!DNA.!

• H3K27me3, Polycomb repression; !

It!is!made!through!different!steps:!

• H3K9me3, heterocromatin regions !

Crosslinking!of!DNA!and!protein!

• • H3K27ac and H3K9ac, increased activation of !

Enrichment! using! monoclonal! antibodies! against! in! the! histone!

• →!

enhancer and promoter regions !

modification! or! the! binding! protein! in! order! to! enrich! the! DNA! !

material!that!is!interacting!with!that!histone! Ancient!genomes!reconstructed!

DNA'purification'and'addition'of'adapters!

• With!NGS!is!also!possible!to!sequence!ancient!genomes,!to!have!a!better!idea!of!the!migration!

Sequencing!

• of!population!in!the!past.!The!sequencing!is!not!only!devoted!to!human!sequencing!but!also!in!

Analysing! the! sequence! we! can! map! it! on! the! genomic! DNA! and!

• H3K4me3, promoter regions;

• →! vegetables!and!organisms!fundamental!for!different!applications!(food…).!

know! which! regions! are! characterized! by! a! defined! histone!

• H3K4me, enhancer regions; !

modification!or!by!the!binding!of!an!interested!transcription!factor!

• H3K36me3, transcribed regions; Methods*to*reveal*genome*3D*structure*

! • H3K27me3, Polycomb repression; Deep!sequencing!is!also!useful!to!understand!the!map'of'interaction'of'chromatin,!because!our!

' • H3K9me3, heterocromatin regions genome!is!organized!in!very!interesting!3D!structure.!!

•

' H3K27ac and H3K9ac, increased activation of Until!the!development!of!the!“C*technologies”!(that!derive!from!the!3C!technology,!based!on!

essing DNA methylation enhancer and promoter regions

' the! PCR),! now! we! have! many! different! modifications! of! these! techniques! that! allow!

Methods to reveal genome 3D structure

Assessing'DNA'methylation' identifying!portion!of!the!DNA.!

You!can!also!immunoprecipitate!modify!DNA,!the!most!famous!are!the!

G sites CpG:! you! can! use! specific! antibodies! against! mC! and! enrich! the! DNA!

rrelates with gene silencing for!these!mC.!

ed in cancer cells You!can!also!use!mAb!against!the!5hmC,!this!method!is!called!MeDIP*

ed with many other diseases: Seq.!

response to to environment !

ylated DNA

n

methyl-cytosine to retrieve

s from sonicated DNA ! !

Exploring'chromatin'packaging' !

Many! techniques! are! fundamental! to! define! the! chromatin! accessibility,! it! is! interesting! to! Dekker, Marti-Renom and Mirny, Nat. Rev. Genet. 2013

RNA'sequencing'applications'

understand!if!one!portion!of!chromatin!is!accessible!or!really!packed!with!histones.!!! Techniques! that! completely! changed! the! analysis! of! cell! identity,! it! is! used! to! define! the!

Until! some! years! ago! only! the! techniques! based! on! the! digestion! of! the! accessible! DNA! wer! differences!between!different!cell!types!(and!now!we!can!do!it!event!on!single!cells).!

used! (DNAseSseq,! MNaseSseq)! or! techniques! bases! on! the! fragmentation! of! the! DNA! using! It!is!fundamental!for!the!identification!of!transcripts,!in!some!cases!also!alternative!splicing.!

physical!methods.! It! can! be! used! in! cancers! to! identify! chimeric! transcripts,! or! it! can! be! used! for! the!

The!technique!ATAC=Seq!requires!less!that!100%!of!the!amount!of!cells!required!by!the!other! identification!and!analysis!of!ncRNAs.!

techniques:!so!we!can!perform!an!experiment!starting!from!only!10000!cells!(against!1M!of! !

the!other!two)!and!this!is!a!possibility!for!people!that!are!working!on!primary!cells.!! !

ATACSSeq!is!based!on!an!enzyme!Tn5!transposase!that!usually!catalyses!the!transposition!of! !

transposomes! that! it! was! modified! in! order! to! have! a! very! active! enzyme:! so! when! you!

! 5! ! 6!

Microarray and RNA-Seq comparison

Microarray'and'RNA?Seq'comparison' 26.01.2017!

Next*generation*sequencing*from*bioinformatic*point*of*view*

Technology RNA-seq Microarray

High run-to run reproducibility Yes Yes *

Dynamic Range comparable to actual transcript NGS*technologies*

>8000 fold Hundred fold

abundance Since! the! human! project! in! which! the! sequencing! is! one! of! the! technologies! used,! the! NGS!

Able to detect alternative splice sites and novel Yes No

isoforms techniques!are!growing!and!there!are!three!areas!in!which!they’re!used:!

De novo analysis of samples without reference Yes No

genome Genomics!

! •

Nowadays!microarray!is!only!used!for!some!approaches!in!which!you!want!to!replicate!some! Transcriptomics!

•

data!in!a!huge!cohort!of!patients!and!you!only!focus!on!few!targets,!because!RNASSeq!is!more! mRNA!sequencing!

o

convenient! for! the! information! that! you! can! retrieve! from! this! kind! of! analysis:! indeed'with' smallRNA!and!ncRNA!sequencing!

o

RNA?Seq'you'can'look'everything'and'not'only'what'you'have'spotted'on'the'microarray'(that!is! Epigenomics!

•

the!big!problem!of!microarray).! ChIP!sequencing!

o

! ATACSseq!

o

! There!are!different!applications!for