Basic Sequence Analysis - Technical University of Denmarkteaching.bioinformatics.dtu.dk/teaching/images/9/... · Basic Sequence Analysis Lars RønnOlsen Assistant Professor, Technical

BasicSequenceAnalysis

LarsRønn OlsenAssistantProfessor, TechnicalUniversityofDenmark

Learningobjectives

Aftertoday,youwillbeableto:

• UnderstandhowBLASTworks

• UseBLASTforsequencesimilaritysearch

• Understand thetheorybehinddenovoassemblyofgenomesfromsequencereads

• Understand thetheorybehindshortreadalignment

• Understandhowmultiplesequencealignmentworks

• Usemultiplesequencealignment toexaminesequencevariability

• Usepublicwebservicestopredictvaccinetargetsinpathogens

Example:vaccinedesignworkflow

Pathogenofinterest:Denguevirus

Generalpathogeninformation:WikiPediaPubMed

Speciesinformation:NCBITaxonomy

Genomicsequencedata:NCBIGenBank

Geneinformation:GeneCardsAmiGO

Genomicsequencedata:Wholegenomesequencing

Proteinsequencedata:NCBIprotein

SwissProt/UniProt

Geneexpressionprofiles:NCBIGEO

Selectionofvaccinetargets

Database

Tool

BLAST- Searchingdatabasesforsequences

BLAST(BasicLocalAlignmentSearchTool) isatooltoqueryadatabase forsequencessimilartoaninputsequence.

Imagineyouhavesequencedagenefromanunknownsample,andyouwouldliketoknowwhatitis.YoucanuseBLASTinNCBItocompareyoursequencetoALLthe197,390,691sequencesinGenBank!

https://blast.ncbi.nlm.nih.gov/Blast.cgi


Example:youwouldliketoknowwhatthefollowingsequenceis:

X)AATGCCG

Youhavethefollowingthreesequencesinyourdatabase:

A)CGTGTGATCB)AATGCCGC)GCTGTGAC




AATGCCG


A)CGTGTGATCB)AATCCCGC)GCTGTGAC


(Tip: use the font “courier new”)

X) AATGCCGB) AATCCCG



X)AATGCCG


A)CGTGTGATCB)AATCCCCGC)GCTGTGAC


X) AATG-CCGB) AATCCCCG



X)AATGCCG


A)AATCCCGB)AATCCCCGC)AATCC


X) AATGCCGA) AATCCCG

X) AATG-CCGB) AATCCCCG

X) AATGCCGC) AAT-CC-

Whichmatchisbest?











SwissProt/UniProt



Database

Tool

Sequencingreadalignment- Toolsfornextgenerationsequencing

Basecalls

Qualitycontrol/trimming

Sequencingwithout reference:Denovoassembly

Rawsequencingoutput Database

Tool

Sequencingwithreference:Short readalignment

GenBank/RefSeq

Geneorgenomesequence

Sequence readarchive

Denovoassembly- Makingsenseofsequencingreadswithoutareferencegeneorgenome

ATGACGTTT

TTTCTGAAA

AAATTCCCC

CCCCTGGCCA)

B)

C)

D)

ATGACGTTT AAATTCCCC

TTTCTGAAA CCCCTGGCC

A) B)C) D)

ATGACGTTTCTGAAATTCCCCCTGGCC


ATGACGCCC

CCCCTGCCC

CCCTTCCCC

CCCCTGGCCA)

B)

C)

D)

ATGACGCCC CCCTTCCCC

CCCCTGCCC CCCCTGGCC

A) B)C) D)

ATGACGCCCCTGCCCTTCCCCCTGGCC

ATGACGCCC

CCCTTCCCC

CCCCTGCCC

CCCCTGGCC

A) C)B) D)

ATGACGTTTTTCCCCCTGCCCCTGGCC

???


ATGACGCCC

CCCCTGCCC

CCCTTCCCC

CCCCTGGCCA)

B)

C)

D)

ATGACGCCC CCCTTCCCC

CCCCTGCCC CCCCTGGCC

A) B)C) D)

ATGACGTTTCTGCCCTTCCCCCTGGCC

ATGACGCCC

CCCTTCCCC

CCCCTGCCC

CCCCTGGCC

A) C)B) D)

ATGACGTTTTTCCCCCTGCCCCTGGCC

???

TTCCCCCTGE)

TTCCCCCTGTTCCCCCTG

Sequencingreadalignment- Toolsfornextgenerationsequencing

Basecalls

Qualitycontrol/trimming

Sequencingwithout reference:Denovoassembly

Rawsequencingoutput Database

Tool

Sequencingwithreference:Short readalignment

GenBank/RefSeq

Geneorgenomesequence

Sequence readarchive

Shortreadalignment- Aligningreadstoareferencegeneorgenome

ATGACGTCAGCTGTTGGCGACATCGTTCGATCAGTCGATTATTCGATAATCGCTCTCTTAGReferenceATGACG TTGGCG CGTTCG AGTCGA TTCGAT TCTCTTReads

ReadDepth“x”

Makeyoursequencesavailable- Otherresearcherscanbenefitimmenselyfromyourwork!

YoursequencereadscanbedepositedintheSequenceReadArchive.

https://www.ncbi.nlm.nih.gov/sra

Makeyoursequencesavailable- Otherresearcherscanbenefitimmenselyfromyourwork!

Yourassembled/mapped sequencescanbedepositedinGenBank.









SwissProt/UniProt



Database

Tool

PredictingTcellepitopes- Toolsforvaccinetargetdiscovery

SequencevariabilityanalysisMultiplesequencealignment

PredictionofepitopesHLAalleleHLAallelefrequency

Selectionofepitopes

Proteinsequences

ImmuneEpitopeDatabase

Epitopesforvaccine

Database

Tool

Multiplesequencealignment- Aligningsequencestodeterminevariability

Multiplesequencealignment isatypeofalgorithmthatallowsyoutocompare2ormoresequencessimultaneously.

Thisishighlyuseful,forexamplewhenanalyzing thevariabilityofacertainproteininapathogen.

Therearemanydifferentalgorithms forthispurpose– oneofthemostfamousbeingClustalW from1997.

Infact,thistoolhasbeencited53,288times(number10inthepapermountain)andisstillcitedheavily.

HOWEVER!ThemainauthorofClustalW,DesHiggins,hasaskedpeopletostopusingandcitingitasthereareupgradesandotherbettertoolsavailabletoday!


Howdoesitwork?VerysimilartoBLAST,exceptallsequencesareconsidered:

AATCCC-GAAATCCCCGTAATCC---T

AATCCCGAAATCCCCGTAATCCT


Inpractice,youcopyordownloadallyoursequencesofinterestinfasta format.

Thefasta formatlookslikethis:

>Thislineistheheader.YoucanwritewhateveryouwanthereATCAGACTGTGCTGATCG…

Youthenpasteorupload thesequencestoamultiplesequencealignmentwebserver(orinstallitlocallyifyouhaveverylargedatasets)andrun thealignment.OneiswebPRANKwhichisavailablethroughEBI(EuropeanBioinformaticsInstitute).

http://www.ebi.ac.uk/goldman-srv/webprank/








Selectionofepitopes

Proteinsequences


Epitopesforvaccine

Database

Tool

PredictionofTcellepitopes- WhatareTcellepitopes?

Virusproteinsarecleavedtoshortpeptides

Someofthesepeptidesbindtothehuman leukocyteantigenprotein(HLA)

Whenviruspeptidesarepresentedonthesurfaceoftheinfectedcell,Tcellskilltheinfectedcells

TheHLAproteincomeindifferentflavors

FindingTcellepitopes- Traditionalapproachtofindingimmunogenicregionsinpathogens

PredictionofTcellepitopes- Computationalapproachtofindingimmunogenicregionsinpathogens

Rappuoli R(2000) Reverse Vaccinology, Curr OpinMicrobiol

PredictionofTcellepitopes- HowtopredictpeptidebindingtoHLA

Therearealotofalgorithms topredictepitopesinpathogensandcancercells.

Among thebestperforming isNetMHC.


http://www.cbs.dtu.dk/services/NetMHC/



MRCVGVGNRRCVGVGNRDCVGVGNRDFVGVGNRDFVGVGNRDFVEVGNRDFVEGGNRDFVEGLNRDFVEGLSDFVEGLSGAFVEGLSGATVEGLSGATWEGLSGATWVGLSGATWVD


Epitopes Notepitopes



MRCVGVGNRDFVEGLSGATWVDVVLFQCLESIEGKAVQHENLKYTVIITVHTGDQHQVG


GVGNRDFVEVGNRDFVEGFVEGLSGATVEGLSGATW

Potentialepitopes




Selectionofepitopes

Proteinsequences


Epitopesforvaccine

Database

Tool

HLAdatabases- Databaseswithinformationaboutthehumanleukocyteantigen

W

K

I

D

HLAcomesindifferent flavors(alleles).

Differentallelesbinddifferentpeptides.

HumanshavesixdifferentHLAalleles(threefromeachparent).

DifferentHLAallelesareprevalentindifferentpopulations– seetheHLAallelefrequencydatabase

http://www.allelefrequencies.net/

YoucanalsoexplorethesequencesoftheHLAsintheHLAalleledatabase

https://www.ebi.ac.uk/ipd/imgt/hla/


W

K

I

D



W

K

I

D





Selectionofepitopes

Proteinsequences


Epitopesforvaccine

Database

Tool

Selectionofepitopes- ChoosingHLAbinders

Combiningvariabilityanalysis(multiple sequencealignment)andHLAbindingpredictionsletsyoupickthebestepitopesforyouvaccine.

Thiscanbedonemanuallyorusing, forexample,theBlockCons tool


http://met-hilab.cbs.dtu.dk/blockcons/


http://met-hilab.cbs.dtu.dk/blockcons/


Afteryouhaveselectedyourpotentialepitopes,youshouldcheckwhethertheyarepresentinhumanproteins,asvaccinationwiththesemayleadtolackofefficacyorpotentiallyautoimmunity.

Howwouldyoudothis?

Answer:useBLASTinGenBank,andsearchforthepeptidesinhumanproteins.




Selectionofepitopes

Proteinsequences


Epitopesforvaccine

Database

Tool

ImmuneEpitopeDatabase(IEDB)- Databaseswithepitopesinanarrayofpathogenspecies

W

K

I

D

TheImmuneEpitopeDatabasecontainsepitopesthatotherresearchershavereportedintheliterature.

Useittoseeifanyonehasworkedexperimentallywithyourpredictedpeptidesbefore.Ifsomeonehasalreadytestedwhetheritgivesrisetoanimmuneresponse,youcanusethistoinformyourdecisiontousethemornot.

ImmuneEpitopeDatabase(IEDB)- Databaseswithepitopesinanarrayofpathogenspecies

W

K

I

D

http://www.iedb.org/

PredictionofTcellepitopes- Computationalapproachtofindingimmunogenicregionsinpathogens

Rappuoli R(2000) Reverse Vaccinology, Curr OpinMicrobiol

1,779 reportsofnovelorimprovedprediction algorithms

4,622 reportsofnovelvaccinetargets

142activeclinical trialsofvarious formsofDNAvaccines

2approved DNAvaccines...protecting horsesagainstWestNilevirusanddogs againstmelanoma.

16yearsofreversevaccinology- Whathaveweachieved?

Therearealotoftoolsoutthere

UseGoogletosearchfortoolsforyourspecificquestions.

Therearealsoonlineforawhereyoucanaskquestionsifyoucannotfindtheanswer.ForexampleBioStars:

https://www.biostars.org/

Youcanalsoexploretoolsinthetoolsregistryat:

https://bio.tools/

Takehomemessages

• Exploringand re-analyzingpublisheddataisincrediblyuseful- whenyousequencesomething, contributetotheresearchcommunityanduploadyourawandprocessedsequencedata!

Documents

Basic Sequence Analysis - Technical University of Denmarkteaching.bioinformatics.dtu.dk/teaching/images/9/... · Basic Sequence Analysis Lars RønnOlsen Assistant Professor, Technical