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Bioinforma)csResources-Genbank-

Lecture&ExercisesProf.B.Rost,Dr.L.Richter,J.Reeb

Ins)tutfürInforma)kI12

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Na)onalCenterforBiotechnologyInforma)on,NCBI

http://nihrecord.nih.gov/newsletters/2013/07_19_2013/images/milestonesPic6.jpg

●  firstideasinthemiddleofthe80s

●  divisionoftheNa)onalLibraryofMedicine(NLM)insidetheNa)onalIns)tutesofHealth(NIH)

●  poli)calmission

●  foundedin1988

●  DavidLipman

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NCBI’spoli)calmissionasdefinedbythebill:1.  design,develop,implement,andmanageautomatedsystems

forthecollec)on,storage,retrieval,analysis,anddissemina)onofknowledgeconcerninghumanmolecularbiology,biochemistry,andgene)cs;

2.  performresearchintoadvancedmethodsofcomputer-basedinforma)onprocessingcapableofrepresen)ngandanalyzingthevastnumberofbiologicallyimportantmoleculesandcompounds;

3.  enablepersonsengagedinbiotechnologyresearchandmedicalcaretousesystemsdevelopedunderparagraph(1)andmethodsdescribedinparagraph(2);and

4.  coordinate,asmuchasisprac)cable,effortstogatherbiotechnologyinforma)ononaninterna)onalbasis.

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SelectedNCBIAccomplishmentsBlastGenBankatNCBI

NCBIwebsite

GenomesOMIM

PubMed

1990

1992

1994

1995

1996

1997

HumanGenomePubMedCentral

EntrezGene/DTDs

NIHPublicAccessGenomeReferenceConsor)um

1000GenomesProject

1999

2000

2003

2005

2007

2008

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NCBIResources●  NCBIcurrentlyhostsavastbunchofresourceshap://www.ncbi.nlm.nih.gov/guide/all/

●  groupedaccordingtovariouscriteria-  metadata,project-centric-  methodoriented-  topicoriented

●  sortedinthesec)ons:databases,downloads,submissions,tools,howtos

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Genbank’sOrigin

●  WalterGoad,LosAlamosNa)onalLaboratory

●  LosAlamosSequenceDatabase1979

●  Crea)onandreleaseofGenBankin1982

●  Endof1982:2000sequences

●  MovetoNCBIin1992http://www.lanl.gov/science-innovation/features/innovations/images/light/thumbnails/21.jpg

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Minutesfrom20thanniversaryofGenBankin2002

“....AmongthemisamemoonLosAlamosNa)onalLaboratorysta)onerydatedMay9,1980,thatreads:Monday,May12at10:30SteveSimoninvitesyouforcakeandcoffeetocelebrate100,000basesnowintheDNAsequencelibrary.”

takenfromhaps://www.genomeweb.com/genbank-turns-20

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GrowthofGenBankandWGS

-doublingapprox.every18months,diagramforrelease207,Apr.2015-currentversion:release213,Apr.2016:211.423.912.047basesinGenbank,1.452.207.704.949basesinWGS-takenfromhap://www.ncbi.nlm.nih.gov/genbank/sta)s)cs

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GrowthofGenBankandWGS

-currentrelease213:193.739.511sequenceinGenbank,338.922.537sequencesinWGS-takenfromhap://www.ncbi.nlm.nih.gov/genbank/sta)s)cs,release207,Apr.2015

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ReferencesforGenBank●  thecurrentcita)onsource:“GenBank”.NucleicAcidsRes.2014Jan;42(Databaseissue):D32-7.doi:10.1093/nar/gkt1030.Epub2013Nov11.

●  PMID:24217914●  partoftheInterna)onalNucleo)deSequenceDatabaseCollabora)on(INSDC)togetherwithEMBLNucleo)deSequenceDatabase(EMBL-Bank),partoftheEuropeanNucleo)deArchive(ENA)andtheDNADataBankofJapan(DDBJ)

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MostGrowingDivisionsDivision Description Release 197

(8/2013) Annual Increase (%)

WGS* Whole-genome shotgun data 500.420.412.665 62.4.

TSA* Transcriptome shotgun data 8.6333123.935 49.9

PHG Phages 119.812.712 42.5

VRL Viruses 1.757.202.472 22.9

BCT Bacteria 10.281.048.518 21.8

ENV Environmental samples 3.743.277.434 10.9

INV Invertebrates 2.737.140.464 9.8

PAT Patented sequences 13.290.161.247 9.7

PLN Plants 5.963.882.822 8.8

GSS Genome survey sequences 23.726.384.753 8.1

VRT Other vertebrates 3.068.956.026 6.3

MAM Other mammals 911.342.025 5.6

... ... ... ...

TOTAL All GenBank sequences 654.613.333.676 45.1

* not distributed with the release; there specific project server sections

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TopOrganisms(Rel.207)Organism Entries Non-WGS base

pair Homo sapiens 20.921.637 17.714.786.437 Mus musculus 9.727.522 9.995.696.539

Rattus norvegicus 2.193.812 6.526.236.496 Bos taurus 2.227.298 5.410.360.312 Zea mays 4.177.175 5.201.714.457 Sus scrofa 3.297.029 4.895.127.638

Danio rerio 1.727.668 3.133.901.682 Triticum aestivum 1.796.780 1.927.718.314

... ... ... Oryza sativa

Japonica Group 1.376.410 1.265.556.227

... ... ... Arabidopsis thaliana 2.578.785 1.202.100.008

... ...

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Distribu)onofSequenceFiles(Rel.207)Division Number of Files

BCT 178 CON 317 ENV 81 EST 478 HTG 142 INV 126 PAT 219 PLN 107 TSA 175 VRL 34

Release 207 consists of 2333 text files in total.

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DatabaseFiles

●  GenBankcomesinasetofcompressedtextfilesavailableviaFTP

●  seejp://jp.ncbi.nih.gov/genbank/gbrel.txt●  2333ASCIIfiles(listedindivisionplusaddi)onallistfiles)intherangeof0.7-520MB

●  uncompressed~709GB●  eachfileconsistsoftwopor)ons

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DatabaseFiles●  Part1:highlyconserveddatabasefileheaders1 10 20 30 40 50 60 70 79 ---------+---------+---------+---------+---------+---------+---------+--------- GBBCT1.SEQ Genetic Sequence Data Bank April 15 2015 NCBI-GenBank Flat File Release 207.0 Bacterial Sequences (Part 1) 51396 loci, 92682287 bases, from 51396 reported sequences ---------+---------+---------+---------+---------+---------+---------+--------- 1 10 20 30 40 50 60 70 79

●  Part1:sequenceentriesforthatdivisiondescribedintheheader

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TheGenBankFlatFileFormat

●  asequenceentryconsistsofmanyrecords(lines)●  eachrecordconsistsoftwoparts

●  Part1:columns1-10/EntryFieldName

●  Part2:remaininglinewiththecontent

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Part1/1●  akeyword,beginningincolumn1oftherecord(e.g.,REFERENCEisakeyword)

●  asubkeywordbeginningincolumn3,withcolumns1and2blank(e.g.,AUTHORSisasubkeywordofREFERENCE)

●  orasubkeywordbeginningincolumn4,withcolumns1,2,and3blank(e.g.,PUBMEDisasubkeywordofREFERENCE)

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Part1/2

●  blankcharacters,indica)ngthatthisrecordisacon)nua)onoftheinforma)onunderthekeywordorsubkeywordaboveit

●  acode,beginningincolumn6,indica)ngthenatureofanentry(featurekey)intheFEATUREStable

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Part1/3●  anumber,endingincolumn9oftherecord:-  Thisnumberoccursinthepor)onoftheentrydescribingtheactualnucleo)desequenceanddesignatesthenumberingofsequenceposi)ons

●  twoslashes(//)inposi)ons1and2,markingtheendofanentry

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Part2●  Thesecondpartofeachsequenceentryrecordcontainstheinforma)onappropriatetoitskeyword

●  inposi)ons13to80forkeywords

●  inposi)ons11to80forthesequence

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EntryFieldTypes(incomplete)●  Locus:Ashortmnemonicnamefortheentry,chosentosuggestthesequence'sdefini)on;mandatorykeyword/exactlyonerecord.

●  Defini4on:Aconcisedescrip)onofthesequence;mandatorykeyword/oneormorerecords

●  Accession:-  theprimaryaccessionnumberisaunique,unchangingiden4fierassignedtoeachGenBanksequencerecord.

-  tobeusedforcita)onsfromGenBank-  mandatorykeyword/oneormorerecords.

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EntryFieldTypes(incomplete)

●  Version:-  compoundiden)fierconsis)ngoftheprimaryaccessionnumberandanumericversionnumberassociatedwiththecurrentversionofthesequencedataintherecord

-  op)onallyfollowedbyanintegeriden)fier(a"GI")assignedtothesequencebyNCBI

-  mandatorykeyword/exactlyonerecord

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EntryFieldTypes(incomplete)

●  DBLINK:providescross-referencestoresourcesthatsupporttheexistenceasequencerecord;op4onalkeyword/oneormorerecords

●  Keywords:shortphrasesdescribinggeneproductsandotherinforma)onaboutanentry;mandatorykeywordinallannotatedentries/oneormorerecords

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EntryFieldTypes(incomplete)

●  Source:Commonnameoftheorganismorthenamemostfrequentlyusedintheliterature;mandatorykeywordinallannotatedentries/oneormorerecords/includesonesubkeyword

●  Organism:Formalscien)ficnameoftheorganism(firstline)andtaxonomicclassifica)onlevels(secondandsubsequentlines);mandatorysubkeywordinallannotatedentries/twoormorerecords

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EntryFieldTypes(incomplete)●  Reference:-  Cita)onsforallar)clescontainingdatareportedinthisentry

-  includessevensubkeywordsandmayrepeat-  mandatorykeyword/oneormorerecords

●  Journal:liststhejournalname,volume,year,andpagenumbersofthecita)on;mandatorysubkeyword/oneormorerecords

●  op)onalsubkeywords:Authors,Consor)um,Title,Medline,Pubmed,Remark

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EntryFieldTypes(incomplete)●  Features:tablecontaininginforma)ononpor)onsofthesequencethatcodeforproteinsandRNAmolecules;sitesofbiologicalsignificance;op4onalkeyword/oneormorerecords

●  Origin:-  specifica)onofhowthefirstbaseofthereportedsequenceisopera)onallylocatedwithinthegenome

-  mandatorykeyword/exactlyonerecord-  followedbysequencedata(mul)plerecords)

●  //:entrytermina)onsymbol;mandatoryattheendofanentry/exactlyonerecord

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DetailedLocusFormatColumns Contents 01-05 'LOCUS'

06-12 spaces

13-28 Locus name

29-29 space

30-40 Length of sequence, right-justified

41-41 space

42-43 bp

44-44 space

45-47 spaces, ss- (single-stranded), ds- (double-stranded), or ms- (mixed-stranded)

48-53 NA, DNA, RNA, tRNA (transfer RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), uRNA (small nuclear RNA), left justified

54-55 space

56-63 'linear' followed by two spaces, or 'circular'

64-64 space

65-67 The division code

68-68 space

69-79 Date, in the form dd-MMM-yyyy (e.g., 15-MAR-1991)

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AccessionFormat●  sixoreightcharacters●  sixcharacterformat:-  singleuppercaseleaer-  5digits

●  eigthcharacterformat:-  twouppercaseleaers-  6digits

●  primaryaccessionnumberalwaysthefirstone

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Features(Incomplete)

●  authorita)vesource:hap://www.insdc.org/documents/feature-table

●  featuretablecontainsinforma)onabout:-  geneandgeneproducts-  regionsofbiologicalsignificance-  canenumeratedifferencesbetweenvariousreports-  providescross-referencestootherdatacollec)ons-  allowshierarchicalrela)onbetweenthefeatures

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Layout●  firstlineofthefeaturetableisaheader●  includesthekeyword‘FEATURES’andthecolumnheader‘Loca)on/Qualifiers’

●  eachfeatureconsistsof:-  descriptorlinecontainingafeaturekeyandaloca)on

-  acon)nua)onlinefortheloca)onmayfollow-  featurequalifiersmayfollowthedescriptorline-  key:column6-20,loca)onstartsincolumn22-  qualifiersonsubsequentlinesatcolumn22star)ngwitha‘/’

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AFewFrequentFeatures●  CDS:sequencecodingforaminoacidsinprotein(includesstopcodon)

●  exon:regionthatcodesforpartofsplicedmRNA●  gene:regionthatdefinesafunc)onalgene,possiblyincludingupstream(promotor,enhancer,etc)anddownstreamcontrolelements,andforwhichanamehasbeenassigned

●  mRNA:messengerRNA

●  .......>60featurescurrently

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Loca)onandQualifiers

●  Loca)on:-  aloca)oncanbe:asinglebase,aspanofbases,asitebetweentwobases,ajoinofsequences,...

-  examples:23,23..56,23^24,join(23..56,87..110)

●  Qualifiers:-  format:fromcolumn22/qualifier_name[=value]-  types:freetext,enumera)onorcontrolledvocabulary,cita)ons,sequences,featurelabels

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DatabaseCrossReferences/db_xref

●  hap://www.ncbi.nlm.nih.gov/genbank/collab/db_xref/

●  Qualifier:/db_xref="database:idenDfier”●  Defini4on:databasecross-reference:pointertorelatedinforma)oninanotherdatabase

●  Scope:allfeaturekeys●  Example:/db_xref="Swiss-Prot:P12345”

●  currently>120databasesavailable

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AnatomyofaGenbankFlatFile

. . .

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AnatomyofaGenbankFlatFile

. . .

Locus line

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AnatomyofaGenbankFlatFile

. . . Accession Number, Version and GI number

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AnatomyofaGenbankFlatFile

. . . Feature table with annotations

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UsefulResourcesfromNCBI

●  Materials:●  Electronicbookshelf

●  hap://www.ncbi.nlm.nih.gov/educa)on/factsheets/

●  jp://jp.ncbi.nih.gov/pub/factsheets/Factsheet_Books.pdf

●  NCBImanuals

●  textbooks

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UsefulResourcesfromNCBI

●  Processes,e.g.Prokaryo)cGenomeAnnota)onPipeline

●  designedforbacterialandarchaealgenomes●  mul)-levelprocessincludingprotein-codinggenepredic)onandfunc)onalgenomeunitlikerRNAs,tRNAs,smallRNAs,pseudogenescontrolregions,repeats,inser)onelementsa.s.f.

●  combina)onofab-iniDopredic)onandhomologybasedmethods

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UsefulResourcesfromNCBI●  referencedatabases:RefSeq●  hap://www.ncbi.nlm.nih.gov/refseq/

●  comprehensive,integrated,non-redundant,well-annotatedsetofsequences,includinggenomicDNA,transcripts,andproteins

●  stablereferenceforgenomeannota)on,esp.subsetofRefSeqGene

●  referencesequences

●  referencecoordinates●  accessibleviaBLAST,EntrezandFTP

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RefSeq●  createdby:-  Eukaryo)cGenomeAnnota)onPipeline-  Prokaryo)cGenomeAnnota)onPipeline-  Manualcura)on-  SubmissiontoINSDCmembers

●  reflectcurrentknowledgeofsequencesdataandbiology

●  formatconsistency●  Accessionnumbercontainsan“_”

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RefSeqGrowth

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DatabasesAccessibleviaEntrez

http://www.ncbi.nlm.nih.gov/gquery/

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Computa)on:BlastatNCBI

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SearchingtheNCBI/Entrez●  provideanintegratedsearchinterfacetothedifferentNCBIdatabases:EntrezProgrammingU)li)es(E-u)li)es)

●  Base-URL:hap://eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/

●  >40databases

●  stableinterfaceofnineserver-sideprograms

●  hap://www.ncbi.nlm.nih.gov/books/NBK25501/

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EntrezGuidelines●  ifyouusetheeu)lsagainsttheguidelinesyoumightbebanned!

●  >100requests:weekendsoroutsideUSpeak)mes(9pm-5am,EST)

●  notmorethan3requestpersecond

●  provideemailandtoolname:&tool=&email=!

●  registra)onwithemailandtoolnamewithNCBImayrelaxtheserestric)ons

●  supportedbyBioPython

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Construc)ngURLs

●  parameter:&lowerCaseName●  excep)on:&WebEnv

●  norequiredorder

●  nullvaluesandinappropriateparameteraregenerallyignored

●  nospaces,use+instead

●  useURLencodingsforspecialcharacterlike:%22for“or%23for#or%40for@

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E-u)li)es●  Einfo●  Esearch

●  EPost

●  ESummary●  EFetch

●  ELink

●  EGQuery

●  ESpell●  ECitMatch

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ESearch

●  textsearch●  eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/esearch.fcgi

●  respondstoatextquerywiththelistofmatchingUIDsinagivendatabase(forlateruseinESummary,EFetchorELink),alongwiththetermtransla)onsofthequery

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ESummary

●  documentsummarydownloads●  eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/esummary.fcgi

●  respondstoalistofUIDsfromagivendatabasewiththecorrespondingdocumentsummaries

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EGQuery

●  globalquery●  eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/egquery.fcgi

●  respondstoatextquerywiththenumberofrecordsmatchingthequeryineachEntrezdatabase

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EInfo

●  databasesta)s)cs●  eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/einfo.fcgi

●  providesthenumberofrecordsindexedineachfieldofagivendatabase,thedateofthelastupdateofthedatabase,andtheavailablelinksfromthedatabasetootherEntrezdatabases

●  without&db:listsallavailabledatabases

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EFetch

●  datarecorddownloads●  eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/efetch.fcgi

●  respondstoalistofUIDsinagivendatabasewiththecorrespondingdatarecordsinaspecifiedformat

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ELink

●  Entrezlinks●  eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/elink.fcgi

●  respondstoalistofUIDsinagivendatabasewitheitheralistofrelatedUIDs(andrelevancyscores)inthesamedatabaseoralistoflinkedUIDsinanotherEntrezdatabase

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ELink

●  checksfortheexistenceofaspecifiedlinkfromalistofoneormoreUIDs

●  createsahyperlinktotheprimaryLinkOutproviderforaspecificUIDanddatabase,orlistsLinkOutURLsandaaributesformul)pleUIDs

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EPost

●  UIDuploads●  eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/epost.fcgi

●  acceptsalistofUIDsfromagivendatabase,storesthesetontheHistoryServer,andrespondswithaquerykeyandwebenvironmentfortheuploadeddataset

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ESpell

●  spellingsugges)ons●  eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/espell.fcgi

●  retrievesspellingsugges)onsforatextqueryinagivendatabase

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ECitMatch

●  batchcita)onsearchinginPubMed●  eu)ls.ncbi.nlm.nih.gov/entrez/eu)ls/ecitmatch.cgi

●  retrievesPubMedIDs(PMIDs)correspondingtoasetofinputcita)onstrings

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Iden)ficators●  recordsareiden)fiedbyanintegerIDcalledUID●  UIDaredatabasespecificlikeGInumbers,PMIDS,MMDB-IDs

●  UIDareaswellinputandoutput

●  especiallyusefulincombina)onwiththeHistoryserver

●  afulldescrip)onofparametersandsyntaxcanbefoundat:hap://www.ncbi.nlm.nih.gov/books/NBK25499/

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SelectedUIDsEntrez Database UID common name E-utility Database Name Books Book ID books Conserved Domains PSSM-ID cdd dbVar dbVar ID dbvar EST GI number nucest Gene Gene ID gene Genome Genome ID genome MeSH MeSH ID mesh NCBI Web Site Web Site ID ncbisearch Nucleotide GI number nuccore PubMed PMID pubmed ... ... ...

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EntrezCoreEngine●  EGQuery,ESearch,andESummary●  twotasks:-  assemblealistofUIDsthatmatchatextquery(ESearch)-  retrieveabriefsummaryrecordcalledaDocumentSummary(DocSum)foreachUIDESummary)

●  EGQuey:globalversionofESearch●  esearch.fcgi?db=database&term=query 
esummary.fcgi?db=database&id=uid1,uid2,uid3,...!

●  expandedintomorecomplicatedEntrezqueries

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EntrezDatabases(EInfo,EFetch,andELink)

●  EInfo:-  providesdetailedinforma)onabouteachdatabase-  includinglistsoftheindexingfieldsinthedatabase-  availablelinkstootherEntrezdatabases

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EntrezDatabases(EInfo,EFetch,andELink)

●  addedvaluetotherawdata:-  supportsavarietyofdisplayformats:EFetchUIDlistsinXMLandplaintext(&retmode)foralldatabases,otherformats(&rettype)aredatabasespecific

-  hap://www.ncbi.nlm.nih.gov/books/NBK25499/table/chapter4.T._valid_values_of__retmode_and/?report=objectonly

-  efetch.fcgi?db=database&id=uid1,uid2,uid3 
&rettype=report_type&retmode=data_mode!

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EntrezDatabases(EInfo,EFetch,andELink)

●  addedvaluetotherawdata:-  linkstorecordsinotherEntrezdatabasesmanifestedaslistofassociatedUIDs

-  UIDsmustbevalidinsourcedatabase(&dbfrom)-  elink.fcgi?dbfrom=protein&db=gene&id=15718680,157427902

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EntrezHistoryServer

●  simple:intheGUIaccessibleviatherespec)vetabs

●  youcanstoretemporarilysetsofUIDsasinputforlaterqueriesthroughothertools

●  eachlistofUIDsisspecifiedby:-  &query_key(integerlabel)-  &WebEnv(cookiestring)

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Crea)onofastoredUIDlist

●  EPost:-  EPostcanbeuseduploadaUIDlist-  returns&query_keyand&WebEnv!

●  ESearch:-  storestheresultsifgiven&usehistory=y!

●  ELink:-  storestheresultsifgiven&cmd=neighbor_history!

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UsageofstoredUIDlists●  Useofstoredlists:esummary.fcgi?db=database&WebEnv=webenv 
&query_key=key!

●  onewebenvironmentcanholdmul)pleresultlists

●  listsinthesamewebenvironmentcanbecombinedwithAND,OR,NOT

●  bydefaulteverycallcreatesanewenvironment

●  ->give&WebEnvinsubsequentcallstostorethelistsinthesamewebenvironment

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SketchingPipelines

●  getDocSummariesorentriesforkeywordsorIDs:-  ESearch->ESummary/EFetch-  EPost->ESummary/EFetch

●  filter/limitarecordset:-  EPost/ELink->ESearch

●  moreadvancedqueries:-  ESearch->ELink->ESummary/EFetch-  EPost->ELink->ESearch->EFetch

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E-u)lityWebinar

●  haps://www.youtube.com/watch?v=iCFVVexp30o