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Page 1: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Assembly,annotation,quantification

Page 2: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Fromreadstotranscripts

denovoassembly

Genomebased Genomeguideddenovo

Reads

Putativetranscripts

Mappingagainstgenome

Readclusters

Page 3: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Transcriptreconstruction

•  Nowweknowwhichexonsareexpressed,but:– Agenecanhavemorethanonetranscript(isoforms)

– Withshortreadsequencing,noreadspansthecompletetranscript

– Whichisoformswerepresentinthesample?

Page 4: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

StringTie

Pertea,etal.(2015)."StringTieenablesimprovedreconstructionofatranscriptomefromRNA-seqreads."NatBiotech33(3):290-295.

Page 5: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Asthereadsgetlonger…

Page 6: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

MappingHisat2

Transcriptannotation

GTFfile

Transcriptidentification

StringTie

Rawreads

FASTQfile

Genomesequence

FASTAfile

Genomeannotation

GFF/GTFfile

Alignedreads

BAMfile

Page 7: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Question

Stringtiecanuseanannotatedgenome,orpredicttranscriptsdenovousingjustthegenomesequence.Whenwouldthelastoptionbeuseful?

Page 8: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Denovoassembly

WithoutareferencegenomeTools:Trinity,Velvet/Oasis,Trans-ABySS

Differentfromdenovogenomeassembly…SplicevariantsReadcoveragedependsonexpression

Page 9: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF
Page 10: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

k-mer

Read: GTAGAGCTGT 5-mers: GTAGA TAGAG AGAGC GAGCT AGCTG GCTGT

Page 11: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

k-mer

Read: GTAGAGCTGT 5-mers: GTAGA TAGAG AGAGC GAGCT AGCTG GCTGT

Page 12: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF
Page 13: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF
Page 14: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF
Page 15: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF
Page 16: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Trinity

Page 17: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

EffectofK-merssizeTworeadscouldbemergedwhentheyhaveaminimumoverlapofkLargerk-mersworkbetterforhighlyexpressedgenesandhashigheraccuracyShorterk-mersworkbetterforlowlyexpressedgenesbutcanleadtoloweraccuracy

Low High

KmersizeworksKmersizewillnotwork

Page 18: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Annotation

Page 19: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Asthereadsgetlonger…

Page 20: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

MappingHisat2

transcriptome

FASTAfile

Transcriptannotation

GTFfile

Transcriptidentification

StringTie

Rawreads

FASTQfile

AssemblyTrinity

Genomesequence

FASTAfile

Genomeannotation

GFF/GTFfile

Alignedreads

BAMfile

Page 21: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Questions

Howwouldadenovoassemblybeaffectedbybadqualityreads?Howcouldyou'repair'transcriptsthatwereassembledinfragments?Howwouldyouidentifyaread'smRNAifyouonlyhavethetranscriptome?

Page 22: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Quantification

MapreadstothegenomeExon/intronstructureAlternativesplicing:whatwerethetranscripts?

Mapreadstothetranscriptome

Differentisoforms

Page 23: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Normalization

NumberofmappedreadspermRNAdependson:TheabundanceofthemRNAThetotalnumberofreadsThelengthofthemRNATechnical“noise”

Page 24: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Normalization

NumberofmappedreadspermRNAdependson:TheabundanceofthemRNAThetotalnumberofreadsThelengthofthemRNATechnical“noise”

Page 25: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

RPKM,FPKM,TPM,CPM

Countthereadsmappingtoatranscript/geneComparethesamegenebetweensamples:normalizeforthetotalnumberofmappedreads-CPM(countspermillion)

Comparedifferentgenes/isoforms:normalizeforthelengthofthetranscript-RPKM,FPKM,TPM(transcriptspermillion)

Page 26: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Correctingfortranscriptlengthandtotalnumberofreads

RPKM =109 x Number of reads mapped to a regionTotal reads x region length

10,000,000300nt

RPKM≈1.7

Allmappedreads

Readsmappedtoregion

Featurelength

RPKM:ReadsPerKilobaseoftranscriptperMillionreads

Page 27: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Correctforoutliers

Page 28: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Sequencecoveragebias

Page 29: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

LiorPachterGenomeInformaticskeynoteCSHL

Readsmappingtoisoforms

Page 30: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Pseudoalignment

"ithasbeenshownthataccuratequantificationdoesnotrequireinformationonwhereinsidetranscriptsthereadsmayhaveoriginatedfrom,butratherwhichtranscriptscouldhavegeneratedthem"

Bray,N.L.,H.Pimentel,P.MelstedandL.Pachter(2016)."Near-optimalprobabilisticRNA-seqquantification."NatBiotech34(5):525-527.

Page 31: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Pseudoalignment

Mappingreadstoatranscriptomebasedonsharedk-mers,onlyusinginformativek-mers.+Veryfast-Doesnotdetectnewtranscripts-DoesnotfindSNPsTools:kallisto,salmon

Bray,N.L.,H.Pimentel,P.MelstedandL.Pachter(2016)."Near-optimalprobabilisticRNA-seqquantification."NatBiotech34(5):525-527.Patro,R.,G.Duggal,M.I.Love,R.A.Irizarry,C.Kingsford(2017)."Salmonprovidesfastandbias-awarequantificationoftranscriptexpression."NatMeth14(4):417-419.

Page 32: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Pseudoalignment:Kallisto

Bray,N.L.,H.Pimentel,P.MelstedandL.Pachter(2016)."Near-optimalprobabilisticRNA-seqquantification."NatBiotech34(5):525-527.

Page 33: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Pseudoalignment

Bray,N.L.,H.Pimentel,P.MelstedandL.Pachter(2016)."Near-optimalprobabilisticRNA-seqquantification."NatBiotech34(5):525-527.

Page 34: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

transcriptome

FASTAfile

Transcriptannotation

GTFfile

counts

csvfile

Rawreads

FASTQfile

Genomesequence

FASTAfile

Genomeannotation

GFF/GTFfile

Alignedreads

BAMfile

Page 35: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

PseudoalignmentKallisto

MappingHisat2

transcriptome

FASTAfile

Transcriptannotation

GTFfile

counts

csvfile

Transcriptidentification

&quantificationStringTie

Rawreads

FASTQfile

AssemblyTrinity

Genomesequence

FASTAfile

Genomeannotation

GFF/GTFfile

Alignedreads

BAMfile

Page 36: Assembly, annotation, quantification Assembly.pdf · annotation GTF file Transcript identification StringTie Raw reads FASTQ file Genome sequence FASTA file Genome annotation GFF/GTF

Handson


Review Free EGASP: the human ENCODE Genome Annotation ...repository.cshl.edu/25307/1/...Genome-Annotation...Automatic genome annotation methods To date, accurate automatic annotation

Crowdsourcing genome annotation at #ccs14

Workflows and Pipelines for NGS analysis: Lessons from ... · Genome annotation. Genome annotation Transcriptome Proteome Structural ... biology Importance of genome annotation Armengaud

Whole Genome Annotation: In Silico Analysis

Era7 Bacterial Genome Annotation

Computational Genome Annotation

Social genome annotation

Genome Annotation - NDSUmcclean/plsc411/Genome... · Genome Annotation . Genome Sequencing • Costliest aspect of sequencing the genome o But Devoid of content • Genome must be

Proteogenomics : Refining and Improving Genome Annotation

Genome Annotation 2010

AGOUTI: improving genome assembly and annotation using ...Keywords: Genome assembly, Scaffolding, Genome annotation, RNA sequencing, RNA-seq Background Findings Genomes sequenced using

Genome Annotation: A Protein-centric Perspective

Apollo Collaborative genome annotation editing

NCBI’s Genome Annotation: Overview

Genome Annotation: A Protein-centric Perspective

Melampsora Genome Annotation and Genome Structure Analysis First Annotation Workshop of the Melampsora Genome Consortium Yao-Cheng Lin Bioinformatics &

Eukaryotic Genome Annotation

Bio305 genome analysis and annotation 2012

Genome annotation 2013

Basics of Genome Annotation

Arabidopsis Genome Annotation

Genome Sequencing Impact on Annotation

The Vertebrate Genome Annotation Database

Gene prediction and Genome Annotation

Genome sequencing and annotation

Bioinformatics and Genome Annotation

Genome Annotation - University of California, Santa Cruz · Genome Annotation Repeat Annotation For Gene Annotation 1 RepeatMasker -pa xx -gccalc -nolow -species aves genome.fasta

Genome Annotation - DTU Health Tech · Genome Annotation and Gene-finding 27621 Prokaryotic Gene Discovery, Metagenomics and Pangenomics. Genome Sequencing. Annotation is the process

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Release 0.3.1 Laurent Gautier ([email protected]) · FASTA, as well as GTF/GFF derived from its genome annotation, for the purpose of running tests and examples. 2.2Installation

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Annotation. Traditional genome annotation BLAST Similarities

Genome Annotation - Cornell University