Expression A nalysis of RNA - seq Data

Expression Analysis of RNA-seq Data Manuel Corpas Plant and Animal Genomes Project Leader manuel.corpas@tgac.ac.uk

Generation of Sequence Mapping Reads Identification of splice junctions Assembly of Transcripts Statistical Analysis 1 Summarization (by exon, by transcript, by gene) 2 Normalization (within sample and between sample) 3 Differential expression testing (poisson test, negative binomial test)

The Tuxedo Tools • Developed by Institute of Genetic Medicine at Johns Hopkins University / University of California, Berkeley / Harvard University • 157 pubmed citations Tophat Fast short read aligner (Bowtie) Spliced read identification (Tophat) Cufflinks package Cufflinks – Transcript assembly Cuffmerge – Merges multiple transcript assemblies Cuffcompare – Compare transcript assemblies to reference annotation Cuffdiff – Identifies differentially expressed genes and transcripts CummeRbund Visualisation of differential expression results

RNA-seq Experimental design • Sequencing technology (Solid, Illumina) • Hiseq 2000, 150 million read pairs per lane, 100bp • Single end (SE) Paired end (PE), strand specific • SE Quantification against known genes • PE Novel transcripts, transcript level quantification • Read length (50-100bp) • Greater read length aids mapping accuracy, splice variant assignment and identification of novel junctions • Number of replicates • often noted to have substantially less technical variability • Biological replicates should be included (at least 3 and preferably more) • Sequencing depth • Dependent on experimental aims

RNA-seq Experimental design Labaj et al. Characterization and improvement of RNA-Seq precision in quantitative transcript expression profiling. Bioinformatics (2011) Toung et al. RNA-sequence analysis of human B-cells. Genome Research (2011) . • Extrapolation of the sigmoid shape suggests 20 % of transcripts not expressed • First saturation effects set in at ~40 million read alignments • ~240 Million reads achieve 84 % transcript recall

RNA-seq Experimental design General guide • Quantify expression of high-moderatly expressed known genes • ~20 million mapped reads, PE, 2 x 50 bp • Assess expression of alternative splice variants, novel transcripts, and strong quantification including low copy transcripts • in excess of 50 million reads, PE, 2 x 100 bp Example • Examine gene expression in 6 different conditions with 3 biological replicates (18 samples) • Multiplexing 6 samples per lane on 3 lanes of the HiSeq (50 bp PE) • Generates ~25 M reads per sample • Assuming ~80% of reads map/pass additional QC (20 M mapped read per sample) • Cost – 3 lanes (£978 x 3 ) 18 libraries (£ 105 x 18), total £4824

Step 1 – Preprocessing reads • Sequence data provided as Fastq files • QC analaysis – sequence quality, adapter contamination (FASTQC) • Quality trimming, adapter removal (FASTX, Prinseq, Sickle)

Step 2 – Data sources • Reads (Fastq, phred 33) • Genomic reference (fasta TAIR10), or pre built Bowtie index • GTF/GFF file gene calls (TAIR10) • http://tophat.cbcb.umd.edu/igenomes.html

Tuxedo Protocol Leaf (SAM1) Leaf (SAM2) Flower (SAM3) Flower (SAM4) Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Reads (FASTQ) TOPHAT (Read Mapping) GTF + Genome Alignments (BAM) GTF CUFFLINKS (Transcript Assembly) Assemblies (GTF) CUFFMERGE (Final Transcript Assembly) Assembly (GTF) GTF CUFFDIFF (Differential expression results) CUFFCOMPARE (Comparion to reference) Visualisation (PDF) CUMMERBUND (Expression Plots)

Step 3 Tuxedo Protocol - TOPHAT Leaf (SAM1) Leaf (SAM2) Flower (SAM3) Flower (SAM4) • Non-spliced reads mapped by bowtie • Reads mapped directly to transcriptome sequence • Spliced reads identified by tophat • Initial mapping used to build a database of spliced junctions • Input reads split into smaller segments • Coverage islands • Paired end reads map to distinct regions • Segments map in distinct regions • Long reads >=75bp used to identify GT-AG, GC-AG and AT-AC splicings) Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Reads (FASTQ) TOPHAT (Read Mapping) GTF + Genome Alignments (BAM)

Step 3 Tuxedo Protocol - TOPHAT Leaf (SAM1) Leaf (SAM2) Flower (SAM3) Flower (SAM4) • -i/--min-intron-length <int> 40 • -I/--max-intron-length <int> 5000 • -a/--min-anchor-length <int> 10 • -g/--max-multihits <int> 20 • -G/--GTF <GTF/GFF3 file> Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Reads (FASTQ) TOPHAT (Read Mapping) GTF + Genome Alignments (BAM)

Step 4 Tuxedo Protocol - CUFFLINKS Leaf (SAM1) Leaf (SAM2) Flower (SAM3) Flower (SAM4) • Accurate quantification of a gene requires identifying which isoform produced each read. • Reference Annotation Based Transcript (RABT) assembly • Sequence bias correction -b/--frag-bias-correct <genome.fa> • multi-mapped read correction is enabled (-u/--multi-read-correct) Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Reads (FASTQ) TOPHAT (Read Mapping) GTF + Genome Alignments (BAM) GTF CUFFLINKS (Transcript Assembly) Assemblies (GTF)

Tuxedo Protocol - CUFFDIFF Leaf (SAM1) Leaf (SAM2) Flower (SAM3) Flower (SAM4) Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Read 1 Read 2 Reads (FASTQ) TOPHAT (Read Mapping) Alignments (BAM) CUFFLINKS (Transcript Assembly) CUFFMERGE (Final Transcript Assembly) Assembly (GTF) GTF + Genome GTF mask file CUFFDIFF (Differential expression results) CUFFDIFF output – FPKM (fragments per kilobase of transcript per million fragments mapped) values, fold change, test statistic, p-value, significance statement.

CUFFDIFF - Summarisation (A) 1 2 3 4 • Cuffdiff output (11 files) • FPKM tracking files (Transcript, Gene, CDS, Primary transcript) • Differential expression tests (Transcript, Gene, CDS, Primary transcript) • Differential splicing tests – splicing.diff • Differential coding output – cds.diff • Differential promoter use – promoters.diff (B) 1 2 4 (C) 1 2 4 A + B + C Grouped at Gene level B + C Grouped at CDS level A + C Grouped at Primary transcript level A, B, C No group at the transcript level Look at difference in distribution (rather than total level)

A test case – Ricinus Communis (Castor bean) • 5 tissues – Aim : identify differences in lipid-metabolic pathways

A test case – RicinusCommunis (Castor bean) • Cufflinks – Cuffcompare Results • RNA-Seq reads assembled into 75090 transcripts corresponding to 29759 ‘genes’ • Compares to the 31221 genes in version 0.1 of the JCVI assembly • 35587 share at least one splice junction (possible novel splice variant). • 2847 were located intergenic to the JCVI annotation and hence may represent novel genes • 218147 splice junctions were identified, 112337 supported by at least 10 reads, >300,000 distinct to the JCVI annotation

Visualisation • Bam files can be converted to wiggle plots • CummeRbundfor visualisation of Cuffdiff output Bam, Wiggle and GTF files viewed in IGV CummeRbundvolcano and scatter plots

Thanks • David Swarbreck (Genome Analysis Team Leader, TGAC) • Mario Caccamo (Head Bioinformatics Division, TGAC)

Expression A nalysis of RNA - seq Data

Expression A nalysis of RNA - seq Data

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