Bioinformatics and OMICs Group MeetingREFERENCE GUIDED RNA SEQUENCING

Hi Name: David Oliver

Advisor: Dr. Shtutman

Research: Understanding the role of COPZ2 silencing in cancer progression using RNA-seq to identify transcriptional changes caused by the loss of COPZ2 and its encoded microRNA.

Experience: Microarray analysis, multiple RNA-seq analyses including long-read (PacBio) and short-read (illumina) sequencing experiments.

Why RNA-seq What’s the question?

◦ Differential Expression◦ Differential splicing

Advantage over other technologies◦ Increased sensitivity◦ Increased reproducibility

RNA-Seq vs Dual- and Single-Channel Microarray Data: Sensitivity Analysis for Differential Expression and Clustering. Alina Sîrbu, Gráinne Kerr, Martin Crane, Heather J. Ruskin. Published: December 10, 2012DOI: 0.1371/journal.pone.0050986

Before You Start Consult a statistician Consult your sequencing core

Actually Doing RNA-seq Minimum Requirements

◦ Have consulted a statistician and your sequencing core◦ Know that your question can be answered using sequencing technology and that the

experimental design is appropriate.

Actually Doing RNA-seq Minimum Requirements

◦ Have consulted a statistician and your sequencing core◦ Know that your question can be answered using sequencing technology and that the

experimental design is appropriate.◦ > 10,000,000 reads per sample

◦ Much more depth required for differential splicing◦ ≥ 3 biological replicates

Actually Doing RNA-seq Minimum Requirements

◦ Have consulted a statistician and your sequencing core◦ Know that your question can be answered using sequencing technology and that the

experimental design is appropriate.◦ > 10,000,000 reads per sample

◦ Much more depth required for differential splicing◦ ≥ 3 biological replicates◦ Access to decent amount of computing power

◦ Can be done on a laptop but it takes ~ 3 weeks (ask me how I know)◦ Basic knowledge of Unix system and R

Actually Doing RNA-seq Minimum Requirements

◦ Have consulted a statistician and your sequencing core◦ Know that your question can be answered using sequencing technology and that the

experimental design is appropriate.◦ > 10,000,000 reads per sample

◦ Much more depth required for differential splicing◦ ≥ 3 biological replicates◦ Access to decent amount of computing power

◦ Can be done on a laptop but it takes ~ 3 weeks (ask me how I know)◦ Basic knowledge of Unix system and R

◦ Or, know someone who is willing to help you.

Actually Doing RNA-seq Suggested Pipeline

◦ Quality assessment:◦ FastQC◦ FastX toolkit

◦ Alignment: ◦ Bowtie2/Tophat2◦ STAR◦ NovoAlign

◦ Counting reads: ◦ FeatureCounts◦ Gencode annotation

◦ Differential expression analysis◦ edgeR

◦ Manipulating sequencing files◦ Samtools, bamtools

Total RNA or mRNA

RNA-Seq

RNA expression levels

Align to genome

NovoAlign

BowTie2

Normalization/Quantification edgeR

Quality Filtering

Raw Reads

Biological System

STAR

fastQC

Read Counting

FeatureCount

Gencode

Target Genome

RNA-seq Walkthrough Check some quality markers Getting the target genome Build aligner-specific indexed genome Perform alignment Do some file manipulation Get the annotation file Count reads Perform normalization and quantification

RNA-seq Walkthrough Check some quality markers Getting the target genome Build aligner-specific indexed genome Perform alignment Do some file manipulation Get the annotation file Count reads Perform normalization and quantification

Total RNA or mRNA

RNA-Seq

Quality Filtering

Raw Reads

Biological System

fastQC

Check some quality markers FastQC

◦ Basic tool for generating reports◦ Java based◦ Does not provide tools for correcting errors (FastX toolkit)◦ http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Other tools◦ FASTX toolkit: For fixing some problems with datasets (adapter trimming,

readthrough error correction, etc)◦ SAMstat: A tool for alignment QC

RNA-seq Walkthrough Check some quality markers Getting the target genome Build aligner-specific indexed genome Perform alignment Do some file manipulation Get the annotation file Count reads Perform normalization and quantification

Total RNA or mRNA

RNA-Seq

Align to genome

Quality Filtering

Raw Reads

Biological System

fastQC

Target Genome

Getting the target genomehttp://genome.ucsc.edu/

RNA-seq Walkthrough Check some quality markers Getting the target genome Build aligner-specific indexed genome Perform alignment Do some file manipulation Get the annotation file Count reads Perform normalization and quantification

Build aligner-specific indexed genome

This step is performed by the aligner and takes a variable amount of time depending on the type of index used and the size of the genome to be indexed.

RNA-seq Walkthrough Check some quality markers Getting the target genome Build aligner-specific indexed genome Perform alignment Do some file manipulation Get the annotation file Count reads Perform normalization and quantification

Total RNA or mRNA

RNA-Seq

Align to genome

NovoAlign

Bowtie2

Quality Filtering

Raw Reads

Biological System

STAR

fastQC

Target Genome

Perform alignmenttophat2 -p 12 --no-coverage-search --b2-N 1 --b2-L 32 --b2-i S,1,0.5 --b2-D 250 --b2-R 25 -o $RNAwork/ $RNAwork/Indexes/hg38_index $RNAwork/sample1.fastq

Reads:

Input : 20889144

Mapped : 18935684 (90.6% of input)

of these: 2674218 (14.1%) have multiple alignments (436 have >20)

90.6% overall read mapping rate.

RNA-seq Walkthrough Check some quality markers Getting the target genome Build aligner-specific indexed genome Perform alignment Do some file manipulation Get the annotation file Count reads Perform normalization and quantification

Do some file manipulation Depending on which aligner you choose to use, the aligned sequences may be output as a BAM or SAM file.◦ Sequence alignment/map format (SAM)

◦ Contains all the alignment information plus room for user-defined information about the alignments◦ Binary alignment/map format (BAM)

◦ A binary version of the SAM file◦ Added benefit of being much smaller and quickly accessed by other software◦ Not all software can manage the conversion from BAM back to SAM

To manipulate these formats i.e. sort, remove duplicates, remove unaligned sequences, use either samtools or bamtools

RNA-seq Walkthrough Check some quality markers

Getting the target genome

Build aligner-specific indexed genome

Perform alignment

Do some file manipulation

Get the annotation file

Count reads

Perform normalization and quantification

Total RNA or mRNA

RNA-Seq

Align to genome

BowTie2 Quality Filtering

Raw Reads

Biological System

fastQC

Read Counting Gencode

Target Genome

Get the annotation file Annotation files are readily available from multiple sources

◦ Gencode ( http://www.gencodegenes.org/releases/ )◦ Ensembl ( http://useast.ensembl.org/info/data/ftp/index.html?redirect=no )◦ Vega ( http://vega.sanger.ac.uk/info/about/data_access.html )◦ RefSeq ( http://www.ncbi.nlm.nih.gov/refseq/ )

These annotation sources mainly vary in the number of non-coding RNAs which have been annotated. ◦ RefSeq < Gencode < Ensembl < Vega

We use Gencode

RNA-seq Walkthrough Check some quality markers

Getting the target genome

Build aligner-specific indexed genome

Perform alignment

Do some file manipulation

Get the annotation file

Count reads

Perform normalization and quantification

Total RNA or mRNA

RNA-Seq

Align to genome

BowTie2 Quality Filtering

Raw Reads

Biological System

fastQC

Read Counting

FeatureCount

Gencode

Target Genome

Count Reads FeatureCounts

◦ We used to use HTseq-Count which was quite nice but we’ve switched to FeatureCounts because it is much, much, much faster.

◦ Also comes as an R package (bioc::Rsubread)

http://www-huber.embl.de/users/anders/HTSeq/doc/count.html

RNA-seq Walkthrough Check some quality markers

Getting the target genome

Build aligner-specific indexed genome

Perform alignment

Do some file manipulation

Get the annotation file

Count reads

Perform normalization and quantification

Total RNA or mRNA

RNA-Seq

Align to genome

BowTie2 Quality Filtering

Raw Reads

Biological System

fastQC

Read Counting

FeatureCount

Gencode

Target Genome

RNA expression levels

Normalization/Quantification edgeR

Perform normalization and quantification

EdgeR:counts <- read.table(file = "All_counts.csv”)counts <- na.omit(counts)counts <- counts[-(which(rowSums(counts) == 0)),]

### start edgeR ###group <- factor(rep(c("DU145.miR1","DU145.miR148a","DU145.miR148b","DU145.miR152"), each =3))y <- DGEList(counts = counts, group = group)### convert count matrix to a DGEList objectdesign <- model.matrix(~0+group) ### Experimental designkeep <- which(rowMeans(cpm(y)) > 10); y <- y[keep,] ### Remove genes with really low counts per milliony$samples$lib.size <- colSums(y$counts) ### this re-calculates the library size after removing samples with low CPMy <- calcNormFactors(y) ### calculate between sample normalizationy <- estimateGLMRobustDisp(y, design) ### calculate within sample normalizations (sort of)fit <- glmFit(y, design) ### fit the “massaged data” to a generalized linear model

### perform Likelihood Ratio Test on each contrast ###lrt.du145.mir148a <- glmLRT(fit, contrast=c(-1,1,0,0,0,0,0,0)) lrt.du145.mir148b <- glmLRT(fit, contrast=c(-1,0,1,0,0,0,0,0))lrt.du145.mir152 <- glmLRT(fit, contrast=c(-1,0,0,1,0,0,0,0))

### generate a user-friendly output table ###tt.du145.mir148a <- topTags(lrt.du145.mir148a, n = Inf, sort.by = "none")tt.du145.mir148b <- topTags(lrt.du145.mir148b, n = Inf, sort.by = "none")tt.du145.mir152 <- topTags(lrt.du145.mir152, n = Inf, sort.by = "none")

Expected Results

du145-148a du145-148b du145-152MAL2 -3.559 -1.869 -4.668CDH1 -2.634 -2.173 -4.030ERRFI1 -1.209 -0.824 -1.595PPP6R1 -1.015 -0.546 -1.082NTSR1 -0.954 -2.126 -1.314ITGA5 -0.865 -0.928 -1.077PPAP2B -0.616 -0.476 -1.413MCAM 0.407 0.702 1.622IGFBP5 1.897 1.398 2.848GPC4 2.106 2.415 3.420CCL2 2.114 2.758 2.956

Long Read (> 1kb) RNA-seq Long read analysis is performed with essentially the same workflow.

For alignment, STAR or GMAP work equally well

Questions?