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Schedule change. Galaxy server going down for maintenance on Thursday. Day 2: AM - Introduction to RNA-Seq (and a touch of miRNA-Seq) Day 2: PM - RNA-Seq practical ( Tophat + Cuffdiff pipeline on Galaxy) Day 3: AM – Introduction to Exome Sequencing and Variant Discovery

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Schedule change

Schedule change

Galaxy server going down for maintenance on Thursday

  • Day 2: AM - Introduction to RNA-Seq (and a touch of miRNA-Seq)

  • Day 2: PM - RNA-Seq practical (Tophat + Cuffdiff pipelineon Galaxy)

  • Day 3: AM –Introduction to Exome Sequencing and Variant Discovery

  • Day 3: PM - Exome sequence analysis practical (Galaxy)


Quick recap

Quick Recap

  • NGS data production becoming commonplace

  • Many applications -> research intent determines technology platform choice

  • High volume data BUT error prone

  • FASTQ is accepted format standard

  • Must assess quality scores before proceeding

  • ‘Bad’ data can be rescued


Introduction to rnaseq

Introduction to RNAseq


The central dogma of molecular biology

The Central Dogma of Molecular Biology

Reverse

Transcription


Rnaseq protocols

RNAseq Protocols

  • cDNA, not RNA sequencing

  • Types of libraries available:

    • Total RNA sequencing (not advised)

    • polyA+ RNA sequencing

    • Small RNA sequencing (specific size range targeted)


Cdna synthesis

cDNA Synthesis


Genome scale applications

Genome-scale Applications

  • Transcriptome analysis

  • Identifying new transcribed regions

  • Expression profiling

  • Resequencing to find genetic polymorphisms:

    • SNPs, micro-indels

    • CNVs

    • Question: Why even bother with exome sequencing then?


Sequencing details

Sequencing details

  • Standard sequencing

    • polyA/total RNA

    • Size selection

    • Primers and adapters

    • Single- and paired-end sequencing

  • Strand-specific sequencing

    • still immature tech

    • Sequencing only + or – strand

    • Mostly paired-end


What about microarrays

What about microarrays??!!!

  • Assumes we know all transcribed regions and that spliceforms are not important

  • Cannot find anything novel

  • BUT may be the best choice depending on QUESTION


Arrays vs rnaseq 1

Arrays vs RNAseq (1)

  • Correlation of fold change between arrays and RNAseq is similar to correlation between array platforms (0.73)

  • Technical replicates almost identical

  • Extra analysis: prediction of alternative splicing, SNPs

  • Low- and high-expressed genes do not match


Rna seq promises pitfalls

RNA-Seq promises/pitfalls

  • can reveal in a single assay:

    • new genes

    • splice variants

    • quantify genome-wide gene expression

  • BUT

    • Data is voluminous and complex

    • Need scalable, fast and mathematically principled analysis software and LOTS of computing resources


Experimental considerations

Experimental considerations

  • Comparative conditions must make biological sense

  • Biological replicates are always better than technical ones

  • Aim for at least 3 replicates per condition

  • ISOLATE the target mRNA species you are after

  • NOT looking for new transcripts can bias expression estimates


Analysis strategies

Analysis strategies

  • De novo assembly of transcripts:

    + re-constructs actual spliced transcripts

    + does not require genome sequence

    easier to work post-transcriptional modifications

    - requires huge computational resources (RAM)

    - low sensitivity: hard to capture low abundance transcripts

  • Alignment to the genome => Transcript assembly

    + computationally feasible

    + high sensitivity

    + easier to annotate using genomic annotations

    - need to take special care of splice junctions


Basic analysis flowchart

Basic analysis flowchart

Illumina

reads

Remove

artifacts

AAA..., ...N...

Clip adapters

(small RNA)

"Collapse"

identical

reads

Align

to the

genome

Pre-filter:

low complexity

synthetic

Count

and

discard

Re-align

with different

number of mismatches

etc

un-mapped

mapped

mapped

un-mapped

Assemble:

contigs (exons)

+ connectivity

Filter out low

confidence

contigs

(singletons)

Annotate


Software

Software

  • Short reads aligners

    • Stampy, BWA, Novoalign, Bowtie, TOPHAT

  • Data preprocessing

    • Fastx toolkit

    • samtools

  • Expression studies

    • Cufflinks package

    • R packages (DESeq, edgeR, more…)

  • Alternative splicing

    • Cufflinks

    • Augustus


The tuxedo protocol

The ‘Tuxedo’ protocol

  • TOPHAT + CUFFLINKS

  • TopHat aligns reads to genome and discovers splice sites

  • Cufflinks predicts transcripts present in dataset

  • Cuffdiff identifies differential expression

    Very widely adopted suite


Tuxedo protocol limitations

‘Tuxedo’ protocol limitations

  • Uses shortread data - Illumina OR SOLiD

  • Requires a sequenced genome

  • No GUI

  • Versions implemented in GALAXY are old(ish)


Read alignment with tophat

Read alignment with TopHat


Splice junctions

Splice junctions

  • In humans, terminal exons are ~1kb long, and since mRNAs are ~2kb,

  • ~half of the reads should originate in initial and internal exons

  • Initial and internal exons are ~200b long

  • => for 75-mer reads, ~20% of reads are supposed to cross splice junctions

R

RNA:

Lexon

Genome:


Splice junctions strategies

Splice junctions strategies

  • Create a splice junctions database joining together donors and acceptors

  • Typically, use known (annotated) splice junctions or known splice sites

  • TopHat: uses putative exons from mapped reads, database is made of canonical splice sites around putative exons


Read alignment with tophat 2

Read alignment with TopHat (2)

  • Uses BOWTIE aligner to align reads to genome

  • BOWTIE cannot deal with large gaps (introns)

  • Tophat segments reads that remain unaligned

  • Smaller segments mostly end up aligning


Read alignment with tophat 3

Read alignment with TopHat (3)

  • When there is a large gap between segments of same read -> probable INTRON

  • Tophat uses this to build an index of probable splice sites

  • Allows accurate measurement of spliceform expression

  • Possibility of detecting gene fusion events


Cufflinks package

Cufflinks package

  • http://cufflinks.cbcb.umd.edu/

  • Cufflinks:

    • Expression values calculation

    • Transcripts de novo assembly

  • Cuffcompare:

    • Transcripts comparison (de novo/genome annotation)

  • Cuffdiff:

    • Differential expression analysis


Cufflinks transcript assembly

Cufflinks: Transcript assembly

  • Assembles individual transcripts based on aligned reads

  • Infers likely spliceforms of each gene

  • Builds ‘transfrags’

    • The smallest number of spliceforms that can be explained by the data

    • NOTE:assembly errors do occur -> sequencing depthhelps


Cufflinks transcript assembly 2

Cufflinks: Transcript assembly (2)

  • Quantifies expression level of each transfrag

  • Filters out those likely to be premature terminations, non-mature mRNAs, etc


Cuffmerge

Cuffmerge

  • Merges transfrags into transcripts where appropriate

  • Also performs a reference based assembly of transcripts using known transcripts

  • Produces single annotation file which aids downstream analysis


Cuffdiff differential expression

Cuffdiff: Differential expression

  • Calculates expression level in two or more samples

  • Expression level relates to read abundance

  • Because of bias sources, cuffdiff tries to model the variance in its significance calculation

    What else is important?


Fpkm rpkm expression values

FPKM (RPKM): Expression Values

  • Fragments Reads Per Kilobase of exon model per Million mapped fragments

  • Nat Methods. 2008, Mapping and quantifying mammalian transcriptomes by RNA-Seq. Mortazavi A et al.

C= the number of reads mapped onto the gene's exons

N= total number of reads in the experiment

L= the sum of the exons in base pairs.


Cufflinks expression analysis

Cufflinks (Expression analysis)

gene_id bundle_id chr left right FPKM FPKM_conf_lo FPKM_conf_hi status

ENSG00000236743 31390 chr1 459655 461954 0 0 0 OK

ENSG00000248149 31391 chr1 465693 688071 787.12 731.009 843.232 OK

ENSG00000236679 31391 chr1 470906 471368 0 0 0 OK

ENSG00000231709 31391 chr1 521368 523833 0 0 0 OK

ENSG00000235146 31391 chr1 523008 530148 0 0 0 OK

ENSG00000239664 31391 chr1 529832 532878 0 0 0 OK

ENSG00000230021 31391 chr1 536815 659930 2.53932 0 5.72637 OK

ENSG00000229376 31391 chr1 657464 660287 0 0 0 OK

ENSG00000223659 31391 chr1 562756 564390 0 0 0 OK

ENSG00000225972 31391 chr1 564441 564813 96.9279 77.2375 116.618 OK

ENSG00000243329 31391 chr1 564878 564950 0 0 0 OK

ENSG00000240155 31391 chr1 564951 565019 0 0 0 OK


Cuffdiff differential expression1

Cuffdiff (differential expression)

  • Pairwise or time series comparison

  • Normal distribution of read counts

  • Fisher’s test

test_idgenelocussample_1sample_2statusvalue_1value_2ln(fold_change)test_statp_valuesignificant

ENSG00000000003TSPAN6chrX:99883666-99894988q1q2NOTEST00001no

ENSG00000000005TNMDchrX:99839798-99854882q1q2NOTEST00001no

ENSG00000000419DPM1chr20:49551403-49575092q1q2NOTEST15.077523.86270.459116-1.395560.162848no

ENSG00000000457SCYL3chr1:169631244-169863408q1q2OK32.562616.5208-0.67854115.81860yes


Visualization genome viewers

Visualization: Genome Viewers

  • Web based:

    • UCSC Genome Browser (http://genome.ucsc.edu/)

  • Standalone

    • Integrated Genome Viewer (http://www.broadinstitute.org/software/igv/)


Rnaseq hands on practical galaxy

RNAseq hands-on practical (Galaxy)

  • Data QC and trimming

  • Aligning reads to reference genome

  • Running CUFFLINKS and looking at some transcripts using the UCSC genome browser

  • Finding differentially expressed genes with CUFFDIFF


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