Transcript detection in RNAseq

[by Joseph Robertson]
RNAseq analysis: Transcript detection (1/2)
What is a jar ?
August 11, 2011

Quick recap: Production informatics
August 11, 2011
Sequencing->Images->Conversion (Demultiplexing)
Resulting file type: FASTQ
“Having raw sequence reads and quality scores”
Sequencing
Image
Fastq
Quality Control
Projects

Objective & Challenges
Objective: study the active transcriptome of the cell
Problems:
The RNA content of a cell is dominated by tRNA, rRNA and housekeeping genes
Flowcell has only a finite real-estate of which most would be occupied by these mainly invariable transcripts
How to focus the sequencing on the “interesting” part of the transcriptome: mRNA and ncRNA ?
August 11, 2011

What RNAseq protocols are there?
RNA seq
total RNA tRNA/rRNA removed + PolyA-tail filtered
Good for studying protein coding genes, e.g.
gene expression, isoforms, expression of variant alleles
RNA editing events
RNA-DNA differences in the human transcriptome provide a yet-unexplored aspect of genome variation.
Small RNAseq:
Total RNA size selection for small RNA molecules
Good for small ncRNA e.g. miRNAs, snoRNA
Duplex-specific thermostable nuclease (DSN) guided RNA seq normalization
Total RNA  high abundant transcripts are digested
Good for studying all transcripts
August 11, 2011
Today
Li M, Wang IX, Li Y, Bruzel A, Richards AL, Toung JM, Cheung VG. Widespread RNA and DNA sequence differences in the human transcriptome. Science. 2011. PMID: 21596952.
Christodoulou DC, Gorham JM, Herman DS, Seidman JG. Construction of normalized RNA-seq libraries for next-generation sequencing using the crab duplex-specific nuclease. CurrProtoc Mol Biol. 2011 PMID: 21472699

RNA-seq workflow
Select PolyA-tail + remove tRNA/rRNA
Fragment RNA
Make cDNA(caution you may loose strand info)
Sequence
Map reads
Identify transcripts
Quantify transcripts
Identify differences between conditions
August 11, 2011
Today
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008 PMID: 18516045.

Production Informatics and Bioinformatics
August 11, 2011
Produce raw sequence reads
Basic Production
Informatics
Map to genome and generate raw genomic features (e.g. SNPs)
Advanced
Production Inform.
Analyze the data; Uncover the biological meaning
Bioinformatics
Research
Per one-flowcell project

Challenges for RNAseq read mapping
Loosing reads because they do not match the ref. genome
Reads spanning exon junctions
RNA editing events
Approaches
Align to ref. transcriptom library
Exon-first e.g. Tophat
Seed-extend methods e.g. GSNAP
August 11, 2011
Sequencing reads
DNA
gRNA
mRNA
editing
event
Garber M, Grabherr MG, Guttman M, Trapnell C. Computational methods for transcriptome annotation and quantification using RNA-seq. Nat Methods. 2011 PMID: 21623353.
Li M, Wang IX, Li Y, Bruzel A, Richards AL, Toung JM, Cheung VG. Widespread RNA and DNA sequence differences in the human transcriptome. Science. 2011. PMID: 21596952.

Exon-first approach
Align reads to ref. genome
Chop up unaligned reads and try to identify matching regions
Find splice junctions around the matches
August 11, 2011

Seed-extend approach
Break reads in smaller k-mers and find matches
Iteratively extend k-mers to identify exact spliced alignment
August 11, 2011

Which method ?
Exon-first: less computationally intensive
The additional exon-junctions found by seed-extend have not (yet) been demonstrated to be real.
August 11, 2011

Challenges for transcript detection
Identifying isoforms is difficult
Transcript abundance is volatile
Most reads are not helpful (reads from exons) or even misleading (incompletely spliced precursor RNA)
Genes can have many isoforms
Approaches
Ignore isoforms
Genome-guided reconstruction, e.g. Cufflinks
Genome-independent reconstruction, e.g. Trinity
August 11, 2011
QBI data

Genome-guided reconstruction
Use reads spanning slice junction to assemble the transcript path
Work out minimal possible set paths so that all reads are visited (graph theory)
If more than one set use read count to pick the most probable
August 11, 2011
Reads aligned to the genome
Isoforms

Genome-independent reconstruction
Break reads into k-mers find their mutual overlap to build a de Bruijn graph
Find probable paths through the graph by using read counts
Map consensus assembly to genome
August 11, 2011
Iyer MK, Chinnaiyan AM. RNA-Seq unleashed. Nat Biotechnol. 2011 PMID: 21747384.

Which method?
De novo methods are very computationally intensive
However, they are able to find alternative isoforms and promoters and structural variation
deletions (yellow)
chimeras (green)
August 11, 2011
Iyer MK, Chinnaiyan AM. RNA-Seq unleashed. Nat Biotechnol. 2011 PMID: 21747384.

What are real transcripts?
Even the most sophisticated computational method can’t tell you what is a real transcript.
August 11, 2011
Roberts et al.
QBI data
Roberts A, Pimentel H, Trapnell C, Pachter L. Identification of novel transcripts in annotated genomes using RNA-Seq. Bioinformatics. 2011 PMID: 21697122.

Solution: biological replicates
Significant findings (here: new isoforms) in small sample sets can be due to
Technical errors
Biological variability
Population outliers
Sequencing experiments are subject to the same issues (even though they are more expensive than arrays)
Replicates are necessary to build confidence in your results!
August 11, 2011
Hansen KD, Wu Z, Irizarry RA, Leek JT. Sequencing technology does not eliminate biological variability. Nat Biotechnol. 2011 PMID: 21747377

Three things to remember
Methods for analyzing RNAseq data are not as mature as expression array analysis tools yet.
Especially identifying transcript isoforms is difficult.
Replicates are crucial to account for the biological variability
August 11, 2011

Next Week:
August 11, 2011
Abstract: This session will follow up from transcript quantification of RNAseq data and discusses statistical means of identifying differentially regulated transcripts, and isoforms and contrasts these against microarray analysis approaches.

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Transcript detection in RNAseq

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