Structure

Primary

• promoters
• intron-exon junctions
• 5' 3' UTRs
• polyA site

Secondary

Tertiary

RNAseq can provide

• 5' TSS
• 5' UTR
• exon-intron boundaries
• 3' UTR
• polyA site
• alternative usage of any of above

Fusion genes: cytogenetic derangements; genomic amplification; translocation deletions

long non-coding RNAs (lncRNAs)

• 200nt

• not overlap protein coding exons
• can control transcription as enhancers (eRNA), competitors (ceRNA) or as noise

small non-coding RNAs

• miRNA (21-23 nt)
• piRNA
• endo-siRNA
• snoRNA
• snRNA
• tRNA (73-93 nt)
• moRNA
• eRNA

Reads coverage:

• Illumina for coverage
• SOLID for accuracy
• Roche 454 / Pacbio for length

Pre-processing: remove low-quality baseds / artifacts, incl. adapters / lib-construct sequences

QC

• base quality: filtering low-quality bases (Trimmomatic / FastX / prinSeq)
• ambiguous bases
• adapters (TagCleaner / CutAdapt)
• read length
• sequence-specific bias
• GC-content
• duplicates (for DGE not recommend to remove)
• sequence contamination
• low-complexity sequences / polyA tails

mapping stats: samtools or RseQC

de novo assembly

different from genome assembly; de Bruijn Graph

• mapping based assembly: Cufflinks and Scripture
• de novo assembly: Velvet + Oases; Trinity (Inchworm-Chrysalis-Butterfly)

Read mapping

• reads per gene: htseq-count / Qualimap / Bedtools / Cufflinks: differ in how to handle multimapping reads
• reads per transcript: Expectation Maximization (EM) Approach: Cufflinks / eXpress
• reads per exon: DEXSeq

DEXSeq

• input from GTF + bam/sam
• counts per exon (from script) -> table in R
• normalise by estimating size factor
• estimation exon-specific dispersion values
• testing for diff exon usage
• can be used for alternative splicing preidction ExonCountSet

DE analysis

the same gene across different cells follow log-normal distribution (qPCR)

different individuals viariability: negative binomial distribution (DESeq / edgeR)

zero inflation hard to fit netative binomial model

tweeDESeq: Possion-Tweedie family

Normalisation

• RPKM/FPKM
• TPM
• TMM

small ncRNAs

miRNA

• miRdeep2
• miRanalyzer

miRNA target (SVMs)

complementary of first 7-8 nt to mRNA

thermodynamic stability positionof particular GC/AU matches

• targetScan
• DIANA-microT

data bases:

• microRNA.org
• miRBase
• piRNABank
• Rfam
• miRGator
• mirWIP
• TarBase
• miRTarBase
• RNAmmer

tRNA prediction

• tRNA-Scan-SE