Next Generation Sequencing (NGS)/Expression quantification

RNA-seq expression quantification Overview Objective: to estimate the relative abundance of transcripts from an rna-seq sample. Biological questions? Inputs: Reference: Annotated Genome (fasta/gff) Transcriptome (fasta) RNA-seq raw reads (fastq, Ilumina). Outputs: Relative abundance per transcript. Experimental Design See (link) RNA sequencing Typical Steps in the Method: (table or workflow or paragraph?) Data generation | Get some data. | Seq (link) Sequencing RNA technologies, Get a reference | You need a reference with all the involved transcripts for which you want relative abundance | RNA-seq read QC | Check the quality of the sequencing itself. | Adaptor trimming / quality filtering / contamination filtering | Reads will contain sequence that is not from the transcript but from the technology. | Mapping | Align your reads to the reference | - Quantification | Get transcript abundance | Workflows Galaxy Workflows Example workflow Real life workflows - - - - - Command line scripts Example workflow: Real life workflows: - - - - - Some tools and resources Get a reference: MetaDB (link)genome assembly and annotation (link) transcriptome denovo assembly .... QC: SeqWiki: FastQC, KAT Trimming: SeqWiki; Trimmomatic, Sickle, Cutadapt.... Mapping: Seqwiki: tools...... ... ... ... ... ... ... .. .... Quant: Seqwiki: tools Discussion and current best practices on specific steps on the method Top X Biostar questions on this topic/method/whatever