User talk:Emilio Palumbo/g2f rna

RNAseq

http://rnaseq.uoregon.edu/img/image1.png

Analyses workflow

An example of a standard workflow we use for RNAseq analyses can be seen [here].

Common analyses that can be performed on RNAseq data are:

• expression quantitative traits loci (eQTL)
• splicing quantitative traits loci (sQTL)
• differantial gene/isoform expression

Mapping

Specific variables to consider when mapping RNAseq:

• intron size
• overhang (number of bases from each side of the junction that should be covered by a certain read)
• splice site consensus (canonical, extended, non-canonical)
• donor/acceptor splice site consensus sequences
• junction “filtering”:
  • chromosome/strand
  • block order
  • min/max distance

Input files

To perform RNAseq analysis we need:

• reference genome sequence
• reference gene annotation
• sequences

Important note: Please make sure the contig names for your reference genome and annotation correspond.

Gemtools

The [GEM mapper] is a mapping program for next generation sequencing developed in collaboration between CRG and CNAG in Barcelona. Many high-performance standalone programs (splice mapper, concersion tool, etc.) are provided along with the mapper; in general, new algorithms and tools can be easily implemented on the top of these.

[Gemtools] is a powerful set of high-level pipelines which greatly simplifies the use of the GEM mapper. Using gemtools one can index references and/or map several kinds of data from a simple command-line interface, without having to type complicated commands. In particular, gemtools contains a fast and accurate pipeline for mapping RNA-sequencing data.

The default gemtools RNAseq pipeline is shown [here].

Element quantification

• exon
• intron
• splice junction
• transcript
• gene

Transcript expression quantification

Transcript quantification is a complex problem. Quantifying the expression of a gene is simple. We just need to count the RNA-seq reads that fall within the exons of this gene. However, to quantify expression of a transcript we can have reads mapping to an exon of the gene where multiple transcripts overlap. The process of assigning a read to a certain transcript is called read deconvolution or isoform expression quantification.

For transcript expression we use [Flux Capacitor] developed at the CRG in Barcelona.

Running the pipeline

The following step are needed to run the gemtools rnaseq pipeline:

• Genome indexing:

gemtools index -i genome.fa

• Transcriptome generation and indexing:

gemtools t-index -i genome.gem -a annotation.gtf -m 80

After those steps completed successfully you can run the pipeline:

gemtools rna-pipeline -f sample.fastq.gz -q 33 -i genome.gem -a annotation.gtf -m 110

To run the Flux Capacitor a bam file sorted by genomic position and indexed is needed. To do this two command hav to be run:

samtools sort my_file.bam my_file_sorted
samtools index my_file_sorted.bam

You can then run the transcript quantifications in the following way:

flux-capacitor -i sample.bam -a annotation.gtf -o sample.gtf