User talk:Emilio Palumbo/g2f rna
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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
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”:
- block order
- min/max distance
To perform RNAseq analysis we need:
- reference genome sequence
- reference gene annotation
Important note: Please make sure the contig names for your reference genome and annotation correspond.
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].
- splice junction
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