User talk:Emilio Palumbo/g2f rna

RNAseq

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

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 analisys we need:

• reference genome sequence
• reference gene annotation
• sequences

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

Gemtools

The [GEM mapper] is a mapping program for next generation sequencing developed in collaboration between CRG and CNAG institutes 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].

Running the pipeline

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

• Genome indexing:

gemtools index genome.fa

• Transcriptome generation and indexing:

gemtools t-index annotation.gtf -m MAX_READ_LENGTH

After those steps completed successfully you can run the pipeline:

gemtools rna-pipeline -f FASTQ_FILE -q QUALITY_OFFSET -i GENOME_INDEX -a ANNOTATION_FILE -t NUMBER_OF_CORES -o OUTPUT_FOLDER -m MAXIMUM_READ_LENGHT_FOR_DENOVO_JUNCTIONS