User talk:Emilio Palumbo/g2f rna

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http://rnaseq.uoregon.edu/img/image1.png
http://rnaseq.uoregon.edu/img/image1.png
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===Analyses workflow===
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An example of a standard workflow we use for RNAseq analyses can be seen [[http://genome.crg.es/~epalumbo/blueprint/crg-pipeline.pdf here]].
===Mapping===
===Mapping===
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===Input files===
===Input files===
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To perform RNAseq analisys we need:
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To perform RNAseq analysis we need:
* reference genome sequence
* reference genome sequence

Revision as of 04:14, 15 November 2013

Contents

RNAseq

image1.png

Analyses workflow

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

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 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
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