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Pipeline for 4-species flaveria developmental gradient de-novo RNAseq experiment

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flaveria_pipeline

Pipeline for processing RNAseq data from Flaveria. Four species, two C3 and two C4.

Flaveria species - C3:

  • Flaveria pringlei
  • Flaveria robusta

Flaveria species - C4:

  • Flaveria trinervia
  • Flaveria bidentis

Data contains samples from six sections of leaves from each species with three replicates of each section.

Steps in pipeline

Fastqc

  • Quality checking raw reads
  • Check for over-represented kmers/sequences.

Trimmomatic

  • Removing adapters?
  • Trimming low quality sequences at the ends of reads. Cutoff set to 10? 15? [1]

Error Correction

  • Read error correction using hamming trees.
  • Reptile initially chosen because it came out best in this paper [2]
  • BayesHammer then used because Reptile has problems [5]

Khmer

  • preparing reads for assembly
  • reduce coverage

Assemblotron

  • optimal transcriptome assembly

Annotation

  • Reciprocal best usearch transcripts against Arabidopsis
  • Round-robin rbusearch between flaveria species to update annotation

Expression quantification

  • mapping reads output from trimmomatic to transcripts
  • using Express[3] or Sailfish[4]

Differential Expression Analysis

  • using EBSeq or possibly BaySeq

How to Run

Type rake


[1] Paper about optimising trimmomatic cutoff

[2] Paper comparing read error correction methods

[3] Paper about Express

[4] Paper about Sailfish

[5] Paper about BayesHammer

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