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

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[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