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In this workshop, we will be performing both genome-guided and genome-free transcript reconstruction.
For genome-guided reconstruction, we will leverage the Tuxedo2 protocol, leveraging HISAT2 tool for aligning RNA-Seq reads to a target genome, and StringTie for reconstructing transcript isoforms.
We will also leverage Trinity for genome-free transcript reconstruction, and compare our de novo assembled transcripts to the genome-guided reconstructions by aligning our Trinity transcripts to the target genome using GMAP and visualizing all the data using IGV.
Our genome and RNA-Seq data can be found at:
% ls ~/workspace/data/trinity/rnaseq/*
.
/home/ubuntu/workspace/data/trinity/rnaseq/minigenome.fa
/home/ubuntu/workspace/data/trinity/rnaseq/samples.txt
/home/ubuntu/workspace/data/trinity/rnaseq/minigenome.gtf
/home/ubuntu/workspace/data/trinity/rnaseq/data:
sA_rep1_1.fastq.gz sA_rep2_2.fastq.gz sB_rep1_1.fastq.gz sB_rep2_2.fastq.gz
sA_rep1_2.fastq.gz sA_rep3_1.fastq.gz sB_rep1_2.fastq.gz sB_rep3_1.fastq.gz
sA_rep2_1.fastq.gz sA_rep3_2.fastq.gz sB_rep2_1.fastq.gz sB_rep3_2.fastq.gz
The genome data correspond to a small subset of the Drosophila genome (~200 genes) and the RNA-Seq data correspond to two conditions (sA & sB), with each containing three biological replicates (rep1,2,3).
The minigenome.gtf contains the reference transcript structure annotations, and the 'samples.txt' file describes the relationships between samples, replicates, and their corresponding paired-end RNA-Seq fastq files.
Create a 'transreco' directory in your workspace where you will perform your analyses.
% cd ~/workspace
% mkdir transreco
% cd transreco
Copy over the genome and rna-seq data to your current working directory 'transreco'.
% cp -r ~/workspace/data/trinity/rnaseq/* .
Load the 'minigenome.fa' into IGV as a new genome, and then load in the annotations 'minigenome.gtf'. Setting the annotations to 'squished' will enable you to see all the different reference isoforms for each gene.
