Arabidopsis - De novo assembly vs Reference Guided Assembly

Goal:

Split into teams and choose one of these strategies:

  1. De novo assembly with trinity, following instructions from earlier

  2. Reference-guided assembly with trinity, following these instructions from Trinity.

    This will follow a slightly altered workflow:

    1. correct reads (same as before)

    2. run skewer (same as before)

    3. map with STAR (new step)

      Note: In addition to the aforementioned options, for GFF3 formatted annotations you need to use –sjdbGTFtagExonParentTranscript Parent.

    4. run trinity (altered command with bam input)

    5. run busco (same as before)

    6. run transrate (same as before)

I have notes for all of these commands if you need help.

Log your results here: https://docs.google.com/spreadsheets/d/12X06LqGM8j4a4oV3_IsM91Hvop6B7L84xSWR71dXK3E/edit?usp=sharing

Get Data

Arabidopsis lyrata RNASeq Flower reads:

wget http://www.hardwoodgenomics.org/sites/default/files/kbs_temp/SRR3993765_1.subsample.fastq
wget http://www.hardwoodgenomics.org/sites/default/files/kbs_temp/SRR3993765_2.subsample.fastq

Arabidopsis thaliana Genome:

wget https://www.arabidopsis.org/download_files/Genes/TAIR10_genome_release/TAIR10_chromosome_files/TAIR10_chr_all.fas

Arabidopsis thaliana Genome Annotation:

wget https://www.arabidopsis.org/download_files/Genes/TAIR10_genome_release/TAIR10_gff3/TAIR10_GFF3_genes.gff

We still have a slight problem - the chromosome names in the fasta file don’t match the chromosome names in the annotation file. This is a surprisingly common problem and breaks any tool that needs both files. So lets fix the names.:

sed -i 's/>\([1-5]\)/>Chr\1/' TAIR10_chr_all.fas
sed -i 's/>mitochondria/>ChrM/' TAIR10_chr_all.fas
sed -i 's/>chloroplast/>ChrC/' TAIR10_chr_all.fas

BUSCO plant:

To get the plant database file:

wget http://buscos.ezlab.org/files/plant_early_release.tar.gz
gunzip plant_early_release.tar.gz

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