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Analyze RNAseq Splicing.md

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Analyze tie-1 for splicing defects

SNP analysis has suggested that there is a splice-acceptor mutation in Solyc02g080120. Here we examine possible splicing defects splicing in the tie-1 GA2 Oxidase gene Solyc02g080120.

fq files for lanes 1,2,3,7 from /iplant/home/shared/ucd.plantbio/maloof.lab/members/amanda/tiernaseqfinal

fq files for lanes 4-6 from

icd /iplant/home/shared/ucd.plantbio/maloof.lab/members/upendra/kazu_palmer_libraries/processed_data/amanda_stuff/JKNE3_4re icd /iplant/home/shared/ucd.plantbio/maloof.lab/members/upendra/kazu_palmer_libraries/processed_data/amanda_stuff/JKNE3_5re icd /iplant/home/shared/ucd.plantbio/maloof.lab/members/upendra/kazu_palmer_libraries/processed_data/amanda_stuff/JKNE3_6re

combine fq files

First rename to prevent clobbering, then move to common directory

in lane 4 directory:

for file in *.fq
    do
        base=`basename $file .fq`
        newName="$base.4.fq"
        mv $file $newName
    done

repeat for all lanes, move files to a common directory and concatenate

next run tophat

bowtie2-build S_lycopersicum_chromosomes.2.40.fa S_lycopersicum_chromosomes.2.40.fa

Working in directory: /home/jnmaloof/ebs5/tie1RNAseqSNPs/newTophat020114

/usr/local/src/tophat-2.0.8b.Linux_x86_64/tophat2 --GTF ../ITAG2.3_gene_models.gff3 --output-dir tophat_M82 ../S_lycopersicum_chromosomes.2.40 ../fqfiles/M82_all.fq

 /usr/local/src/tophat-2.0.8b.Linux_x86_64/tophat2 --GTF ../ITAG2.3_gene_models.gff3 --output-dir tophat_tie1 ../S_lycopersicum_chromosomes.2.40 ../fqfiles/Tie_all.fq

View in IGV.

It appears that the first intron of Solyc02g080120 is not spliced in tie-1. If true this would lead to a premature stop codon.

Quantify splicing defect

Working in newTophat020114 directory

determine the number of reads spanning the splice junction or spliced in M82 and tie-1

For the first of these I am just going to hand count in IGV

location M82 splice M82 span tie-1 splice tie-1 span
5' junction 25 1 0 33
3' junction 25 2 0 15

determine coverage before, in, and after splice junction in M82 and tie-1

Use samtools to calculate coverage:

cd "/Users/jmaloof/Documents/Lab Notebook support/2013/tie1_phyE/RNAseq/newTophat020114/tophat_tie1"
samtools depth -r SL2.40ch02:39009924-39009943 accepted_hits.bam > tie1_5prime.tsv
samtools depth -r SL2.40ch02:39009944-39010179 accepted_hits.bam > tie1_intron.tsv
samtools depth -r SL2.40ch02:39010180-39010199 accepted_hits.bam > tie1_3prime.tsv
samtools idxstats accepted_hits.bam > tie1_hits.tsv

cd ../tophat_M82
samtools depth -r SL2.40ch02:39009924-39009943 accepted_hits.bam > M82_5prime.tsv
samtools depth -r SL2.40ch02:39009944-39010179 accepted_hits.bam > M82_intron.tsv
samtools depth -r SL2.40ch02:39010180-39010199 accepted_hits.bam > M82_3prime.tsv
samtools idxstats accepted_hits.bam > M82_hits.tsv

For subsequent analysis see Analyze RNAseq Splicing.R