Pipe1.md

de_novo_RNA-seq_analysis

trimming

wget http://genomedata.org/rnaseq-tutorial/illumina_multiplex.fa

trimmomatic PE -threads 30 -phred33 [corrected_input1].fastq.gz [corrected_input2].fastq.gz [trimmed_ourput]_1P.fastq.gz [trimmed_ourput]_1U.fastq.gz [trimmed_ourput]_2P.fastq.gz [trimmed_ourput]_2U.fastq.gz ILLUMINACLIP:illumina_multiplex.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

fastqc and multiqc (optional)

fastqc [trimmed_ourput]_1P.fastq.gz [trimmed_ourput]_2P.fastq.gz multiqc .

assembly do novo transcriptome

Trinity --seqType fq --single [trimmed_ourput_sample1]_1P.fastq.gz, [trimmed_ourput_samp1en]_1P.fastq.gz --CPU 8 --max_memory 20G --min_contig_length 150

OR for paird end

Trinity --seqType fq --left [trimmed_ourput_sample1]_1P.fastq.gz,[trimmed_ourput_samp1en]_1P.fastq.gz --right [trimmed_ourput_sample1]_2P.fastq.gz,[trimmed_ourput_samp1en]_2P.fastq.gz --CPU 8 --max_memory 20G --min_contig_length 150

OR SPAdes

STAR alignment

STAR --runMode genomeGenerate --runThreadN 30 --genomeDir Trinity_index --genomeFastaFiles Trinity.fasta --genomeSAindexNbases 10

STAR --genomeDir Trinity_index --runThreadN 30 --readFilesIn [trimmed_ourput]_1P.fastq.gz [trimmed_ourput]_2P.fastq.gz --readFilesCommand zcat --quantMode GeneCounts --outFileNamePrefix [output_name].sorted.bam --outSAMtype BAM SortedByCoordinate

count reads and expression matrix based star BAM

Create a stringtie samples file

sample 1 path_to_sample1.gtf sample 2 path_to_sample2.gtf sample 3 path_to_sample2.gtf

stringtie [output_name].sorted.bam -p 30 -o [sample].gtf -A [sample].gene_abundances.tsv

wget https://ccb.jhu.edu/software/stringtie/dl/prepDE.py3

python3 prepDE.py3 -i sample_f.txt

DE Analyis

Create a samples.txt

condition1 condition1_R1 condition1 condition1_R2 condition1 condition1_R3
condition2 condition2_R1 condition2 condition2_R2 condition2 condition2_R3

edgeR, DESeq2 using Tinity DE_analysis script

run_DE_analysis.pl --matrix stringtie.gene/transcipt.counts.matrix --samples_file samples.txt --reference_sample [condition1/condition2] --method edgeR --output edgeR_genes run_DE_analysis.pl --matrix stringtie.gene/transcipt.counts.matrix --samples_file samples.txt --reference_sample [condition1/condition2] --method DESeq2 --output DESeq2_genes

OR

R code from Protocol: Using StringTie with DESeq2

https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual

Extracting differentially expressed transcripts and generating heatmaps

Extract those differentially expressed (DE) transcripts that are at least 4-fold differentially expressed at a significance of <= 0.05 in any of the pairwise sample comparisons:

analyze_diff_expr.pl --matrix stringtie.gene/transcipt.counts.matrix --samples samples.txt -P 0.05 -C 2

Functional Enrichment Analysis

Plaza (https://bioinformatics.psb.ugent.be/plaza/)

TAIR GO Term Enrichment for Plants

PANTHER (http://pantherdb.org/webservices/go/overrep.jsp)