3_assembly_de_novo.txt

#warming_AK

#Adriana L. Romero-Olivares' protocol for processing RNAseq data of soil 
#Section 3
#File name: 3_assembly_de_novo
#Step: Asssembly with Trinity 

#this protocol assembles a de novo reference meta-transcriptome 
#uses only mDNA, output from sortmeRNA, all_outputfiler1r2_non_rRNA, after unmerging mRNA.r1.fq and mRNA.r2.fq
#you can input all samples separately or concatenate them in a single r1 and r2 file 
#if need to concatenate samples use 

cat *.r1.fq > reads.r1.fq
cat *.r2.fq > reads.r2.fq

#to build reference de novo meta-transcriptome you will have to costumize Trinity's script, the example below is very specific for my data  
#use at least 1 gb of max_memory per million pair-end reads
#output HAS to say Trintiy otherwise will mark error 
#EXAMPLE 

module load trinity/r2016-2.3.2
module load samtools/1.3
module load java/1.8.0.51
module load bowtie2/2.2.7

Trinity --seqType fq --full_cleanup --max_memory 300G --min_kmer_cov 2 --no_bowtie --CPU 24 --SS_lib_type RF --left Trinity_input_files/all_C1-5.r1.fq,Trinity_input_files/all_C3-2.r1.fq,Trinity_input_files/C4-5_repeat.r1.fq,Trinity_input_files/all_C5-5.r1.fq,Trinity_input_files/all_W1-3.r1.fq,Trinity_input_files/all_W3-1.r1.fq,Trinity_input_files/all_W4-3.r1.fq,Trinity_input_files/all_W5-5.r1.fq --right Trinity_input_files/all_C1-5.r2.fq,Trinity_input_files/all_C3-2.r2.fq,Trinity_input_files/C4-5_repeat.r2.fq,Trinity_input_files/all_C5-5.r2.fq,Trinity_input_files/all_W1-3.r2.fq,Trinity_input_files/all_W3-1.r2.fq,Trinity_input_files/all_W4-3.r2.fq,Trinity_input_files/all_W5-5.r2.fq --output Trinity_ALL_10/Trinity_ALL_f

#output will be a single fasta file: Trinity_ALL_f.Trinity.fasta
#this file will be used for downstream analyses