Msc_Metatranscriptomics

Metatranscriptomics data analysis

This repository describes step by step the analysis of the Metatranscriptome dataset

A continuous effort to create a reliable pipeline for;

1. Quality control

2. Removing host RNAs

3. Taxonomic assignment

4. Denovo assembly

5. Differential gene expression

6. Functional annotation

7. Pathway analysis

codes to be automated for the pipeline

Quality control

fastp -q 5 -l 25 -3 -M 5 -i ${sampleid}_R1_001.fastq.gz -I ${sampleid}_R2_001.fastq.gz -o ./clean_data/${sampleid}_R1_001.clean_fastq.gz -O ./clean_data/${sampleid}_R2_001.clean_fastq.gz --html ./clean_data/fastqc_results/${sampleid}_fastq_trim_report.html

Removing host RNAs

bbsplit.sh in1=${sampleid}_R1_001.clean_fastq.gz in2=${sampleid}_R2_001.clean_fastq.gz ref=../refferences/coral.fna.gz basename=./contaminats_free/out_%.fq.gz outu1=./contaminats_free/${sampleid}_R1_001.contfree_fastq.gz outu2=./contaminats_free/${sampleid}_R2_001.contfree_fastq.gz

Taxonomic assignment

kraken2 --use-names --threads 20 --db ../databases/minikraken_8GB_20200312 --fastq-input --report ../kraken_results/mysample.kreport2 --gzip-compressed --paired ${sampleid}_R1_001.contfree_fastq.gz ${sampleid}_R2_001.contfree_fastq.gz > ../kraken_results/${sampleid}.krk

Taxonomy visualization

rcf -n /home/cley/recentrifuge/taxdump -k ${sampleid} -c 1 -o ../recentrifuge_results/${sampleid}.html

Denovo assembly

Trinity --seqType fq --max_memory 100G -–left all_reads_R1.fa.fq –-right all_reads_R2.fa.fq --SS_lib_type RF --CPU 48 --output trinity_results

Differential expression

DE_Updated.Rmd

Functional annotation and pathways analysis

pathways_Plotting