Metagenomic Data Analysis Pipeline

This repository contains the steps and scripts for analyzing metagenomic data, from raw reads to functional and taxonomic annotations. Below is a detailed breakdown of the pipeline.

Steps

1. Filtering Steps
2. Metagenomic Assembly
3. Gene Prediction and Quantification
4. Non-Redundant Gene-Set Construction
5. Functional Annotation
   • KEGG Analysis
   • CAZy Analysis
   • Antibiotic Resistance Analysis
   • Xenobiotics Degradation Analysis
6. Taxonomic Analysis
7. Genome Reconstruction
8. Statistical Analysis

1. Filtering Steps

FastQC

Run FastQC to assess the quality of raw reads:

qsub 1_Fastqc_only.sh

Ambiguity Filtering

Filter out reads with ambiguous bases:

qsub 2_Ambiguity.sh
perl ~/Softwares/NGSQCToolkit_v2.3.3/Trimming/AmbiguityFiltering.pl -i ../sample_R1.fastq -irev ../sample_R2.fastq -c 1 -t5 -t3

Homopolymer Removal

Remove homopolymers using Prinseq:

qsub 3_Prinseq.sh
prinseq-lite.pl -fastq ../sample_R1.fastq_trimmed -fastq2 ../sample_R2.fastq_trimmed -custom_params "AAT 10;T 70%;A 15;G 70%;C 15"

Barcode Removal

Trim adapters and low-quality bases using Trimmomatic:

qsub 4_Trimmomatic.sh
java -jar $TRIMMOMATIC/trimmomatic.jar PE -phred33 input_R1.fastq input_R2.fastq output_R1_P.fq output_R1_U.fq output_R2_P.fq output_R2_U.fq ILLUMINACLIP:$TRIMMOMATIC/adapters/TruSeq2-PE.fa:2:40:15 LEADING:10 TRAILING:10 SLIDINGWINDOW:10:20 MINLEN:80

Host Contamination Removal

Remove host-derived reads using Bowtie2:

qsub 5_Host_contaRemoval.sh
bowtie2 -x ~/Databases/Human/Homo_sapiens.GRCh38.cdna_BowtieIndex -1 input_R1.fastq -2 input_R2.fastq -S output.sam
samtools view -bS output.sam > output.bam
samtools view -b -f 12 -F 256 output.bam > output_unmapped.bam
samtools sort -n output_unmapped.bam > output_sorted.bam
bedtools bamtofastq -i output_sorted.bam -fq output_host_removed_r1.fastq -fq2 output_host_removed_r2.fastq

2. Metagenomic Assembly

SPAdes Assembly

Assemble reads using SPAdes:

qsub 6_Spades_Assembly.sh
spades.py --meta -1 input_R1.fastq -2 input_R2.fastq -o assembly_output

Contigs Filtering

Filter contigs based on length:

perl Filter_contigs_read_length.pl

3. Gene Prediction and Quantification

Gene Prediction

Predict genes using Prodigal:

qsub 7_Prodigal.sh
prodigal -i contigs.fasta -o genes.gff -a proteins.faa -p meta

Gene Quantification

Quantify genes using BWA and SAMtools:

qsub newBWA.sh
bwa index genes.fna
bwa mem genes.fna input_R1.fastq input_R2.fastq | samtools view -Sb - > output.bam
samtools sort output.bam -o sorted_output.bam
samtools index sorted_output.bam

4. Non-Redundant Gene-Set Construction

CD-HIT

Create a non-redundant gene set:

qsub 9_cdhit.sh
cd-hit-est -i genes.fna -o genes_cdhit.fna -c 0.95 -n 10 -aS 0.9 -d 0 -T 48 -M 60000

5. Functional Annotation

KEGG Analysis

Annotate genes using KEGG:

blastp -query genes.faa -db KEGG_DB (pre-built) -out kegg_out.txt -num_threads 24

CAZy Analysis

Annotate genes using CAZyDB ((pre-built)):

blastp -query genes.faa -db CAZyDB -out cazy_out.txt -num_threads 24

Antibiotic Resistance Analysis

Annotate genes using ARDB ((pre-built)):

blastp -query genes.faa -db ARDB -out ardb_out.txt -num_threads 24

Xenobiotics Degradation Analysis

Annotate genes for xenobiotics degradation:

blastp -query genes.faa -db Xenobiotics_DB(pre-built) -out xenobiotics_out.txt -num_threads 24

6. Taxonomic Analysis

Taxonomic Assignment

Assign taxonomy using BLAST against NCBI and HMP databases (pre-built):

blastn -query genes.fna -db NCBI_HMP_DB -out taxonomic_out.txt -num_threads 50

7. Genome Reconstruction

Cross-Assembly

Assemble reads using Megahit:

qsub Megahit_human_assembly.sh

Binning and Quality Assessment

Bin contigs using MetaBAT and assess quality using CheckM:

qsub binning_quality_check.sh

8. Statistical Analysis

Perform statistical analyses on metagenomic data:

Assemble reads using Megahit:

Rscript statistical_analysis.R

This pipeline provides a comprehensive workflow for metagenomic data analysis, covering data preprocessing, assembly, annotation, taxonomic classification, and genome reconstruction. Please make sure all dependencies are correctly installed before running the scripts.

• All intermediate scripts can be found in the Final_Scripts folder!!

Contact

For any questions, please contact: 👉 Ashok K. Sharma; ashoks773@gmail.com