Metagenomic Analysis Pipeline for Illumina and Nanopore Sequences

1. Data Preprocessing

Objective: Ensure high-quality input data by removing artifacts, adapters, and low-quality reads.

Illumina-specific:

#Tools: 
    FastQC (quality assessment).
    Trimmomatic or Cutadapt (adapter trimming, quality filtering).
#Steps:
    1. Run FastQC to visualize raw read quality (e.g., per-base quality scores, adapter content).
    2. Trim adapters and low-quality bases (e.g., Q<20), remove reads shorter than 50 bp.
    3. Re-run FastQC post-trimming for QC validation.

#Command Example for bash

trimmomatic PE -phred33 input_R1.fastq input_R2.fastq output_R1_paired.fq output_R1_unpaired.fq output_R2_paired.fq output_R2_unpaired.fq ILLUMINACLIP:adapters.fa:2:30:10 SLIDINGWINDOW:4:20 MINLEN:50


Nanopore-specific:

#Tools: 

    NanoFilt (filtering and trimming).
    Porechop (adapter removal).

#Steps:

    1. Filter reads by quality (e.g., Q>10) and length (e.g., >500 bp) using NanoFilt.
    2. Remove adapters with Porechop.
    3. Assess quality with NanoStat.

#Command Example for bash

    porechop -i input.fastq -o trimmed.fastq 
    nanofilt -q 10 -l 500 trimmed.fastq > filtered.fastq


2. Host/Contaminant Removal (Optional)

Objective: Remove host or non-target sequences (e.g., human DNA).

Tools: 

    1. Bowtie2 (Illumina) or Minimap2 (Nanopore) for alignment to a reference host genome.
    2. Samtools for filtering unmapped reads.

Steps:

    1. Index the host genome (e.g., human GRCh38).
    2. Map reads to the host genome.
    3. Extract unmapped reads (metagenomic fraction).

#Command Example for bash 

Illumina:
    bowtie2 -x host_index -1 output_R1_paired.fq -2 output_R2_paired.fq | samtools view -b -f 4 -o unmapped.bam
    samtools fastq unmapped.bam > cleaned.fastq
    
Nanopore:
    minimap2 -ax map-ont host_index filtered.fastq | samtools view -b -f 4 -o unmapped.bam
    samtools fastq unmapped.bam > cleaned.fastq