Snakemake PacBio Assembly & Reassembly Pipelines 🐍

This repository contains two robust Snakemake pipelines tailored for the genome assembly, quality assessment, and targeted reassembly of PacBio sequencing data (specifically HiFi reads).

🚀 Pipelines Overview

1. Primary Assembly Pipeline (Pacbio-assembly-snakemake.smk)

An automated end-to-end workflow for de novo genome assembly of PacBio HiFi reads and subsequent taxonomic/quality evaluation.

Key Steps:

1. Quality Control: Validates input FastQ reads using FastQC.
2. De Novo Assembly: Assembles long reads using Hifiasm, optimized for diploid/haploid phasing.
3. Contig Extraction: Parses .gfa graphs to extract primary contigs (.fasta).
4. Completeness Assessment: Evaluates assembly completeness using BUSCO against the insecta_odb10 lineage (customizable).
5. Taxonomic Profiling: Queries contigs against the NCBI nt database using blastn.
6. Contamination Screening: Generates Taxon-Annotated GC-Coverage (TAGC) plots using BlobTools by integrating the assembly, BAM coverage, BLAST hits, and BUSCO results.

2. Targeted Reassembly Pipeline (reassembly_snakemake.smk)

A highly specialized pipeline designed for extracting subset reads aligned to a specific region/reference and reassembling them using SPAdes.

Key Steps:

1. Reference Indexing: Indexes the target/reference genome using BWA.
2. Alignment: Maps paired-end reads (P1, P2) to the reference genome via BWA-ALN / BWA-SAMPE.
3. Read Extraction: Extracts reads of interest from the resulting SAM files utilizing a custom Perl script (extract.fasta.from.sam.using.list.pl).
4. Targeted Assembly: Concatenates extracted reads and reassembles them ab initio utilizing SPAdes (--careful, --only-assembler) incorporating trusted contigs.

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💻 Installation & Requirements

Ensure you have Conda and Snakemake installed on your HPC or local machine.

Dependencies

The pipelines rely on the following bioinformatics tools being available in your environment or $PATH:

• fastqc
• hifiasm
• busco
• blastn
• blobtools
• bwa (v0.6.2+)
• spades.py (v3.6.1+)
• perl

Tip: It is highly recommended to manage these dependencies using a Conda environment.

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⚙️ Usage

Running the Primary Assembly Pipeline

1. Edit the input variables at the top of Pacbio-assembly-snakemake.smk:

   input_fastq = "your_reads.fastq.gz"
   threads = 92
   taxdump = "/path/to/taxdump"
   busco_lineage = "/path/to/lineage_odb10"

2. Execute the workflow:

   snakemake -s Pacbio-assembly-snakemake.smk --cores 92

Running the Targeted Reassembly Pipeline

1. Ensure your inputs (FASTA references and subset lists) are accurately defined within the rule parameters of reassembly_snakemake.smk.
2. Execute the workflow:

   snakemake -s reassembly_snakemake.smk --cores 30

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📬 Contact & Support

Nasir Mahmood Abbasi
Computational Biologist & Single-Cell Transcriptomics Expert
🌐 Portfolio: nmabbasi.github.io
📚 Educational Portal: The Omics Hub

If you use these pipelines in your research, please consider citing or acknowledging this repository. For bug reports or feature requests, feel free to open an issue!