Shotgun Metagenomics Analysis

Analysis of metagenomic shotgun sequences including assembly, speciation, ARG discovery and more

Description

The input for this analysis is paired end next generation sequencing data from metagenomic samples. The workflow is designed to be modular, so that individual modules can be run depending on the nature of the metagenomics project at hand. More modules will be added as we develop them - this repo is a work in progress!

These scripts have been written ...

This is a genomics pipeline to do a single germline sample variant-calling, adapted from GATK Best Practice Workflow.

This workflow is a reference pipeline for using the Janis Python framework (pipelines assistant).

• Alignment: bwa-mem
• Variant-Calling: GATK HaplotypeCaller
• Outputs the final variants in the VCF format.

Resources

This pipeline has been tested using the HG38 reference set, available on Google Cloud Storage through:

• https://console.cloud.google.com/storage/browser/genomics-public-data/references/hg38/v0/ ...

Combined workflow for large genome assembly

The tutorial document for this workflow is here: https://doi.org/10.5281/zenodo.5655813

What it does: A workflow for genome assembly, containing subworkflows:

• Data QC
• Kmer counting
• Trim and filter reads
• Assembly with Flye
• Assembly polishing
• Assess genome quality

Inputs:

• long reads and short reads in fastq format
• reference genome for Quast

Outputs:

• Data information - QC, kmers
• Filtered, trimmed reads
• Genome assembly, assembly graph, ...

MetaDEGalaxy: Galaxy workflow for differential abundance analysis of 16s metagenomic data

Assess genome quality; can run alone or as part of a combined workflow for large genome assembly.

• What it does: Assesses the quality of the genome assembly: generate some statistics and determine if expected genes are present; align contigs to a reference genome.
• Inputs: polished assembly; reference_genome.fasta (e.g. of a closely-related species, if available).
• Outputs: Busco table of genes found; Quast HTML report, and link to Icarus contigs browser, showing contigs aligned to a reference ...

Assembly polishing subworkflow: Racon polishing with long reads

Inputs: long reads and assembly contigs

Workflow steps:

• minimap2 : long reads are mapped to assembly => overlaps.paf.
• overaps, long reads, assembly => Racon => polished assembly 1
• using polished assembly 1 as input; repeat minimap2 + racon => polished assembly 2
• using polished assembly 2 as input, repeat minimap2 + racon => polished assembly 3
• using polished assembly 3 as input, repeat minimap2 + racon => ...

Assembly with Flye; can run alone or as part of a combined workflow for large genome assembly.

• What it does: Assembles long reads with the tool Flye
• Inputs: long reads (may be raw, or filtered, and/or corrected); fastq.gz format
• Outputs: Flye assembly fasta; Fasta stats on assembly.fasta; Assembly graph image from Bandage; Bar chart of contig sizes; Quast reports of genome assembly
• Tools used: Flye, Fasta statistics, Bandage, Bar chart, Quast
• Input parameters: None required, but recommend ...

Trim and filter reads; can run alone or as part of a combined workflow for large genome assembly.

• What it does: Trims and filters raw sequence reads according to specified settings.
• Inputs: Long reads (format fastq); Short reads R1 and R2 (format fastq)
• Outputs: Trimmed and filtered reads: fastp_filtered_long_reads.fastq.gz (But note: no trimming or filtering is on by default), fastp_filtered_R1.fastq.gz, fastp_filtered_R2.fastq.gz
• Reports: fastp report on long reads, html; fastp report ...

Kmer counting step, can run alone or as part of a combined workflow for large genome assembly.

• What it does: Estimates genome size and heterozygosity based on counts of kmers
• Inputs: One set of short reads: e.g. R1.fq.gz
• Outputs: GenomeScope graphs
• Tools used: Meryl, GenomeScope
• Input parameters: None required
• Workflow steps: The tool meryl counts kmers in the input reads (k=21), then converts this into a histogram. GenomeScope: runs a model on the histogram; reports estimates. k-mer ...