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Organelle Assembly and Annotation

The organelle.nf nextflow script will take any number of samples with paired-end reads in FASTQ format and output an annotated mitochondrial and chloroplast genome. If part of the pipeline is unsuccessful for a sample then these errors are ignored and no assembly or annotation files will be produced for that sample.

Pipeline flowchart:

graph TD;
    FASTQ-->|bowtie2| m1[Align to Mitochondrial Seeds];
    FASTQ-->|bowtie2| c1[Align to Chloroplast Seeds];
    subgraph I 
        c1-->|Unicycler| c2[Assemble Plastome];
        c2-->|Custom Python Script| c3[Annotate Plastome];
    end
    subgraph I 
        m1-->|Unicycler| m2[Assemble Mitogenome];
        m2-->|Custom Python Script| m3[Annotate Mitogenome];
    end
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Dependencies (version tested)

  • Nextflow (24.04.4)
  • Java (18.0.2.1)
  • Python (3.10)
  • bowtie2 (2.5.3)
  • SAMtools (1.19.2)
  • Unicycler (0.5.0)
  • BLAST+ (2.15.0)
  • biopython

Conda Environment

Create environment using conda:
conda env create -f ./nextflow-pipelines/env/organelle.yml

Activate conda environment:
conda activate organelle or source activate organelle

Prepare reference files

Download seed sequences for species of interest

# Activate conda environment
conda activate organelle

# Create directory to store references
mkdir references

# Download organelle genomes (replace -id with your accession IDs)
efetch -db nucleotide -id MW357900.2,MW357901.2,MH281621.1 -format fasta > references/mitochondrion-seeds.fa
efetch -db nucleotide -id OQ417768.1,OQ417769.1,MH281627.1 -format fasta > references/chloroplast-seeds.fa

Download GenBank references

# Activate conda environment
conda activate organelle

# Download one or more GenBank Reference files (replace -id with your accession IDs)
efetch -db nucleotide -id MW357900.2 -format gb > references/mitochondrion-reference.gb
efetch -db nucleotide -id OQ417768.1 -format gb > references/chloroplast-reference1.gb
efetch -db nucleotide -id OQ417769.1 -format gb > references/chloroplast-reference2.gb
efetch -db nucleotide -id MH281627.1 -format gb > references/chloroplast-reference3.gb

Usage

#!/bin/bash

# Activate conda environment
conda activate organelle

# Variables
cpus=20
reads=/path/to/reads/directory
ref=/path/to/references/directory
outdir=/path/to/output/directory

# Index reference seed sequences
bowtie2-build ${ref}/mitochondrion-seeds.fa ${ref}/mitochondrion-seeds.fa
bowtie2-build ${ref}/chloroplast-seeds.fa ${ref}/chloroplast-seeds.fa

# Run pipeline
nextflow run ~/nextflow-pipelines/src/organelle.nf \
    --reads ${reads} \
    --suffix "_{1,2}.fq.gz" \
    --mitogenome \
    --mito_seed ${ref}/mitochondrion-seeds.fa \
    --mito_ref ${ref}/mitochondrion-reference.gb \
    --plastome \
    --plastid_seed ${ref}/chloroplast-seeds.fa \
    --plastid_ref ${ref}/chloroplast-reference1.gb,${ref}/chloroplast-reference2.gb,${ref}/chloroplast-reference3.gb \
    --outdir ${outdir} \
    --cpus ${cpus}
Parameter                                   Description
--reads path to input directory containing FASTQ files
--suffix string denoting the suffix after a sample name and the forward (read1) and reverse (read2) designation (e.g. for read pair sample_1.fq.gz and sample_2.fq.gz set the parameter to --suffix "_{1,2}.fq.gz")
--mitogenome run the mitogenome pipeline (this or --plastome required)
--mito_seed file path to the mitochondrion seeds sequences (.fa)
--mito_ref file path to the mitochondrion GenBank references (.gb). Multiple reference files separated by a comma.
--plastome run the plastome pipeline (this or --mitogenome required)
--plastid_seed file path to the chloroplast seeds sequences (.fa)
--plastid_ref file path containing GenBank references (.gb). Multiple reference files separated by a comma.
--outdir path to output directory
--test prints out a tuple of the sample ID and paths to the input paired reads (dry run)
--nextflow_pipelines_path path to nextflow pipelines directory (default: ${HOME}/nextflow-pipelines)
--cpus integer denoting the number of cpus (default: 16)

Example input:

$ ls reads/
SampleID_01_1.fq.gz SampleID_02_1.fq.gz
SampleID_01_2.fq.gz SampleID_02_2.fq.gz

Output

The output of organelle.nf are two directories called mitochondrial_genomes/ and chloroplast_genomes/ that are automatically created in the --outdir path. In these directories are subdirectories for each sample which contain the assembly (assembly.fasta) and annotation (sampleID.cds.fasta) output files.

Example output:

$ ls ${outdir}/mitochondrial_genomes
SampleID_01 SampleID_02

$ ls ${outdir}/chloroplast_genomes
SampleID_01 SampleID_02

Extract annotated gene sequences

Samples that run successfully will have a coding sequences (CDS) FASTA file in their output directory. The code below allows users to extract specific genes that were annotated (e.g. COX1 or psbA).

$ cat mitochondrial_genomes/*/*.cds.fasta | seqkit grep -r -p "COX1" > COX1.fasta
$ cat chloroplast_genomes/*/*.cds.fasta | seqkit grep -r -p "psbA" > psbA.fasta