indoor-plants

Chloroplast genome assembly

• A bash script to assemble a chloroplast genome from long and short sequencing reads.

How to use

Example:

• Copy plastid-assembly.sh into the directory where you will run the analysis
• Activate your conda environment with the tools installed.
• Run: bash plastid-assembly.sh -a adapters.fasta -b baits.fasta R1.fq R2.fq nano.fq

Basic process

• Get chloroplast-only reads out of all the reads
• Filter and downsample if required
• Assemble
• Polish with long reads
• Polish with short reads

Inputs

• Long reads, e.g. nanopore
• Short reads, e.g. Illumina R1 and R2
• An illumina adapters file, for trimming
• A baits.fasta file - sequences from a similar chloroplast genome to use to extract matching chloroplast reads from all the reads. I used three gene sequences from a similar species; the genes are likely to be fairly evenly spaced to allow matching reads to span the whole genome.

What the script does:

• extract chloroplast nanopore reads from all long reads
• keep a subset of these reads (longest, to x250 coverage) using filtlong
• assemble these with flye
• polish that assembly with the long reads themselves
• trim and filter illumina reads with fastp
• extract illumina chloroplast reads from all illumina reads
• downsample to X300 coverage with rasusa
• polish the flye assembly with the short reads, with pilon
• do an additional assembly with Unicycler, which uses both long and short reads
• do an additional assembly with miniasm, using long reads
• polish that assembly with minipolish and the long reads
• polish again with the short reads
• compare the assemblies with mummer - dnadiff, to get delta files (then view in http://www.assemblytics.com/)
• calculate stats for all read sets and assemblies with seqkit stats

Outputs

• Polished chloroplast genome assemblies using long and short reads, from Flye, Unicycler and Miniasm/Minipolish.
• Upload the assembly fasta files to https://chlorobox.mpimp-golm.mpg.de/geseq.html if you want to annotate the genomes.

Tools

Installed with conda.

minimap2
samtools
filtlong
flye
racon
fastp
rasusa
bwa
pilon
unicycler
miniasm
minipolish
mummer
seqkit

A conda env yml file is included this repo, with a record of the tools and versions used in the analysis, but this environment is platform-dependent (linux).