ejongepier / compgen-caulerpa-chloroplast

README to Caulerpa chloroplast comparative genomics project

This repo contains the code and documentation for the comparative genomics of the Caulerpa chloroplast genomes. How these genomes were obtained is detailed elsewhere (https://github.com/ejongepier/compgen-caulerpa-fin). Results are stored in minio bucket fnwi/202203-compgen-chloroplast-jongepier/.

Usage

Preliminary chloroplast assembly

The purpose is to get a quick assembly to use for read mapping and filtering. The filtered reads will be used in the final assembly

sbatch scripts/getorganelle.sh -f fnwi/202201-compgen-caulerpa-jongepier/results/qc/qc/sickle/${spp}_R1_sickle.fq.gz -r fnwi/202201-compgen-caulerpa-jongepier/results/qc/qc/sickle/${spp}_R2_sickle.fq.gz -o fnwi/202203-compgen-chloroplast-jongepier/results/assembly -d embplant_pt -p ${spp}-chloroplast

Post process by changing the fasta header, doubling the sequence and building the bowtei2 db. The purpose of doubling the seq is to allow for concordant mapping accross the circularized junction.

Filter chloroplast reads

The purpose is to get the chloroplast reads as input to metaspades

sbatch scripts/assembly-based-filtering-illumina.sh -f fnwi/202201-compgen-caulerpa-jongepier/results/qc/qc/sickle/${spp}_R1_sickle.fq.gz -r fnwi/202201-compgen-caulerpa-jongepier/results/qc/qc/sickle/${spp}_R2_sickle.fq.gz -d fnwi/202203-compgen-chloroplast-jongepier/results/assembly/$spp-chloroplast-getorganelle-doubled -p ${spp} -o fnwi/202203-compgen-chloroplast-jongepier/results/readfilt
sbatch scripts/assembly-based-filtering-minion.sh -i fnwi/202011-metagenomics-caulerpa-anastasiabarilo/data/wgs/${spp}-minion-wgs-filtered.fastq.gz -d  fnwi/202203-compgen-chloroplast-jongepier/results/assembly/$spp-chloroplast-getorganelle-doubled -p ${spp} -o fnwi/202203-compgen-chloroplast-jongepier/results/readfilt

Cloroplast meta-assembly

Identify chloroplast ORFs

ORFs were identified with EMBOSS getorfs utility, and annotated using blastp against NCBI nr. This analyses was run on blobfish.

Get chloroplast genome assemblies:

mkdir -p data/assemblies
mc cp fnwi/202201-compgen-caulerpa-jongepier/results/masurca/clen-masurca-organel/clen-chloroplast-genome.fa.gz data/assemblies/
mc cp fnwi/202201-compgen-caulerpa-jongepier/results/getorganelle/cpro-chloroplast-genome.fa.gz data/assemblies/

Get ORFs:

SPP=clen
mkdir -p results/getorfs/
gzip -d data/assemblies/${SPP}-chloroplast-genome.fa.gz
getorf -circular Y -reverse T -sequence data/assemblies/${SPP}-chloroplast-genome.fa -outseq results/getorfs/${SPP}-chloroplast-orfs.faa

Get NCBI db:

cd ~/Databases/ncbi-nr
wget ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/nr.gz
gzip -d nr.gz
mv nr nr.faa
makeblastdb -dbtype 'prot' -in nr.faa
echo `date` > VERSION.txt
cd -

Blast orfs against nr:

blastp -db ~/Databases/ncbi-nr/nr -query results/getorfs/${SPP}-chloroplast-orfs.faa -outfmt 6

Cleanup

gzip results/getorfs/${SPP}-chloroplast-orfs.faa
mc cp results/getorfs/${SPP}-chloroplast-orfs.faa fnwi/202203-compgen-chloroplast-jongepier/results/getorfs/
rm -r data results

Annotate chloroplast genomes

Chloroplast genomes were annotated with the webutility of GeSeq.

Poolseq variant calling

sbatch scripts/depth-pileup-illumina.sh -f fnwi/202201-compgen-caulerpa-jongepier/results/assembly-based-filt/clen_R1_clen-chloroplast-noncirc-genome-mapped.fastq.gz -r fnwi/202201-compgen-caulerpa-jongepier/results/assembly-based-filt/clen_R2_clen-chloroplast-noncirc-genome-mapped.fastq.gz -d fnwi/202201-compgen-caulerpa-jongepier/results/masurca/clen-masurca-organel/clen-chloroplast-genome -p clen -o fnwi/202203-compgen-chloroplast-jongepier/results/pileup

Authors

• Evelien Jongepier ([email protected])