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phytophthora's Introduction

Phytophthora

Scripts used in the analysis of Phytophthora genomes

ands used during analysis of phytophthora genomes. Note - all this work was performed in the directory: /home/groups/harrisonlab/project_files/idris

The following is a summary of the work presented in this Readme: Data organisation:

  • Preparing data
    Draft Genome assembly
  • Data qc
  • Genome assembly
  • Repeatmasking
  • Gene prediction
  • Functional annotation Genome analysis
  • Homology between predicted genes & published effectors

#Data organisation

Data was copied from the raw_data repository to a local directory for assembly and annotation.

  cd /home/groups/harrisonlab/project_files/idris
  mkdir -p raw_dna/paired/P.cactorum/415/F
  mkdir -p raw_dna/paired/P.cactorum/415/R
  mkdir -p raw_dna/paired/P.cactorum/416/F
  mkdir -p raw_dna/paired/P.cactorum/416/R
  mkdir -p raw_dna/paired/P.fragariae/A4/F
  mkdir -p raw_dna/paired/P.fragariae/A4/R
  mkdir -p raw_dna/paired/P.fragariae/SCRP245_v2/F
  mkdir -p raw_dna/paired/P.fragariae/SCRP245_v2/R
  mkdir -p raw_dna/paired/P.fragariae/Bc23/F
  mkdir -p raw_dna/paired/P.fragariae/Bc23/R
  mkdir -p raw_dna/paired/P.fragariae/Nov5/F
  mkdir -p raw_dna/paired/P.fragariae/Nov5/R
  mkdir -p raw_dna/paired/P.fragariae/Nov77/F
  mkdir -p raw_dna/paired/P.fragariae/Nov77/R
  mkdir -p raw_dna/paired/P.fragariae/ONT3/F
  mkdir -p raw_dna/paired/P.fragariae/ONT3/R
  mkdir -p raw_dna/paired/P.cactorum/2003_3/F
  mkdir -p raw_dna/paired/P.cactorum/2003_3/R
  mkdir -p raw_dna/paired/P.idaei/SCRP370/F
  mkdir -p raw_dna/paired/P.idaei/SCRP370/R
  mkdir -p raw_dna/paired/P.rubi/SCRP249/F
  mkdir -p raw_dna/paired/P.rubi/SCRP249/R
  mkdir -p raw_dna/paired/P.rubi/SCRP324/F
  mkdir -p raw_dna/paired/P.rubi/SCRP324/R
  mkdir -p raw_dna/paired/P.rubi/SCRP333/F
  mkdir -p raw_dna/paired/P.rubi/SCRP333/R
  mkdir -p raw_dna/paired/P.cactorum/2003_4/F
  mkdir -p raw_dna/paired/P.cactorum/2003_4/R
  mkdir -p raw_dna/paired/P.cactorum/PC13_15/F
  mkdir -p raw_dna/paired/P.cactorum/PC13_15/R


  RawDat=/home/groups/harrisonlab/raw_data/raw_seq/raw_reads/150716_M01678_0023_AB0YF
  cp $RawDat/Pcactorum415_S2_L001_R1_001.fastq.gz raw_dna/paired/P.cactorum/415/F/.
  cp $RawDat/Pcactorum415_S2_L001_R2_001.fastq.gz raw_dna/paired/P.cactorum/415/R/.
  cp $RawDat/Pcactorum416_S1_L001_R1_001.fastq.gz raw_dna/paired/P.cactorum/416/F/.
  cp $RawDat/Pcactorum416_S1_L001_R2_001.fastq.gz raw_dna/paired/P.cactorum/416/R/.
  cp $RawDat/PfragariaeA4_S3_L001_R1_001.fastq.gz raw_dna/paired/P.fragariae/A4/F/.
  cp $RawDat/PfragariaeA4_S3_L001_R2_001.fastq.gz raw_dna/paired/P.fragariae/A4/R/.
  RawDat=/home/groups/harrisonlab/raw_data/raw_seq/raw_reads/150925_M01678_0029_AC669
  cp $RawDat/Pfrag-SCRP245_S3_L001_R1_001.fastq.gz raw_dna/paired/P.fragariae/SCRP245_v2/F/.
  cp $RawDat/Pfrag-SCRP245_S3_L001_R2_001.fastq.gz raw_dna/paired/P.fragariae/SCRP245_v2/R/.
  cp $RawDat/Pfrag-Bc23_S2_L001_R1_001.fastq.gz raw_dna/paired/P.fragariae/Bc23/F/.
  cp $RawDat/Pfrag-Bc23_S2_L001_R2_001.fastq.gz raw_dna/paired/P.fragariae/Bc23/R/.
  RawDat=/home/groups/harrisonlab/raw_data/raw_seq/raw_reads/150918_M01678_0028_AC60K
  cp $RawDat/Pfrag-Nov-5_S2_L001_R1_001.fastq.gz raw_dna/paired/P.fragariae/Nov5/F/.
  cp $RawDat/Pfrag-Nov-5_S2_L001_R2_001.fastq.gz raw_dna/paired/P.fragariae/Nov5/R/.
  cp $RawDat/Pfrag-Nov-77_S3_L001_R1_001.fastq.gz raw_dna/paired/P.fragariae/Nov77/F/.
  cp $RawDat/Pfrag-Nov-77_S3_L001_R2_001.fastq.gz raw_dna/paired/P.fragariae/Nov77/R/.
  cp $RawDat/Pfrag-ONT-3_S1_L001_R1_001.fastq.gz raw_dna/paired/P.fragariae/ONT3/F/.
  cp $RawDat/Pfrag-ONT-3_S1_L001_R2_001.fastq.gz raw_dna/paired/P.fragariae/ONT3/R/.
  RawDat=/home/groups/harrisonlab/raw_data/raw_seq/raw_reads/160404_M004465_0008-ALVUT
  cp $RawDat/Pc20033_S2_L001_R1_001.fastq.gz raw_dna/paired/P.cactorum/2003_3/F/.
  cp $RawDat/Pc20033_S2_L001_R2_001.fastq.gz raw_dna/paired/P.cactorum/2003_3/R/.
  cp $RawDat/SCRP370_S3_L001_R1_001.fastq.gz  raw_dna/paired/P.idaei/SCRP370/F/.
  cp $RawDat/SCRP370_S3_L001_R2_001.fastq.gz  raw_dna/paired/P.idaei/SCRP370/R/.
  RawDat=/home/groups/harrisonlab/raw_data/raw_seq/raw_reads/160412_M04465_0010-AMLCU
  cp $RawDat/SCRP249_S1_L001_R1_001.fastq.gz raw_dna/paired/P.rubi/SCRP249/F/.
  cp $RawDat/SCRP249_S1_L001_R2_001.fastq.gz raw_dna/paired/P.rubi/SCRP249/R/.
  cp $RawDat/SCRP324_S2_L001_R1_001.fastq.gz raw_dna/paired/P.rubi/SCRP324/F/.
  cp $RawDat/SCRP324_S2_L001_R2_001.fastq.gz raw_dna/paired/P.rubi/SCRP324/R/.
  cp $RawDat/SCRP333_S3_L001_R1_001.fastq.gz raw_dna/paired/P.rubi/SCRP333/F/.
  cp $RawDat/SCRP333_S3_L001_R2_001.fastq.gz raw_dna/paired/P.rubi/SCRP333/R/.
  RawDat=/home/groups/harrisonlab/raw_data/raw_seq/raw_reads/160415_M004465_00011-AMLCL
  cp $RawDat/20033_S4_L001_R1_001.fastq.gz raw_dna/paired/P.cactorum/2003_4/F/.
  cp $RawDat/20033_S4_L001_R2_001.fastq.gz raw_dna/paired/P.cactorum/2003_4/R/.
  cp $RawDat/PC1315_S5_L001_R1_001.fastq.gz raw_dna/paired/P.cactorum/PC13_15/F/.
  cp $RawDat/PC1315_S5_L001_R2_001.fastq.gz raw_dna/paired/P.cactorum/PC13_15/R/.

#Data qc

programs: fastqc fastq-mcf kmc

Data quality was visualised using fastqc:

# 415
for RawData in $(ls raw_dna/paired/P.cactorum/415/*/*.fastq.gz); do
echo $RawData;
ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
qsub $ProgDir/run_fastqc.sh $RawData;
done
# 416
for RawData in $(ls raw_dna/paired/P.cactorum/416/*/*.fastq.gz); do
echo $RawData;
ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
qsub $ProgDir/run_fastqc.sh $RawData;
done
# A4
for RawData in $(ls raw_dna/paired/P.fragariae/A4/*/*.fastq.gz); do
echo $RawData;
ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
qsub $ProgDir/run_fastqc.sh $RawData;
done
# SCRP245_v2
for RawData in $(ls raw_dna/paired/P.fragariae/SCRP245_v2/*/*.fastq.gz); do
echo $RawData;
ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
qsub $ProgDir/run_fastqc.sh $RawData;
done
# Bc23
for RawData in $(ls raw_dna/paired/P.fragariae/Bc23/*/*.fastq.gz); do
echo $RawData;
ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
qsub $ProgDir/run_fastqc.sh $RawData;
done
# Nov5
for RawData in $(ls raw_dna/paired/P.fragariae/Nov5/*/*.fastq.gz); do
echo $RawData;
ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
qsub $ProgDir/run_fastqc.sh $RawData;
done
# Nov77
for RawData in $(ls raw_dna/paired/P.fragariae/Nov77/*/*.fastq.gz); do
echo $RawData;
ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
qsub $ProgDir/run_fastqc.sh $RawData;
done
# ONT3
for RawData in $(ls raw_dna/paired/P.fragariae/ONT3/*/*.fastq.gz); do
echo $RawData;
ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
qsub $ProgDir/run_fastqc.sh $RawData;
done

Trimming was performed on data to trim adapters from sequences and remove poor quality data. This was done with fastq-mcf

for Strain in 414; do
echo $Strain
Read_F=$(ls raw_dna/paired/P.*/$Strain/F/cact414_130517_S2_L001_R1_001.fastq.gz)
Read_R=$(ls raw_dna/paired/P.*/$Strain/R/cact414_130517_S2_L001_R2_001.fastq.gz)
IluminaAdapters=/home/armita/git_repos/emr_repos/tools/seq_tools/ncbi_adapters.fa
ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/rna_qc
qsub $ProgDir/rna_qc_fastq-mcf.sh $Read_F $Read_R $IluminaAdapters DNA
done
  for Strain in 415 416 A4; do
    echo $Strain
    Read_F=$(ls raw_dna/paired/P.*/$Strain/F/*.fastq.gz)
    Read_R=$(ls raw_dna/paired/P.*/$Strain/R/*.fastq.gz)
    IluminaAdapters=/home/armita/git_repos/emr_repos/tools/seq_tools/ncbi_adapters.fa
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/rna_qc
    qsub $ProgDir/rna_qc_fastq-mcf.sh $Read_F $Read_R $IluminaAdapters DNA
  done
  for Strain in SCRP245_v2 Bc23; do
    echo $Strain
    Read_F=$(ls raw_dna/paired/P.*/$Strain/F/*.fastq.gz)
    Read_R=$(ls raw_dna/paired/P.*/$Strain/R/*.fastq.gz)
    IluminaAdapters=/home/armita/git_repos/emr_repos/tools/seq_tools/ncbi_adapters.fa
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/rna_qc
    qsub $ProgDir/rna_qc_fastq-mcf.sh $Read_F $Read_R $IluminaAdapters DNA
  done
  for Strain in Nov5 Nov77 ONT3; do
    echo $Strain
    Read_F=$(ls raw_dna/paired/P.*/$Strain/F/*.fastq.gz)
    Read_R=$(ls raw_dna/paired/P.*/$Strain/R/*.fastq.gz)
    IluminaAdapters=/home/armita/git_repos/emr_repos/tools/seq_tools/ncbi_adapters.fa
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/rna_qc
    qsub $ProgDir/rna_qc_fastq-mcf.sh $Read_F $Read_R $IluminaAdapters DNA
  done

Data quality was visualised once again following trimming:

  for RawData in $(ls qc_dna/paired/P.cactorum/415/*/*.fq.gz); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done
  # 416
  for RawData in $(ls qc_dna/paired/P.cactorum/416/*/*.fq.gz); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done
  # A4
  for RawData in $(ls qc_dna/paired/P.fragariae/A4/*/*.fq.gz); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done
  # SCRP245_v2
  for RawData in $(ls qc_dna/paired/P.fragariae/SCRP245_v2/*/*.fq.gz); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done
  for RawData in $(ls qc_dna/paired/P.fragariae/SCRP245_v2/*/SCRP245_v2_no_adapt.fq); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done
  # Bc23
  for RawData in $(ls qc_dna/paired/P.fragariae/Bc23/*/*.fq.gz); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done
  # Nov5
  for RawData in $(ls qc_dna/paired/P.fragariae/Nov5/*/*.fq.gz); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done
  # Nov77
  for RawData in $(ls qc_dna/paired/P.fragariae/Nov77/*/*.fq.gz); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done
  # ONT3
  for RawData in $(ls qc_dna/paired/P.fragariae/ONT3/*/*.fq.gz); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done

``bash qsub /home/armita/git_repos/emr_repos/tools/seq_tools/rna_qc/qc_trimmomatic.sh raw_dna/paired/P.fragariae/SCRP245_v2/F/Pfrag-SCRP245_S3_L001_R1_001.fastq.gz raw_dna/paired/P.fragariae/SCRP245_v2/R/Pfrag-SCRP245_S3_L001_R2_001.fastq.gz /home/armita/git_repos/emr_repos/tools/seq_tools/ncbi_adapters.fa qc_dna/paired/P.fragariae/SCRP245_v2

```bash
  for RawData in $(ls qc_dna/paired/P.fragariae/SCRP245_v2/*/*.fq); do
    echo $RawData;
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc;
    qsub $ProgDir/run_fastqc.sh $RawData;
  done

kmer counting was performed using kmc. This allowed estimation of sequencing depth and total genome size:

  # for Strain in 415 416 A4 SCRP245_v2 Bc23 Nov5 Nov77; do
  for Strain in ONT3; do
    echo $Strain
    Trim_F=$(ls qc_dna/paired/P.*/$Strain/F/*.fq.gz)
    Trim_R=$(ls qc_dna/paired/P.*/$Strain/R/*.fq.gz)
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/dna_qc
    qsub $ProgDir/kmc_kmer_counting.sh $Trim_F $Trim_R
  done

** Estimated Genome Size is:

** Esimated Coverage is:

#Assembly Assembly was performed using: Velvet / Abyss / Spades

A range of hash lengths were used and the best assembly selected for subsequent analysis

  for Strain in 415 416 A4 SCRP245_v2 Bc23 Nov5 Nov77 ONT3; do
    F_Read=$(ls qc_dna/paired/P.*/$Strain/F/*.fq.gz)
    R_Read=$(ls qc_dna/paired/P.*/$Strain/R/*.fq.gz)
    CovCutoff='10'
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/assemblers/spades
    Species=$(echo $F_Read | rev | cut -f4 -d '/' | rev)
    OutDir=assembly/spades/$Species/$Strain
    echo $Species
    echo $Strain
    qsub $ProgDir/submit_SPAdes.sh $F_Read $R_Read $OutDir correct $CovCutoff
    # qsub $ProgDir/submit_dipSPAdes.sh $F_Read $R_Read $OutDir correct $CovCutoff
  done

Quast

  for Strain in $(ls -d assembly/spades/*/* | rev | cut -f1 -d'/' | rev); do
    ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/assemblers/assembly_qc/quast
    Assembly=$(ls -d assembly/spades/*/$Strain/filtered_contigs/contigs_min_500bp.fasta)
    Species=$(echo $Assembly | rev | cut -f4 -d'/' | rev)
    OutDir=$(ls -d assembly/spades/*/$Strain/filtered_contigs)
    qsub $ProgDir/sub_quast.sh $Assembly $OutDir
  done

Assemblies were summarised to allow the best assembly to be determined by eye.

** Assembly stats are:

  • Assembly size:
  • N50:153669
  • N80:
  • N20:
  • Longest contig:687738 **

As SPADes was run with the option to autodetect a minimum coverage the assembly was assessed to identify the coverage of assembled contigs. This was done using the following command:

	BestAss=assembly/spades/N.ditissima/R0905_v2/filtered_contigs/contigs_min_500bp.fasta
	cat $BestAss | grep '>' | cut -f6 -d'_' | sort -n | cut -f1 -d '.' | sort -n | uniq -c | less

From this it was determined that SPades could not be trusted to set its own minimum threshold for coverage. In future an option will be be used to set a coverage for spades. In the meantime contigs with a coverage lower than 10 were filtered out using the following commands:

	Headers=assembly/spades/N.ditissima/R0905_v2/filtered_contigs/contigs_min_500bp_10x_headers.txt
	cat $BestAss | grep '>' | grep -E -v 'cov_.\..*_' > $Headers
	FastaMinCov=assembly/spades/N.ditissima/R0905_v2/filtered_contigs/contigs_min_500bp_10x_headers.fasta
	cat $BestAss | sed -e 's/\(^>.*$\)/#\1#/' | tr -d "\r" | tr -d "\n" | sed -e 's/$/#/' | tr "#" "\n" | sed -e '/^$/d' | grep -A1 -f $Headers | grep -v -E '^\-\-' > $FastaMinCov
	~/git_repos/emr_repos/tools/seq_tools/assemblers/assembly_qc/remove_contaminants/remove_contaminants.py --inp ../neonectria_ditissima/assembly/spades/N.ditissima/R0905_v2/filtered_contigs/contigs_min_500bp_10x_headers.fasta  --out assembly/spades/N.galligena/R0905_v2/filtered_contigs/contigs_min_500bp_10x_filtered_renamed.fasta  --coord_file editfile.tab

We run Quast again.

	ProgDir=/home/armita/git_repos/emr_repos/tools/seq_tools/assemblers/assembly_qc/quast
	Assembly=assembly/spades/N.ditissima/R0905_v2/filtered_contigs/contigs_min_500bp_10x_headers.fasta
	OutDir=assembly/spades/N.ditissima/R0905_v2/contigs_min_500bp_10x_headers
	qsub $ProgDir/sub_quast.sh $Assembly $OutDir

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