Assembly and Annotation of Four C. jejuni Strains

Raw Data

Strain	Accession	Type
MS2167	SRR20020687	Illumina
MS2074	SRR20020688	Illumina
MS2058	SRR20020689	Illumina
MS2005	SRR20020690	Illumina
MS2167	SRR20020691	Nanopore
MS2074	SRR20020692	Nanopore
MS2058	SRR20020693	Nanopore
MS2005	SRR20020694	Nanopore

Download SRA data

ROOT=$(git rev-parse --show-toplevel)

for SRA in "${sra[@]}"; do

    URL=ftp://ftp.sra.ebi.ac.uk/vol1/fastq/${SRA:0:6}/

    if [ ${#SRA} -gt 9 ]; then
        TMP=00${SRA:9}
        URL+=${TMP: -3}/
    fi

    URL+=$SRA

    wget -O $ROOT/0-raw/${SRA}_1.fastq.gz $URL/${SRA}_1.fastq.gz
    wget -O $ROOT/0-raw/${SRA}_2.fastq.gz $URL/${SRA}_2.fastq.gz

done

Assemble

Canu

ROOT=$(git rev-parse --show-toplevel)
cd $ROOT

PATH=$ROOT/apps/canu-1.9/bin:$PATH

name=${names[$SLURM_ARRAY_TASK_ID]}

canu -d $ROOT/1-assemble/1-canu/$name -p "$name" \
    genomeSize=2m useGrid=false \
    -nanopore-raw $ROOT/0-raw/${data[$name]}_1.fastq.gz

Trim

ROOT=$(git rev-parse --show-toplevel)
DIR=$ROOT/1-assemble
cd $DIR

name=${names[$SLURM_ARRAY_TASK_ID]}
assembly=$DIR/1-canu/$name/$name.contigs.fasta

ml igbb samtools blast
ml singularity

mkdir $DIR/2-trim/$name

grep '>' -A10 --no-group-separator $assembly |
    blastn -query - -subject $assembly -outfmt '6 std qcovs' -out $DIR/2-trim/$name/blast.out

awk '$1 == $2 && $3 >= 95 && $13 >= 95 && $9 > 1000 && $9 < $10 && $1 != last {
     printf "%s:1-%d\n", $1, $9; last=$1}' $DIR/2-trim/$name/blast.out \
         > $DIR/2-trim/$name/trim.lst

xargs samtools faidx $assembly \
    < $DIR/2-trim/$name/trim.lst \
    > $DIR/2-trim/$name/trim.fa

sed 's/:.*//' $DIR/2-trim/$name/trim.lst |
    grep -f - -v $assembly.fai |
    cut -f 1 > $DIR/2-trim/$name/unchanged.txt

cat $DIR/2-trim/$name/unchanged.txt |
    xargs samtools faidx $assembly >> $DIR/2-trim/$name/trim.fa

Fix start

>NC_000913.3:c3883729-3882326 Escherichia coli str. K-12 substr. MG1655, complete genome
GTGTCACTTTCGCTTTGGCAGCAGTGTCTTGCCCGATTGCAGGATGAGTTACCAGCCACAGAATTCAGTA
TGTGGATACGCCCATTGCAGGCGGAACTGAGCGATAACACGCTGGCCCTGTACGCGCCAAACCGTTTTGT
CCTCGATTGGGTACGGGACAAGTACCTTAATAATATCAATGGACTGCTAACCAGTTTCTGCGGAGCGGAT
GCCCCACAGCTGCGTTTTGAAGTCGGCACCAAACCGGTGACGCAAACGCCACAAGCGGCAGTGACGAGCA
ACGTCGCGGCCCCTGCACAGGTGGCGCAAACGCAGCCGCAACGTGCTGCGCCTTCTACGCGCTCAGGTTG
GGATAACGTCCCGGCCCCGGCAGAACCGACCTATCGTTCTAACGTAAACGTCAAACACACGTTTGATAAC
TTCGTTGAAGGTAAATCTAACCAACTGGCGCGCGCGGCGGCTCGCCAGGTGGCGGATAACCCTGGCGGTG
CCTATAACCCGTTGTTCCTTTATGGCGGCACGGGTCTGGGTAAAACTCACCTGCTGCATGCGGTGGGTAA
CGGCATTATGGCGCGCAAGCCGAATGCCAAAGTGGTTTATATGCACTCCGAGCGCTTTGTTCAGGACATG
GTTAAAGCCCTGCAAAACAACGCGATCGAAGAGTTTAAACGCTACTACCGTTCCGTAGATGCACTGCTGA
TCGACGATATTCAGTTTTTTGCTAATAAAGAACGATCTCAGGAAGAGTTTTTCCACACCTTCAACGCCCT
GCTGGAAGGTAATCAACAGATCATTCTCACCTCGGATCGCTATCCGAAAGAGATCAACGGCGTTGAGGAT
CGTTTGAAATCCCGCTTCGGTTGGGGACTGACTGTGGCGATCGAACCGCCAGAGCTGGAAACCCGTGTGG
CGATCCTGATGAAAAAGGCCGACGAAAACGACATTCGTTTGCCGGGCGAAGTGGCGTTCTTTATCGCCAA
GCGTCTACGATCTAACGTACGTGAGCTGGAAGGGGCGCTGAACCGCGTCATTGCCAATGCCAACTTTACC
GGACGGGCGATCACCATCGACTTCGTGCGTGAGGCGCTGCGCGACTTGCTGGCATTGCAGGAAAAACTGG
TCACCATCGACAATATTCAGAAGACGGTGGCGGAGTACTACAAGATCAAAGTCGCGGATCTCCTTTCCAA
GCGTCGATCCCGCTCGGTGGCGCGTCCGCGCCAGATGGCGATGGCGCTGGCGAAAGAGCTGACTAACCAC
AGTCTGCCGGAGATTGGCGATGCGTTTGGTGGCCGTGACCACACGACGGTGCTTCATGCCTGCCGTAAGA
TCGAGCAGTTGCGTGAAGAGAGCCACGATATCAAAGAAGATTTTTCAAATTTAATCAGAACATTGTCATC
GTAA

ROOT=$(git rev-parse --show-toplevel)
DIR=$ROOT/1-assemble
cd $DIR

ml singularity

name=${names[$SLURM_ARRAY_TASK_ID]}
assembly=$DIR/2-trim/$name/trim.fa

mkdir $DIR/3-fixstart/$name

singularity exec -B$ROOT --cleanenv $ROOT/apps/circlator.simg \
    circlator fixstart \
    --genes_fa $DIR/3-fixstart/dnaa.fa \
    --ignore $DIR/2-trim/$name/unchanged.txt \
    $assembly \
    $DIR/3-fixstart/$name/fixstart

Filter

ROOT=$(git rev-parse --show-toplevel)
DIR=$ROOT/1-assemble
cd $DIR

ml igbb samtools bwa

name=${names[$SLURM_ARRAY_TASK_ID]}
assembly=$DIR/3-fixstart/$name/fixstart.fasta

mkdir $DIR/4-filter/$name

bwa index $assembly

bwa mem -t $SLURM_CPUS_PER_TASK $assembly \
    $ROOT/raw/${data[$name]}_{1,2}.fastq.gz |
    samtools view -bS -F4 - |
    samtools sort -o $DIR/4-filter/$name/frags.bam -T $DIR/4-filter/$name/frags.tmp -

samtools index $DIR/4-filter/$name/frags.bam

ROOT=$(git rev-parse --show-toplevel)
DIR=$ROOT/1-assemble
cd $DIR

ml igbb samtools bwa

name=${names[$SLURM_ARRAY_TASK_ID]}
assembly=$DIR/3-fixstart/$name/fixstart.fasta

ml igbb samtools
bedtools () { $ROOT/apps/bedtools-2.29.2 $@; }

samtools faidx $assembly

bedtools genomecov -ibam $DIR/4-filter/$name/frags.bam > $DIR/4-filter/$name/genome.cov

awk '$1 != "genome" && $2 == 0 && $5 == 1 {s[$1]++}
     {_[$1]++}
     END {
       s["genome"]++;
       for(k in _)
         if(!s[k])
           print k
     }' $DIR/4-filter/$name/genome.cov > $DIR/4-filter/$name/filter.lst


xargs samtools faidx $assembly \
    < $DIR/4-filter/$name/filter.lst\
    > $DIR/4-filter/$name/filtered.fa

Pilon

ROOT=$(git rev-parse --show-toplevel)
DIR=$ROOT/1-assemble
cd $DIR

ml igbb samtools bwa

name=${names[$SLURM_ARRAY_TASK_ID]}
assembly=$DIR/4-filter/$name/filtered.fa

ml igbb bwa samtools

mkdir $DIR/5-pilon/$name

java -Xmx16G -jar $ROOT/apps/pilon-1.23.jar \
    --genome $assembly \
    --frags $DIR/4-filter/$name/frags.bam \
    --outdir $DIR/5-pilon/$name/ \
    --output pilon \
    --changes

Coverage

ROOT=$(git rev-parse --show-toplevel)
DIR=$ROOT/1-assemble
cd $DIR

ml igbb samtools bwa minimap2
ml singularity
bedtools () { $ROOT/apps/bedtools-2.29.2 $@; }

name=${names[$SLURM_ARRAY_TASK_ID]}
assembly=$DIR/5-pilon/$name/pilon.fasta

mkdir $DIR/6-coverage/$name

minimap2 -x map-ont -d $assembly.mmi $assembly
bwa index $assembly
samtools faidx $assembly

minimap2 -ax map-ont -t $SLURM_CPUS_PER_TASK -a --sam-hit-only \
    $assembly.mmi $ROOT/0-raw/${nanopore[$name]}_1.fastq.gz |
    samtools sort -m10G -o $DIR/6-coverage/$name/nanopore.bam \
        -T $DIR/6-coverage/$name/nanopore.tmp -

bwa mem -t $PBS_NUM_PPN -R "@RG\tID:$name\tPL:illumina\tSM:$name\tLB:$name" \
    $assembly $ROOT/0-raw/${illumina[$name]}_{1,2}.fastq.gz |
    samtools fixmate -O bam - - |
    samtools sort -o $DIR/6-coverage/${name}/illumina.bam \
        -T $DIR/6-coverage/${name}/illumina.tmp -

samtools index $DIR/6-coverage/${name}/illumina.bam

for type in nanopore illumina; do
    bedtools genomecov -ibam $DIR/6-coverage/$name/$type.bam > $DIR/6-coverage/$name/$type.gencov
    bedtools genomecov -bga -ibam $DIR/6-coverage/$name/$type.bam > $DIR/6-coverage/$name/$type.perbase

    bedtools makewindows -g $assembly.fai -n 1 |
        bedtools coverage -sorted -a - -b $DIR/6-coverage/$name/$type.bam -g $assembly.fai \
            > $DIR/6-coverage/$name/$type.hist

    bedtools makewindows -g $assembly.fai -w 100 |
        bedtools coverage -sorted -a - -b $DIR/6-coverage/$name/$type.bam -g $assembly.fai \
            > $DIR/6-coverage/$name/$type.coverage
done

for file in 6-coverage/MS*/*.gencov ; do
    echo $file;
    awk '{t[$1]+=$2*$3; c[$1]+=$3;} END {for(i in t) print i, t[i]/c[i];}' $file;
done

MS2005	Illumina	Nanopore
genome	587.654	732.671
tig00000001:1-1748626_pilon	571.423	736.11
tig00000002:1-5175_pilon	6746.58	404.858
tig00000005_pilon	0.0600298	15.5255
MS2058	Illumina	Nanopore
genome	885.409	465.478
tig00000002:1-40652_pilon	1085.76	231.062
tig00000006:1-1750266_pilon	889.547	472.265
tig00000007_pilon	520.681	409.633
MS2074	Illumina	Nanopore
genome	986.434	690.612
tig00000001:1-1621348_pilon	986.434	690.612
MS2169	Illumina	Nanopore
genome	1007.31	950.819
tig00000001:1-1702611_pilon	1007.31	950.819

Annotation

Prokka

ROOT=$(git rev-parse --show-toplevel)
DIR=$ROOT/2-annotation
cd $DIR


ml singularity
prokka () { singularity exec -B $ROOT $ROOT/apps/prokka-v1.13.sif prokka $@;}

for name in "${names[@]]}"; do
    assembly=$ROOT/1-assembly/5-pilon/$name/pilon.fasta

    prokka --cpus 12 \
        --outdir $DIR/1-prokka/$name \
        --prefix $name \
        --locustag $name \
        $assembly
done

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Assembly and Annotation of Four C. jejuni Strains

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Assembly and Annotation of Four C. jejuni Strains

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