APEC 211 & NMEC-O75 Workflow

Assemble Illumina data de novo

1. Trim reads using trimmomatic

   • Notes: do NOT use a SLURM script on SciNet
   • module load trimmomatic
   • java -jar /software/apps/trimmomatic/64/0.36/trimmomatic-0.36.jar PE AP211_R1_001.fastq.gz AP211_R2_001.fastq.gz AP211_R1_paired.fq.gz AP211_R1_unpaired.fq.gz AP211_R2_paired.fq.gz AP211_R2_unpaired.fq.gz ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 LEADING:6 TRAILING:6 SLIDINGWINDOW:4:15 MINLEN:50
   • Learn more: Trimmomatic, Next-Gen Sequence Analysis Tutorial

2. Examine trimming (FastQC)

   • module load fastqc
   • fastqc AP211_R*_paired.fq.gz
   • Download to view interactive HTML file
   • Examine any categories on the report, if red consider more-strict trimming by modifying step 1.
   • Learn more: FastQC

3. Assemble data (Spades)

   • module load spades
   • spades.py -1 AP211_R1_paired.fq.gz -2 AP211_R2_paired.fq.gz -o MiSeq_assembled_AP211 --careful --cov-cutoff auto
     • Here we are assembling the paired reads generated by trimmomatic
   • Learn more: Spades

4. Examine assembly (Quast)

   • module load quast
   • quast.py MiSeq_assembled_AP211/contigs.fasta
   • Download report.pdf to view report found in quast_results/results*
   • Learn more: Quast

Identify Plasmid Replicons

1. One useful program for identification of antibiotic, virulence, and plasmid replicons is ABRicate.

   module load abricate

   • Plasmid replicons: abricate --db plasmidfinder MiSeq_assembled_AP211/contigs.fasta >> ../abricate_output/plasFinder.csv

2. Download .csv files and open with Excel or R!

3. Learn more: ABRicate

Long-read data

1. Assemble fastq files (Canu):
   • module load canu or module load canu/gcc/64/1.5
   • canu -p AP211 -d AP211_output genomeSize=5.05m -pacbio-raw *.fastq
     • -p: prefix for files
     • -d: directory for output
     • genomeSize: provide an approximate genome size (using Illumina data)--yields a better assembly
     • pacbio-raw: reads are uncorrected PacBio reads
     • AP211_output/AP211.contigs.fasta: Assembly in a FASTA format

• Align Illumina assembly to PacBio assembly for "quick" verification.
• Learn more: Canu, Next-Gen Sequence Analysis Tutorial

Circularization

• Long-read assemblies and corrected long reads imported into Geneious
• Reads mapped to assemblies
• L.R. assemblies were circularized before they were polished with Pilon

Polish long-read data with short reads

• Load modules:

module load bwa
module load samtools
module load pilon
module load quast

• Index, Map, and Align:

bwa index AP211.contigs.fasta

bwa mem -t 32 AP211.contigs.fasta ../AP211_Illumina/AP211_R1_paired.fq.gz ../AP211_Illumina/AP211_R2_paired.fq.gz | samtools sort > aln.bam

samtools index aln.bam
samtools faidx AP211.contigs.fasta
samtools sort -O bam -T ./tmp -o aln.bam AP211.aln.sam

bwa mem AP211.contigs.fasta ../AP211_Illumina/AP211_R1_paired.fq.gz ../AP211_Illumina/AP211_R2_paired.fq.gz > AP211.aln.sam
samtools index aln.bam

• Run Pilon & Quast output:

java -jar /software/apps/pilon/64/1.18/pilon-1.18.jar --genome AP211.contigs.fasta --frags aln.bam --output pilonAP211.fasta --fix bases --changes --verbose

quast.py pilonAP211.fasta.fasta

Learn more: BWA, Samtools, Pilon, Quast,