4 Download Workshop Data

In this workshop you will be working with isolate ERR531380, which was sequenced using the Illumina platform.

How was the strain isolated?

The P. aeruginosa strain was isolated from wound swabs by inoculation onto cysteine lactose electrolyte deficient agar (CLED) and cetrimide agar and incubation for 24 h at 37°C. Identification was confirmed by resistance to C-390 and the VITEK 2 GN identification card. For storage and DNA extraction a single colony was purified from the primary culture plate.

Isolates were stored on Biobank beads at −20°C prior to DNA extraction. Organisms were resuscitated on CLED agar plates and genome DNA either extracted directly using the MOBIO UltraClean Microbial DNA Kit, or from overnight LB broth culture using a Qiagen Genomic-Tip 100G.

How was the isolate sequenced?

Genomic DNA was prepared from single colony picks using the MOBIO Ultraclean microbial kit (MOBIO, Carlsbad, USA). 1 ng input DNA, as quantified by Qubit (Life Technologies, Carlsbad, USA) was used to prepare genomic libraries for sequencing using the Illumina Nextera XT DNA sample kit as per manufacturer’s protocol (Illumina, San Diego, USA). Libraries were sequenced on the Illumina MiSeq using a paired-end protocol resulting in read lengths between 150 and 300 bases.

Download the workshop data

Create a new folder on your computer desktop called BM327_Workshop
Download the files from the sections below into this new folder.

4.1 Sequencing reads for your isolate

Illumina paired-end reads, forward direction(ERR531380_1.fastq.gz)
Illumina paired-end reads, reverse direction(ERR531380_2.fastq.gz)

4.2 Pseudomonas aeruginosa reference strain for SNP comparison

You will be using the PAO1 strain of P. aeruginosa (Stover et al. (2000)) for some comparisons. The genome sequence with accession GCF_000006765.1 was downloaded from NCBI.

P. aeruginosa PA01 genome sequence (.fasta)(GCF_000006765.1_ASM676v1_genomic.fna)

4.3 Room 1 isolate assembly for SNP comparison

The isolate with accession ERR531413, sampled from the shower in room 1 of the burns unit, is also used as a reference genome for some comparisons.

SPAdes assembly of isolate 1031 (ERR531413), from the room 1 shower hose(ERR531413_asm.fasta)

Your Working Directory

When you have downloaded all the files required for the workshop, the contents of the BM327_Workshop folder should resemble the image in Figure 4.1.

macOS folder called BM327_Workshop, containing the downloaded files required for this workshop. — Figure 4.1: macOS `BM327_Workshop` folder with all files downloaded.