Sequencing CRISPR Array from Phage-infected Plasmids

Plasmid DNA was extracted from liquid cultures after phage infection experiments (see protocols above) and used as a template for Phusion PCR to amplify the CRISPR array. After bands of the CRISPR array were extracted and purified from a 2% agarose gel, samples were prepared for sequencing with the TrueSeq Nano DNA Library Prep protocol (Illumina) and subject to Illumina MiSeq sequencing. Data analysis was performed in Python, similar to that described previously (33 (link)). In short, the spacer sequences were extracted and the number of reads for each spacer was recorded from the raw Illumina FASTQ files. For each unique spacer sequence matching the ФNM4γ4 genome, various characteristics were determined and recorded, including the spacer's frequency within the sequencing population (number of reads of that spacer compared to total spacer reads sequenced), the position of its protospacer in the ФNM4γ4 genome, the strand of the genome on which the protospacer is found, and the PAM sequence flanking the protospacer (see Supplementary File 1).