Preparation of Phage Illumina Sequencing Library

Phage sequencing library preparation

Luke Wheeler

This protocol outlines how to isolate phage genomic ssDNA from amplified phage pools and use a two step PCR system to build a barcoded DNA library for high-throughput sequencing. The details of this protocol are based on the barcoded primers that we have available from the UO genomics core facility. 

1. Use the Qiagen M13 spin kit to isolate phage genomic DNA from amplified, purified phage. This procedure can be done directly on phage glycerol stocks. Alternatively, DNA can be extracted using the protocol outline in the NEB PhD kit manual. Elute the DNA in either ddH2O or the elution buffer that comes with the Qiagen kit.
2. Design “primary PCR” primers to amplify the randomized region from the isolated phage DNA. The primers should anneal to regions that flank the region of interest. For example: to amplify the randomized 12-mer (36bp) region of the NEB PhD-12 kit library I designed a forward primer that anneals immediately upstream of the randomized region and a reverse primer that uses the same annealing region as the kit 96seq       sequencing primer (~96 bases downstream of the randomized region). These primers must also contain flanks that will generate annealing templates for the barcoded primers used in the secondary PCR.
3. Amplify the region of interest from isolated phage ssDNA using the primary PCR primers. I do 25uL reactions using Q5 high-fidelity polymerase (NEB: M0491L) with the included high-GC enhancer (reaction specifications and thermal cycler settings will vary depending on the primers, polymerase used, etc.). Purify the amplified PCR product by either gel extraction or a PCR cleanup kit (i.e. Qiagen). This purified product will be the template for the secondary PCR. 
4. Use barcoded primers that contain Illumina adapter regions to amplify from the product of the first PCR. Assign a uniqe pair of barcodes to each sample that is going to be included. If many samples will be submitted in one lane, then they can be multiplexed and later identified based on the unique barcode assignments. These primers must anneal to the flanks introduced by the primary PCR (I have designed the specifics of my pipeline around primers that are available to me via the UO genomics core facility). Again, I use Q5 high-fidelity polymerase with the included high-GC enhancer. Purify the desired product of the secondary PCR by agarose gel extraction ( I use a higher-than-normal ~1.8% agarose gel to ensure tight bands and excellent separation of the desired product from). Elute the PCR products in ddH2O.
5. Pool together samples at a desired ratio. Dilute the multiplexed library (need a 50uL sample @ ~10nM) for sequencing. Along with sample, submit ~10uL of 100uM forward primer for sequencing (Illumina NextSeq Hi output single-end reads)