SPRI beads in a polyethylene glycol (PEG) solution, such as AMPure XP, preferentially bind longer DNA fragments, making them useful for removing free primers and primer dimers from reactions. As the PEG concentration decreases, a wider range of short fragments can no longer bind to the beads, and primer dimers are more completely eliminated. However, if the PEG concentration is too low, the range of fragment sizes eliminated could include DNA of interest. For hamPCR, it is important that the SPRI cleanup does not affect the ratio of the host and microbial amplicons, which could lead to systematic bias and noise. To determine an acceptable PEG concentration, we tested cleanups with SPRI (AMPure XP) beads at different SPRI: DNA ratios (resulting in a range of PEG concentrations) on a standard DNA size ladder (GeneRuler DNA Ladder Mix, Thermo Scientific, Waltham, MA, USA), and quantified abundance of the purified fragments with a Bioanalyzer (Agilent, Santa Clara, CA, USA). Using the pure, uncleaned ladder, we calculated the ratio of each peak’s abundance to the adjacent larger peak (200: 300 bp, 300: 400 bp, etc.), and used this set of abundance ratios as a baseline (no SPRI, far left). With each successive decrease in SPRI: DNA ratio (from left to right), we looked for a decrease in the abundance ratio between adjacent bands, which would indicate elimination of the smaller fragment. Of highest interest is the 300: 400 bp ratio (black), because the smallest tagged templates in hamPCR are around 300 bp. We determined SPRI: DNA ratios less than 1.0 endangered the 300: 400 ratio, and thus decided to conservatively use a SPRI: DNA solution of 1.1: 1 or higher for the cleanup of tagged products.