Figure 3. Comparison of PCR-free kits for the preparation of Illumina-compatible sequencing libraries. Libraries were prepared in triplicate from 200 ng of fragmented PhiX174 DNA (average fragment size 289 bp) using three different PCR-free kits: KAPA HyperPrep (KAPA), NxSeq AmpFREE Low DNA (NxSeq) and NEBNext Ultra II (NEBNext).(a) Size distribution profiles of DNA libraries. The quality of DNA libraries was determined on Agilent Bioanalyzer 2100 instrument. One representative electrophoregram per kit is shown (KAPA in blue, NxSeq in green and NEBNext in red). The black arrow indicates the localization of free adapter dimers. (b) Concentration of adapter-ligated fragments determined by qPCR KAPA. The concentration of DNA fragments that are ligated with two adapters was determined by qPCR KAPA, after the first (post-purification 1) and the second (post-purification 2) SPRI beads purification step. The concentration obtained in a total volume of 50 µL after the first purification step was normalized to the final volume of the libraries (20 µL). Bars represent the mean concentration ± standard deviation of libraries from 3 replicates. Concentrations obtained were compared by a one-tailed Mann-Whitney test. ns, p > 0.05; *, p ≤ 0.05.