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Background  With the advent of Next-Generation Sequencing technologies (NGS), a large amount of short read data has been generated. If a reference genome is not available, the assembly of a template sequence is usually challenging because of repeats and the short length of reads. When NGS reads cannot be mapped onto a reference genome alignment-based methods are not applicable. However it is still possible to study the evolutionary relationship of unassembled genomes based on NGS data.    Results  We present a parameter-free alignment-free method, calledU  n  d  e    r    2¯, based on variable-length patterns, for the direct comparison of sets of NGS reads. We define a similarity measure using variable-length patterns, as well as reverses and reverse-complements, along with their statistical and syntactical properties. We evaluate several alignment-free statistics on the comparison of NGS reads coming from simulated and real genomes. In almost all simulations our methodU  n  d  e    r    2¯outperforms all other statistics. The performance gain becomes more evident when real genomes are used.    Conclusion  The new alignment-free statistic is highly successful in discriminating related genomes based on NGS reads data. In almost all experiments, it outperforms traditional alignment-free statistics that are based on fixed length patterns.

Assembly-free genome comparison based on next-generation sequencing reads and variable length patterns