The emerging genome-wide hairpin bisulfite sequencing (hairpin-BS-Seq) technique enables the determination of the methylation pattern for DNA double strands simultaneously. Compared with traditional bisulfite sequencing (BS-Seq) techniques, hairpin-BS-Seq can determine methylation fidelity and increase mapping efficiency. However, no computational tool has been designed for the analysis of hairpin-BS-Seq data yet. Here we present HBS-tools, a set of command line based tools for the preprocessing, mapping, methylation calling, and summarizing of genome-wide hairpin-BS-Seq data. It accepts paired-end hairpin-BS-Seq reads to recover the original (pre-bisulfite-converted) sequences using global alignment and then calls the methylation statuses for cytosines on both DNA strands after mapping the original sequences to the reference genome. After applying to hairpin-BS-Seq datasets, we found that HBS-tools have a reduced mapping time and improved mapping efficiency compared with state-of-the-art mapping tools. The HBS-tools source scripts, along with user guide and testing data, are freely available for download.Published version