This protocol describes the use of a flexible Snakemake software workflow for reproducible analysis of genome re-sequencing experiments. It confirms sample relationships from the raw sequencing reads with alignment-free methods (kwip and mash) and performs alignment, variant calling, and variant annotation against one or several reference genomes; read alignments with bwa and/or NextGenMap, variant calling with freebayes and/or bcftools mpileup. If a genome annotation is available, variants are annotated with snpEff.