Schematic overview of steps in genotyping-by-sequencing (GBS) library construction, sequencing, and analysis. (1) Genomic DNA is quantified using fluorescence-based method. (2) Genomic DNA (gDNA) is normalized in a new plate. Normalization is needed to ensure equal representation of all samples and equal molarity of gDNA and adapters. (3) A master mix with restriction enzyme(s) and buffer is added to the plate and incubated. (4) The DNA barcoded adapters are added along with ligase and ligation buffers. (5) Samples are pooled and cleaned. (6) The GBS library is polymerase chain reaction (PCR) amplified. (7) The amplified library is cleaned and evaluated on a capillary sizing system. (8) Libraries are sequenced. Data analysis: Following a sequencing run, FASTQ files containing raw data from the run are used to parse sequencing reads to samples using the DNA barcode sequence. Once assigned to individual samples, the reads are aligned to a reference genome. In the case of species without a complete reference genomic sequence, reads are internally aligned (alignment of all sequence reads will all other reads from that library) and single nucleotide polymorphisms (SNPs) identified from 1 or 2 bp sequence mismatch. Various filtering algorithms can then be used to distinguish true biallelic SNPs from sequencing errors.