PipMaker: input and output files. Files for submission to PipMaker include Sequences (required), Repeats (recommended), Underlay (optional), and Exon annotations (optional). The Repeats file is made by simplifying RepeatMasker output using the program rmask2repeats (from the PipTools program package). The simplified version is shown. The coordinates in the Repeats file and Underlay file correspond to the coordinates in the Pip plot. PipMaker generates three multiple output files. The Pip plot shown is a subregion of the human ST7interval compared with the orthologous baboon, cow, mouse, or fugu sequences. Each panel represents a pairwise comparison between human sequence and that of the indicated species. Each alignment consists of a series of horizontal lines that represent the gap-free aligning segments that are graphed on a vertical scale of 50%–100% and relative to the coordinates in the human sequence on the horizontal axis. Icons across the top panel represent annotations for the reference human sequence and include triangles for various repeats and rectangles for exons and CpG islands. The names of gene (ST7) and direction of transcription are indicated above the alignments. The Nucleotide-Level View shows the multiple alignment of the multispecies comparison at the nucleotide level. Dots represent nucleotides that are the same. Dashes represent gaps. The interval shown includes ST7 exon sequences, and thus some nucleotides are conserved between humans and fugu. The Concise output gives a coordinate-based format of the aligning segments. The gap-free interval in the first sequence and the corresponding interval in the second sequence are listed, along with the percent identity and length. The program strong-hits reads the concise file format and returns alignments that fulfill user-specified thresholds (length of the alignment and the percent identity).