Karlin-Altschul Sum statistics to the evaluation of multiple, gapped alignment scores in all BLAST search modes. Sum statistics were originally developed analytically for the evaluation of multiple, ungapped alignment scores. The empirical use of Sum statistics in the treatment of gapped alignment scores was validated in collaboration with Stephen Altschul, from 1994-1995. In May 1996, WU-BLAST version 2.0 with gapped alignments was publicly released in the form of a drop-in upgrade for existing users of ungapped NCBI BLAST and WU-BLAST (both at version 1.4, after having forked in 1994). Little NIH funding was received for his WU-BLAST development, with an average of 20% FTE starting in November 1995, and ending shortly after the September 1997 release of the NCBI gapped BLAST (“blastall”). As an option to WU-BLAST, Gish implemented a faster, more memory-efficient and more sensitive two-hit BLAST algorithm than was used by the NCBI software for many years. In 1999, Gish added support to WU-BLAST for the Extended Database Format (XDF), the first BLAST database format capable of accurately representing the entire draft sequence of the human genome in full-length chromosome sequence objects. This was also the first time any BLAST package introduced a new database format transparently to existing users, without abandoning support for prior formats, as a result of abstracting the database I/O functions away from the data analysis functions. WU-BLAST with XDF was the first BLAST suite to support indexed-retrieval of NCBI standard FASTA-format sequence identifiers (including the entire range of NCBI identifiers); the first to allow retrieval of individual sequences in part or in whole, natively, translated or reverse-complemented; and the first able to dump the entire contents of a BLAST database back into human-readable FASTA format. In 2000, unique support for reporting of links (consistent sets of HSPs; also called chains in some later software packages) was added, along with the ability for users to limit the distance between HSPs allowed in the same set to a biologically relevant length (e.g., the length of the expected longest intron in the species of interest) and with the distance limitation entering into the calculation of E-values. Between 2001-2003, Gish improved the speed of the DFA code used in WU-BLAST. Gish also proposed multiplexing query sequences to speed up BLAST searches by an order of magnitude or more (MPBLAST); implemented segmented sequences with internal sentinel bytes, in part to aid multiplexing with MPBLAST and in part to