Dear all, I have been using the pairwiseAlignment() function in the Biostrings library for creating global alignments. However, I noticed that the way it behaves is not quite like I expected. For example, I wanted to create a global alignment of sequences "HEIAKGKAL" and "HEIAKGKALIIIIEALKCLA" so I typed: > x1 <- "HEIAKGKAL" > y1 <- "HEIAKGKALIIIIEALKCLA" > pairwiseAlignment(x1,y1, substitutionMatrix = BLOSUM62, gapOpening = -1,gapExtension=-1) Global PairwiseAlignedFixedSubject (1 of 1) pattern: [1] HEIAKGKAL subject: [1] HEIAKGKAL score: 37 The output that I got seems to be a local alignment, as it doesn't contain the whole of the two input sequences. Therefore, I am wondering if this function is using the Needleman- Wunsch algorithm to make the global alignment, or not? It looks to me like this actually the result of the Smith-Waterman algorithm for local alignment. By the way, I still get the same result when I explicitly use the "type=global" option: > pairwiseAlignment(x1,y1, substitutionMatrix = BLOSUM62, gapOpening = -1 ,gapExtension=-1,type='global') Global PairwiseAlignedFixedSubject (1 of 1) pattern: [1] HEIAKGKAL subject: [1] HEIAKGKAL score: 37 Just to check that I am not going crazy, I tried aligning the same sequences using needle from the EMBOSS package (which does Needleman-Wunsch global alignments) at http://mobyle.pasteur.fr/cgi-bin/portal.py?form=needle with gapOpening and gapExtension penalties of -1, and the BLOSUM62 matrix. It gave the best global alignment as: x1 1 HEIAKGKAL------- 9 ||||||||| y1 1 HEIAKGKALEALKCLA 16 Score: 45.0 This is something like I would have expected pairwiseAlignment() to give me, ie. a global alignment spanning the full lengths of the two sequences. Please could you explain why pairwiseAlignment is not giving me a global alignment? I am very confused, and am wondering if I am being very stupid and have misunderstood something (probably!).. Regards, Avril Avril Coghlan Dept. Microbiology University College Cork Ireland