As far as I know mafft is the only program where one can align short sequences to a big one. The command to do that is mafft --addfragments shorties big_one > result.

By default mafft will put gaps where it can not find a match. To prohibit this behaviour we can increase the penalties for gap opening and extension as this: mafft --op 10 --ep 10 --addfragments shorties big_one > result.

The defaults are:

  • –op # : Gap opening penalty, default: 1.53
  • –ep # : Offset (works like gap extension penalty), default: 0.0

Warning: I do not know what are the limits for –op and –ep. If both are set to 15, it works pretty well.