Use Mauve. Mauve is MegAlign Pro’s only genome-level aligner and only algorithm capable of producing a multi-block alignment or an alignment when one or more of the sequences are rearranged relative to one another. Mauve uses MUSCLE to create multiple alignments for each block that contains more than a single sequence. The main disadvantage with Mauve is that its fine-scale gapping not as good as MegAlign Pro’s four gene-level alignment methods described below.

Mauve was originally developed by Aaron Darling, Bob Mau, and Nicole Perna in 2010 at the Genome Evolution Laboratory at the University of Wisconsin-Madison.

Links:

• MegAlign Pro tutorial “Genomic alignment with Mauve”
• MAUVE website

I have fewer than fifty “short” (< 1kb) DNA, RNA, or protein sequences

Start with ClustalW. The Clustal W alignment algorithm is faster than Clustal Omega (reference), though its maximum accuracy is only obtained if you select the default “Slow-Accurate” option in MegAlign Pro’s ClustalW settings panel. A disadvantage to this method is that it does not always handle end gaps ideally. ClustalW was developed by JD Thompson et al. in 1994 at the European Molecular Biology Laboratory, Heidelberg, Germany.

Links:

• ClustalW website

I need to do a gene-level alignment of up to 30,000 DNA, RNA, or protein sequences

Start with MAFFT. The MAFFT alignment algorithm is based on Fourier transformation and has several editable options. At least one paper found it to be the most accurate of the four gene-level algorithms (reference). Another found that it gave “structurally consistent alignments” for RNA data specifically (reference). When using long sequences, the algorithm performs best if the sequences are closely related. MAFFT was developed by Katoh M & Kumar M (2002) at the Computational Biology Research Center.

Links:

• MAFFT website

I need to do a gene-level alignment of up to thousands of taxa and have DNA, RNA, or protein sequences