MULTIPLE SEQUENCE ALIGNMENT SOFTWARE

Performing a multiple sequence alignment in MegAlign Pro is easy.

1. From the Welcome screen, choose New Alignment.
2. Select the sequences you wish to align.

MegAlign Pro will perform the alignment using the alignment method and parameters determined to be the most appropriate for your data.

Alternatively,

1. From the Welcome screen, choose New Alignment with options.
2. Select the sequences you wish to align.
3. Specify the alignment algorithm, change other settings as desired, then click Align to begin the alignment.

The short answer is that you should not use MegAlign Pro to align ABI/trace or other raw read files, but should instead assemble the reads using SeqMan Ultra or SeqMan NGen.

The terms sequence alignment and sequence assembly mean very different things but are often used interchangeably. This can lead to confusion for many researchers. Below, we define each term and which Lasergene application to use for that workflow.

MegAlign Pro is sequence alignment software. This type of software is used to check sequence similarity or evolutionary relatedness between two or more organisms or strains. Examples of sequences that you might want to align would include a) sequences from different strains of the same virus, with the goal of seeing how the virus is mutating over time, and b) histone gene sequences from different species of plants and animals, with the goal of creating a phylogenetic tree showing their evolutionary relationships.

Common sequence types used for sequence alignment are .fasta and .genbank.

Some multiple sequence alignment software applications, including MegAlign Pro, allow you to specify a reference sequence to use in the alignment. In this case, the reference is treated as the “standard” sequence that other sequences will be compared to. Residues in other sequences can be colored/displayed/hidden to show visually which residues match (or do not match) the reference at each position.

By contrast, sequence assembly software is used to create longer consensus sequences from short fragments of a single sample of DNA, commonly known as “sequencing reads” or just “reads.”

Common sequence types used for sequence assembly include .fasta, .fas, .fastq, and .abi. Within Lasergene, SeqMan Ultra is used to assemble .abi trace data files, while SeqMan NGen is used to assemble all other file types, including sequences produced using Illumina, PacBio and Oxford Nanopore (ONT) sequencing technologies. MegAlign Pro is not used for any type of sequence assembly.

As with sequence alignment, a reference sequence can also be specified in sequence assembly. But in this situation, the reference is used as a template or scaffold, allowing DNA fragments to be assembled into larger contigs faster and more accurately than assembling them de novo.

After performing an assembly, MegAlign Pro can calculate variants between other sequences and the currently-specified reference sequence, or between all sequences and the consensus.

Details about the variants in your alignment, including position, feature, impact, and translation change, can be evaluated in the Variants table, which you can access by clicking the Variants tab or by choosing View > Variants > Show.

Lasergene’s sequence alignment software, MegAlign Pro, supports multiple ways to modify an alignment. You can:

• Trim ends at the beginning or end of an alignment.
• Realign a range of aligned sequences using different parameters or a different alignment engine.
• Merge two existing completed alignments together.
• Add all/selected sequences from the “Unaligned Sequences” area into the current alignment, while retaining all of the gaps of the original, pre-merge alignment.
• Realign existing aligned sequences plus selected unaligned sequences

MegAlign Pro’s feature mapping (Features > Map Features) commands let you map a single annotation or all annotations from a source sequence to a target sequence. The sequences involved must have been previously aligned. During the process, you may optionally filter annotations so as to include/exclude specific gene types. Feature mapping is most commonly used to map desired annotations from a “completely annotated” sequence to a closely-related but “incompletely annotated” one.

Sequences that consist of more than one chromosome, contig, or fragment are called multi-segment sequences. When you enter a group of sequences into MegAlign Pro using the File > Add Sequences End-to-end command instead of the standard File > Add Sequences command, the entire data set will be concatenated end-to-end and treated as a single multi-segment sequence.

To import gapped sequences from an existing MegAlign Pro file, use File > Open and choose the .msa file. Once the project is open, select all the sequences, then use Align > Unalign Selected.

To import gapped nucleotide sequences from another type of file, use SeqMan Pro to remove the gaps. Launch SeqMan Pro, then drag and drop the gapped sequence file(s) onto the SeqMan Pro window. Select all sequences, then choose Contig > Export Sequences > Single File. Select either FastA or GenBank Flat File format and choose a file name and location. Make sure that Include gaps is left unchecked, and then click Save. The sequence(s) in the newly saved file can now be added to MegAlign Pro in the usual way.

For gene-level alignment of either protein or nucleotide sequences, use  Clustal Omega, Clustal W, or MAFFT. They offer editable options for speed, capacity, algorithm, etc., and are the only methods available for “profile” alignments. The disadvantages to gene-level aligners is that sequences must be on same strand, and that large rearrangements (e.g., inversions, translocations) are not allowed. For genome-level alignment of nucleotide sequences, use Mauve. Mauve has high capacity and uses MUSCLE to create multiple alignments for each block that contains more than a single sequence.