What is Sequlator?

Sequlator is a free multiple sequence alignment editor with a pairwise alignment function. Sequlator can be used merely as a tool for viewing and manipulating multiple alignments of nucleotide sequences, but the main purpose of Sequlator is to facilitate the assembly and analysis of sequences produced by sequencing of newly isolated recombinant plasmids or PCR fragments. During molecular cloning of gene transcripts or DNA fragments undesired spontaneous mutations may be introduced into recombinant vectors. Therefore, before further amplification and use of isolated plasmids in experiments, plasmid regions spanning the insert and ligation sites should be sequenced and checked for the presence of mutations. After samples have been processed by a capillary Sanger sequencing service, the sequencing results are delivered in FASTA format.

  • With Sequlator, the received sequencing reads, the expected vector or PCR template sequence, primer sequences, and restriction enzyme recognition sites can be easily assembled into an alignment.
  • In the resulting multiple sequence alignment, the overlapping sequencing reads can be further manipulated. You can delete irrelevant fragments of the alignment. You can change the order of the sequences, rename the sequences, add additional sequences, or remove flawed or dispensable sequences. Or, you can edit any individual sequence - delete fragments of poor quality or insert new bases or gaps.
  • Finally, any differences from the reference sequence can be revealed in the consensus, and the sequencing reads can be inspected for the presence of mutations.

If you are a molecular biologist routinely designing, constructing and sequencing vectors you might facilitate your work by using Sequlator. Similarly, Sequlator can be used for the analysis of sequences produced by bisulfite sequencing (also known as bisulphite) or gene walking.

Sequlator is written in Java and functions on all Windows (tested on Vista, 7, 8), Linux (tested on Ubuntu), Mac computers with installed Java (If you use the Internet you have it installed).

The two videos below demonstrate the main features of Sequlator software.

How to view and edit a multiple sequence alignment

The tutorial video below briefly demonstrates the Sequlator functions for the manipulation of multiple alignments saved in the most commonly used .msf (GCG multiple sequence format) format. With Sequlator several alignments of nucleotide sequences can be visualized and manipulated simulataneously in separate windows. As an example, an alignment of 26 related repetitive mobile elements from Arabidopsis genome was used. The length of the alignment is 10690 bases. The alignment was produced by ClustalW2 multiple alignment tool available on The European Bioinformatics Institute website. The alignment can be futher used for design of RT-PCR primers specific for transcripts of a unique template but insensitive to other members of the aligned retrotransposon family.

The Sequlator functions shown in the video:

In addition to watching, please try out manipulating the same retrotransposon alignment in an applet of Sequlator software.

How to align sequencing reads with the reference sequence

In addition to its multiple alignment editor functions, Sequlator has a function of pairwise local alignment of one sequence relative to another. This function can be very useful for aligning sequencing reads from samples of either plasmids or isolated PCR products with the expected template sequence. For example, if you reverse transcribed RNA and cloned a several kilobase-long cDNA into some protein expression vector, the next step would be to sequence the region including the insert and make sure that the plasmid is free of mutations. With Sequlator the sequencing results can be easily aligned with the reference sequence and mutation can be easily spotted.

The tutorial video below demonstrates how sequencing results of two independent clones of the same construct are manually aligned and compared to the expected vector sequence. A spontaneous mutation is detected in one of the clones, whereas the second clone appears to be free of mutations. The clones were produced by cloning 1.2kb insert into NheI and HindIII restriction sites of a vector. Each clone was sequenced with forward and reverse primers so that the produced sequences overlap. For the sake of simplicity of the video, all sequences, namely the predicted vector sequence, four sequencing reads, NheI and HindIII target sites were copied to a Notepad file in advance.

The video demonstrates the following useful Sequlator functions: