Working From Home – Comparing sequences

During the Covid-19 pandemic we want to ensure that you have access to the MacVector license that you would use in the lab, especially if you are working from home. If you use MacVector, even an older version, and are having trouble activating it (or installing it) at home email MacVector Support and we will help. If you are in anyway connected with COVID19 research, then please have our thanks, and have an annual license free of charge.

For the next few weekly tips we want to help familiarize you with the wide range of functionality in MacVector. You may have just been using MacVector to design PCR primers, but since you do not have a PCR machine on the kitchen table, then here’s what else MacVector can do for you. This week we’ll cover different types of alignments and what you would use them for:

Multiple Sequence Alignments

The tool to which most people first turn to for aligning sequences is the Multiple Sequence Alignment tool. This allows you to align multiple DNA or protein sequences using either Muscle, Clustalw or T-Coffee. This functionality is most suited for protein alignments, or for nucleic acid sequences where you are interested in examining phylogenetic relationships. Remember that you can also align DNA sequences based on their protein translations.

  • FILE > NEW > PROTEIN ALIGNMENT
  • EDIT > ADD SEQUENCES FROM FILE..
  • Click on the Align toolbar button

Confirming a small sequencing project against a reference.

At some point all molecular biologists have to verify that a sub cloning procedure has worked or a new construct is correct. The Align to Reference tool is perfect for quickly verifying a set of sequencing reads against a reference

  • Go to ANALYZE > ALIGN TO REFERENCE
  • Add your reads and click ALIGN

Aligning cDNA/mRNA sequences against a genomic template.

Align to Reference can also be used to align cDNA clones against a genome sequence. The steps are similar – use the genomic sequence as the reference, then add one or more cDNA clones to the alignment.

  • Go to ANALYZE > ALIGN TO REFERENCE
  • Add your reads
  • Click ALIGN with the algorithm set to cDNA ALIGNMENT

Using a Dot Plot to quickly compare two sequences.

Dot Plot is great for identifying weak regions of similarity between two sequences. Dot Plots are also the best way of identifying sequence rearrangements

  • Open the two sequences to compare.
  • ANALYZE > CREATE DOT PLOT

Comparing two genomes.

Compare Genomes will compare two related annotated genomes (or smaller sequences) to identify and list, identical, similar and weakly similar features along with missing features.

  • Open the two sequences to compare.
  • ANALYZE > COMPARE GENOMES

Internet BLAST

Use this to identify and align a sequence to the databases at the NCBI using the BLAST algorithm. You can download any hits directly to your Desktop including all annotations.

  • Open your sequence
  • DATABASE > INTERNET BLAST SEARCH
  • Choose the database and click OK
  • To retrieve hits select the hits and click TO DESKTOP
  • To download and save hits, select hits and click TO DISK

Aligning a sequence against a folder of local sequences

Align to Folder allows you to scan a local folder full of sequences and align them using the FastA alignment algorithm. Its kind of like a local BLAST, but more sensitive.

  • Open your Sequence.
  • Run DATABASE > ALIGN TO FOLDER
  • Choose a folder of sequences and click ALIGN

Sequence Assembly

This requires our optional Assembler add-on. Use this if you want to align ten or more DNA sequences with the idea of assembling them into a longer sequence with a consensus (de novo) or for aligning reads against a reference (resequencing).

  • Go to FILE > NEW > ASSEMBLY PROJECT
  • Add your sequencing reads and also any reference sequences.

Primer Database

Primer Database.lets you automatically map your primer collection to any sequence you open. Primers can have tails and mismatches.

  • Go to ANALYZE > PRIMER DATABASE SEARCH

or

  • Go to MACVECTOR > PREFERENCES > SCAN DNA FOR.. PRIMER DATABASE
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