"DNADynamo is by far and away the best and quickest software for checking sequences"- Professor Greg Towers, University College London
A guide to basic DNA Dynamo sequence analysis functions.
So you've sent off some DNA to be sequenced and received back some .abi or .scf files containing the trace data and base calls. Now you
want to analyse your sequencing data. DNA Dynamo can help you accomplish this quickly and efficiently, without the need to spend hours
analysing each separate data trace manually. By auto assembling translated multiple alignments of your sequencing data, where each
base in the alignment is linked to it's chromatogram peak, and presenting an interactive graphic map of sequencing discrepancies, open
reading frames and features maps, as well as interfacing with NCBIs Blast and VecScreen services, you can get a handle on your
sequence data within minutes. Intuitive sequence editing buttons allow you to manually edit your chromatogram trace base calls and then
save the edited and assembled sequence for future reference. By spending a few minutes working out how to use DNA Dynamo for
sequence analysis you will save yourself a vast amount of time and effort in the future.
DNA Dynamo works in two modes for sequence assembly, depending on whether you know what the expected sequence should be (eg
sequencing a construct after subcloning or confirming the result of a site directed mutagenesis experiment) or the sequence is unknown
(eg sequencing inserts from a two-hybrid screen, degenerate PCR or other similar library screens).
A) Guide/Reference Mode - to confirm your sequence against the expected result or find
For example, you have just PCR sub-cloned Your Favorite Gene (YFG) into a vector, and you need to confirm that no errors have been
introduced during the PCR reaction, so you have sent of a few minipreps to your sequencing service together with sequencing primers to
sequence in from both ends, and received back a bunch of .abi files.
1) open a DNA Dynamo file that contains the wildtype sequence of YFG in the main sequence window. From the 'Sequencing' menu,
select 'Open And Align Sequence Data Files to this Windows Sequence', and then select the .abi files that represent the sequence of one
clone. DNA Dynamo will align the sequences in the .abi files to the sequence of the 'guide' dna in the main sequence window, and display
the aligned sequences in a sequencing editor window. The result of such an alignment, where two .abi files were aligned against expected
sequence, is shown below. Note that DNA Dynamo automatically determines whether the sequence in the abi files is better aligned as
forward or reverse - and this is indicated on the graphics map by arrows - though these are not visible at this resolution.
Discrepancies between the expected sequence and the abi sequence files are highlighted on the graphic map. In the example
shown above, a patch of bad sequence is indicated with an arrow. Clicking on this section of the graphic map displays the
corresponding sequence and chromatogram trace in the display areas. It can be seen in the illustration below that the area of
'bad sequence' is due to the fall off of sequencing quality at the end of the read, as opposed to 'real' errors in the sequence.
Such ends of bad sequence can easily be removed using the 'Trim' buttons on the sequence editing panel.
By placing the cursor on the right hand side of the area of low sequence quality, and pressing the '<Trim' button, the
sequence to the left of the cursor is removed from the alignment. Note that the characters '<' and '>' present on the
editing buttons indicate on which side of the cursor the editing effect will take place.
After removing both sections of bad sequence, it can bee seen that the sequencing data in the .abi files corresponds
exactly to the expected 'guide' sequence.
Another useful feature of the sequence editor is the ability to highlight oligo hits or annotations on the 'guide' sequence. This
can be very useful for example when determining if a site directed mutagenesis reaction has been successful, as you can
compare the sequence in your .abi file directly to both the guide sequence and sequence of the mutagenesis oligo
B) Consensus Mode - assembling unknown sequences.
To open an abi file without aligning the sequence against a guide, simply start from a DNADynamo window that does not
contain any sequence in its main sequence window. You may open a single file simply by using the 'Open' option in the 'File'
menu. Alternatively you can open multiple files using the 'Import and Align Multiple Files' option under the 'Sequencing'
menu. When multiple files are opened, DNA Dynamo attempts to assemble a continuous stretch of sequence (contig) from
the separate sequences. The consensus of the assembled sequences is shown together with each individual sequence and
trace. In the example shown below, 5 .abi files were assembled to a consensus, and an Open Reading Frame found and
set. Stretches of sequence from the consensus can be sent directly to NCBI for Blast analysis. Note that DNA Dynamo is
suitable for small scale sequencing projects and is not intended for the assembly of large stretches of sequence.
As a point of interest, the first previously unknown sequence that was assembled and published using DNADynamo's
consensus mode sequence assembly functions was the African Green Monkey TRIM5alpha gene (Lv1 retroviral
restriction factor activity) in Greg Towers Lab - PNAS
DNA Dynamo contains many more functions. See the help pages for more advice, or feel free to use the 'Ask a Question'
option in the File menu, or email firstname.lastname@example.org directly to ask any questions you may have about
DNADynamo functionality. We will get back to you as soon as we can.
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