Wednesday, 7 June 2017

The joys of recieving DNA sequences.

by Nicholas Galuszynski

The struggle is real! Image courtesy Sketching Science.
So you've done it!
After hours (that felt like years) battling with DNA extraction methods and PCR protocols you have finally received the sacred e-mail, "please see attached sequences..." and it all starts seeming worthwhile, maybe.
But now what, you've done the lab work but what are you supposed to do with these .ab1 files? What are .ab1 files any way? I WANT MY SEQUENCES!!!

So firstly, .ab1 files are are the output format for raw DNA data produced by Applied Biosystems' Sequencing Analysis Software. These files contain an electropherogram as well as the DNA base sequence, which can be viewed using a DNA viewer program. Secondly, there are many ways of opening these files, however, freely available software options for handling your .ab1 files, normally have limited capabilities compared to their expensive counterparts such as CodonCode Aligner

So I've recently been using the trial version of Codon to check out some Cyclopia sequences, and damn this program is easy to use once you know where and what the different functions do. After checking out a few tutorial video on YouTube, I was able to do the basics such as import my sequences, create contigs based on names and visually inspect and correct these contigs. If you are starting out with Codon, watch those videos! From that base you can start trouble shooting and finding your own ways to do things, which is made much easier with the wide range to tutorials available from the manufacturers.

Oddly enough, one of the most useful features of the software, comparing all the contigs you've assembled, needs to be added to the toolbar. But this is easily explained by CodonCode. Before you get too into creating contigs and aligning them all to each other, it is often really useful to run a base call on your freshly relieved sequences. This can improve the quality of your sequence, resulting in better alignment of the forward and reverse sequence. I tended to have a lot of strange indels pop up in the sequences for a cDNA region, possible due to sequencing errors in highly repetitive A and T rich sections. These made contig comparisons difficult and I ended up selecting the best quality sequence as a reference sequence (make a reference seq by selecting the sample and pressing ctrl+alt+m), I then selected the Clustal-Omega method of aligning the contigs as apparently this option handles these gaps a bit better than Muscle that tends to delete them. But maybe deleting them isn't the worst option if all you're interested in is detecting SNPs and if that is your desire, you can then use Codon to find primer sites around those SNPs without even having to open another program. That is however, until the end of the 30 day trial after which you'll have to buy a licence or like reinstall windows or something.

So CodonCode is a pretty useful tool for the molecular biologist, offering a lot of functionality in one neat package. This however, comes at a price so make the best use of your trial and check out the helpful link's in this post before you activate your copy. 

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