Arking:JCAOligoTutorial6

Sequencing Analysis

So, you've made your basic or composite, and you think you've found a colony that contains the right product. You now want to confirm that it's right. You need to sequence to find out exactly what the sequence is of the thing in your tube. In general, sequencing is cheaper and better when outsourced to a company or core facility. So, you send out your sample, and they send you back a sequence file. In this part of the tutorial, we're going to go through how sequencing works and how to analyze the data they send you. Before we start, you will need ApE (A Plasmid Editor) for this. So, if you haven't already done so, download it from http://www.biology.utah.edu/jorgensen/wayned/ape/. Additionally, you will want to replace the feature annotation database within ApE with an updated version. To do this, find directory in which you installed ApE on your computer and locate the file "Default_Features.txt". On my computer, it's: C:\Program Files\ApE\Accessory Files\Features. Replace this file with This Version.

How sequencing works

What you really need to know

Sequencing starts with a sample plasmid DNA or PCR product and one DNA oligonucleotide. This is what you send to the sequencing facility. They do something called "cycle sequencing" to your sample, and email you back some data. You can expect between 400bp and 1000bp of "true" data that begins somewhere around 20-50 bp into the read. Where "good" and "bad" data starts and ends varies sample to sample, and we'll get into that later. The sequence you read corresponds to the region 3' of where your oligo anneals. So, you must pick the appropriate oligo to send the sequencers based on what region of the plasmid you are interested in.

Overview of the process

(From http:// www.biochem.arizona.edu/classes/bioc471/pages/Lecture21/AMG9.1a.gif)
So, the facility is going to run a reaction similar to a PCR on your sample and then take the products generated in that sample and load them into an instrument. The reaction is going to contain many little fragments of your sequencing, and the machine will separate them to single base pair resolution using capillary electrophoresis. Capillary electrophoresis is basically like the agarose gels we run in lab, but the gel is in a long narrow tube. It detects each little DNA as it comes off the column by fluorescence, and the spectrum of fluorescence (the "Chromatogram") can be interpretted by software as a string of A's, C's, T's and G's referred to as the "calls". They will send you both a text file of calls and a chormatogram file.

How the cycling reaction works

From http:// www.ejbiotechnology.info/content/vol1/issue1/full/3/bip/Fig1.gif The cycling reaction starts by denaturing your sample plasmid and annealing the oligo to its homolous sequence. The reaction contains essentially a PCR reaction (dNTPs, thermostable DNA polymerase, buffer), so the polymerase starts adding bases to the 3' end of the oligo. However, there are two additional components -- ddNTPs (dideoxynucleotides, in "A" of the figure) and dyes. The dye will make the synthesized products visible in the electrophoresis instrument. The ddNTPs are chain terminators. Because they lack a 3' hydroxyl group, whenever one of these gets incorporated into a growing DNA the synthesis cannot proceed further resulting in a truncated product. For each cycling reaction, one of the 4 ddNTPs is added. So, in the reaction with ddATP, the chains get termiated at every A, and so on for all 4 reactions. The "cycling" aspect of this is that the process of denaturing, annealing, and extending is repeated so that there is linear (but not exponential) amplification of the original plasmid template. If we were to load these cycling reactions on a normal gel like we have in lab, you'd see something like the gel at left (from http:// www.cambio.co.uk/images/html_images/sequitherm_cycle1.gif).

In practice, since the sample is run by capillary electrophoresis, you end up with a chromatogram plotting fluorescence intensity versus time like this:

(From http:// site.hylabs.co.il/upload/infocenter/info_images/08062004105243@Sequencing-quality-.gif)

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If you have any comments or want to report a potential error in the tutorial, please email me (Chris Anderson) at JCAnderson2167-at-gmail.com