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Hello, in this presentation, we will be

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looking at genomics in livestock and in

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particular sequencing. We will use the

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example of the laboratory process of

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Sanger sequencing on an automatic

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sequencer. This presentation is part

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of module 1, Animal Genetics. The

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creation of this presentation was

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supported by an Erasmus+

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KA2 grant within the ISAGREED

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project, Innovating the content and

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structure of study programmes in the

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field of animal genetics and food

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resources management using

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digitization.

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The actual laboratory procedure of Sanger

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sequencing involves several steps.

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The first is the preparation of a suitable

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sample, i.e. DNA. The

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sample can be DNA cloned in a vector

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and obtained by isolation from bacterial

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cells. Universal primers are

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used for sequencing and therefore, the

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result is usually of high quality.

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However, cloned DNA is not

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always available. More often, we use

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the PCR product as template.

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Here, the purity of the PCR product is

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critical (the PCR product must be

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purified), as well as

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its quantity. If we have a good quality

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sample, we can proceed to the second

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step, the actual sequencing reaction.

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This is an enzymatic reaction similar to

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PCR, but only one primer

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and commercially available reaction

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mixture containing specific fluorescently

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labeled ddNTPs (each

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base a different color), colled

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terminators, must be used.

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The result is a mixture of fragments,

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each terminated by a terminator, which

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indicated by its collour the base at

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the corresponding site in the sequence.

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For a detailed description of the method,

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see the lecture on this topic.

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This is followed by removal of the

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free nucleotides

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and precise capillary electrophoresis.

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The final step is the software evaluation

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of the data and assembly of the

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final sequence.

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One option for removing free

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nucleotides, especially free clolor-labeled

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terminators, is to use an

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emulsion of a porous reagent that

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absorbs these nucleotides and thus

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purifies the solution. The reagent

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must be thoroughly remixed by vortexin

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before adding it to the sequencing

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mixture and than removed with a sheared

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tip.

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To do this, the sequencing mixture and

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emulsion should be allowed to shake for

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30 minutes.

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After centrifugation, the

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supernatant is removed into a sequencing

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plate and is ready for analysis in

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the sequencer.

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The basis of the automatic sequencer is

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a fluorescence capillary ectophoresis.

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This picture shows the basic components

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of the instrument: the anode section

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with the polymer pump on the left, the

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detection chamber in the middle, the

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capillary array on the top right, and

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the sample plate and the cathode section on

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the bottom. Prior to each

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separation, the pump pushes fresh

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polymer into the capillaries, then the

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sample is electro-injected into the

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capillary, and separation proceeds

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from the cathode to the anode.

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The fluorescent signal is picked up in

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the detection section, with shorter DNA

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fragments arriving before longer

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ones.

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This picture shows a close-up of the

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pump, polymer bag, anode

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and anode buffer container.

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Glass capillaries filled with polymer are

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the basis of precise DNA

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separation. Polymer is a

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special gel with separation

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ability. The DNA travels through the

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capillary and separates according to

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size. Smaller fragments reach

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the window earlier than longer ones

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and the instrument reads the

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corresponding signal (fluorescent

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color).

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Insertion of fresh anode

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electrophoresis buffer must be done

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very carefully. You can see the

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block with the pump, which always fills

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the capillary with fresh polymer before the

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run. The bag on the right contains

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a separation polymer.

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Auto sampler extends and is ready to

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load the sample plate.

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Snap the plate retainer (cover)

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onto the plate, septa, and plate

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base.

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First, we insert a cartridge with cathode

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buffer (left side)

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and washing solution (right)

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and subsequently plate assembly.

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After closing, the sequencer will

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automatically move to the correct

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position.

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We can insert a maximum of two

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plates with samples, i.e. a total

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of 192

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samples.

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After the wasching step,

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the electrodes are immersed in the

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samples and electro-injection into the

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capillaries take place.

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Then the electrodes are placed in the

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cathode buffer and electrophoresis

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begins.

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The reading of the fluorescent signal

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takes place gradually as the relevant

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fragments turn to the sensor.

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After the electrophoresis is finished,

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we will check the raw data.

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The evaluation is carried out using

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sequencing analysis software. The

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color of the peak corresponds to the

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corresponding base in the sequence shown

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above. Here we also see

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an example of a heterozygote.

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It can be recognized by the fact that

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there are peaks of two different colors

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at the same position.

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Specific software can be used to compare

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the sequences of a large number of

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samples and thereby find

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differences: polymorphism.

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In the mentioned bee samples,

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there are two types of polymorphism:

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division on the left and single

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nucleotide on the right.

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Evaluation of this result allowed us to

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distinguish different mitochondrial

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DNA haplotypes in our bee

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population that are related to their

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origin.

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We can export a finished sequence

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in a suitable format for further use.

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For comparing finished sequences,

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e.g., the Clustal program is suitable.

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We can thus find disease-causing

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mutations, alleles influencing

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performance traits in farm animals,

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determine genetic diversity in

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animal populations, kinkship, and

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many other applications.

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And that's all for this short

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presentation explaining the basics of

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DNA sequence determination. I

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believe that the illustrative examples of

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this video will help you understand one

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of the basic laboratory methods

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without which modern genetics and

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genomics cannot be. Thank you for

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your attention.