Genome sequencing

Presentation contains reqiured detail for understanding of topic with related diagrams

1. G e n o m e s e q u e ncing P r e s e n t e d b y , Aakifah Amreen H . E 4th sem MSc (BT)

2. C o n t e n t I ntroduction R e a s o n M e t h o d s of s e q u e ncing Sangers s e q u e ncing Automated s e q u encing A B E R a t i o n a l e R e f e r e nce

3. Introduction Genome sequencing refers to sequencing the entire genome of an organism, instead of sequencing it gene by gene. I nvolves revealing the order of bases present in the entire genome of an organism Sequencing the entire genome will provide a wealth of data which can be studied completely Studying the functions of genes one by one is time-consuming, it leads to redundant work by many scientists, slow progress, and only incomplete information is obtained. Due to the advances in DNA sequencing technology, genome sequencing has been made possible

4. W h y s h o u l d w e d o g e n o me s e q u e ncing ? Genes do not work alone in cells; genome sequencing helps to understand the functions of all the genes which are present in the cells. Gene function is not directly controlled by the promoter alone; it i s controlled by many other regulatory elements such as the response elements, the enhancers, the silencers, etc. Whole genome sequencing gives information about the related DNA elements involved in gene expression.

5. The presence of vast amounts of non-coding DNA in the eukaryotic genome without any known function . Genome sequencing and its characterization will provide some clue to the function of non-coding DNA. The genomes of individuals of a single species are similar but not identical, in the case of humans, about 2 per cent of the genomic DNA sequence varies among individuals.

6. M e t h o d s of G e n o m e S e q u e n c i n g F l o u r e s cen ce s e q u encing A u t o m a t e d s e q u encing Shot g u n s e q u encing C l o n e b y c l o n e s e q u encing

7. Sangers s e q u encing Sanger sequencing was developed by the British biochemist Fred Sanger and his colleagues in 1977. Regions of DNA up to about 900 base pairs in length are routinely sequenced P r i n c i p l e when given enough time and enough starting material, at least one DNA sequence of every possible length will be produced with a tagged nucleotide at the end. The tagged nucleotide will always terminate the sequence because dideoxynucleotides are missing the 3'-OH group that is necessary to continue the chain

8. Require d c o m p o n e nts A DNA polymerase enzyme A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts as a "starter" for the polymerase The four DNA nucleotides (dATP, dTTP, dCTP, dGTP) The template DNA to be sequenced Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of dye

9. P r o c e d u r e The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides. The mixture is first heated to denature the template DNA , then cooled so that the primer can bind to the single-stranded template. Once the primer has bound, the temperature is raised again, allowing DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide.

10. This process is repeated in a number of cycles. By the time the cycling is complete, it’s virtually guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the target DNA in at least one reaction. That is, the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA The ends of the fragments will be labeled with dyes that indicate their final nucleotide. fragments run through a long, thin tube containing a gel matrix in a process called capillary gel electrophoresis. Short fragments move quickly through the pores of the gel, while long fragments move more slowly. As each fragment crosses the “finish line” at the end of the tube, it’s illuminated by a laser, allowing the attached dye to be detected.

11. A u t o mated s e q u encing The 1970’s technologies of Sanger’s chain termination, and Maxim and Gilbert’s chain degradation, were improved upon and automated with ABI’s fully automated sequencing machine in 1987. Since that time sequencing has gotten faster and cheaper; next generation sequencing has taken it to the level of complete genome sequencing in a matter of hours.

12. A u t o m a t ed s e q u e ncing Automated DNA sequencing instruments (DNA sequencers) can sequence upto 384 DNA samples in a single batch (run) in up to 24 runs a day. DNA sequencers carry out capillary electrophoresis for size seperation,detection and recording of dye fluorescence,and data output as fluorescent peak trace chromatograms. A number of commercial and non-commercial software packages can trim low-quality DNA traces automatically. These programmes score the quality of each peak and remove low- quality base peaks (generally located at the ends of the sequence).

13. This procedure uses the principle of the Sanger chain-termination method. Instead of labeling dATP in the original Sanger method, each of the dideoxynucleotides used in the reaction is labeled with a different fluorescent marker. S i n c e 4 d i f f erent f l u o r o p h o r e s a r e u s e d , a l l 4 r e a c t i o ns c a n b e r u n i n t h e s a m e t u b e , g r e a t l y i n c r e a s i ng t h e s p e e d a n d e a s e o f s e q u encing . A f t e r restriction , D N A f r a g m ents a r e separated b y

14. - c a p i l l ary g e l e l e c t r o p h o r e s i s u s i n g s m a l l , g e l f i l l e d c a p i l l ary t u b e s , c l u s t e r e d t o g e ther and r e a d w i t h a l a s e r s c a n n i n g s y s t e m . E l e c t r o p h e r o g r a m : A s e a c h c a p i l l ary t u b e i s moved i n t o t h e p a t h o f t h e l a s e r b e a m , f l o u r e s c e n t l y labeled nucleotides a r e d e t e c t e d o n e a t a t i m e , p r o d u c i n g a c o l o u r e d Electropherogram

15. A u t o m ated D N A s e q u e n c e r

16. A B E

17. R a t i o n a l e o f t h e s t u d y Analysis of WGS data could be used for epidemio logical investigations such as tracing of transmission chains. Identification of Mixed Infections. Mixed M. tuberculosis infections are described as TB disease caused by more than one distinct M. tuberculosis strain. Traditionally they are identifed based on at least two distinct patterns on MIRU- VNTR results

18. .Prediction of Drug Resistance and Understanding o f Mechanisms of Drug Resistance. Unlike other molecular methods that typically target specifc genes for determination of drug resistance, WGS allows for the interrogation of the entire M. tuberculosis genome for mutations conferring drug . resistance.

19. C o n c l usion Beginning in the 1970s, the Sanger process made it possible for researchers to sequence stretches of DNA at speeds never before possible. Further refinement and automation of this process continued to increase sequencing rates, thereby allowing researchers to reach major milestones in the Human Genome Project well ahead of schedule

20. References https://www.nature.com/scitable/topicpage/dna-sequencing-technologies-690/ http://www.mun.ca/biology/scarr/4241_AutomatedSequencing.html https://www.biocompare.com/Molecular-Biology/7127-Automated-DNA-Sequencing/ https://www.sciencedaily.com/releases/2019/04/190424153613.htm