Nextgen Explanation

Nextgen Explanation
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  Next Generation Sequencing for   UMMIES Looking at a presentation without the explanation from the author is sometimes difficult to understand. This document contains extra information for some slides that need it. Making a presentation, showing it once and keeping it for myself is a waste of time. That is why the  presentation is available on the internet. I hope you find it informative.Enoy, !ndy  Slide 1 : Choice of the title : I do not mean that you are dummies when we are talking about Next Generation Sequencing. Maybe you are maybe you are not. !hat I mean is that I still find myself a dummy when talking about Next Generation Sequencing. #en after a $%days training to use the Ion&orrent 'GM and a (%days course about Next Generation Sequencing I still ha#e the feeling that I only see the )ti* of the iceberg+. !ith this *resentation I,m trying to gi#e you the basic *rinci*les and work flow so if you know what is ha**ening you can understand what can go wrong. -nce you know the basics it is easier to read more detailed *ublications. Slide 2 : &he to*ics I will talk about. &he orkflow   and #ifferent platforms  will be ex*lained simultaneously.  !pplications  $un types  and #ata analysis  will be discussed briefly. Slide 5 :  &he first *ublication of a ( b* sequence from a / b* fragment. 0e#olutionary in that time background noise in the sequencing world of today. Slide 6 :   1 limited list of a#ailable *latforms. &he ones in gray are not yet a#ailable but according to the manufacturers they will be a#ailable within the next year.'ur*le : Next Generation Systems : need an am*lification ste*.0ed : &hird Generation Systems : single molecule sequencing. Slide 8 :  2irst ex*lain the Next Generation Sequencers  3*ur*le list on slide 45 :  1ll systems require a library *re*aration. Slide 9 : 6ibrary *re*aration : % g7N1 3or 0N15 is fragmented randomly 3e.g. by sonication en8ymatic ...5.% 1da*ter and *rimer are ligated to the fragments 3I use the term )*rimer+ for the *art that will be Next Generation Sequencing for UMMIES Andy Vierstraete (2012) 99(  used when sequencing and the term )ada*ter+ for the *art that will anneal to the bead or the slide. 1ctually they are both *rimer sequences5. Notice that the ligation is random some fragments will contain ada*ters on both sides some will contain *rimers on both sides the good ones are those with an ada*ter on one side and a *rimer on the other side. 1 barcode can be included.% Some *latforms do a si8e selection so that only fragments with the ideal length are used. Slide 10 : &here are two ty*es of am*lification : mulsion 'C0 or )'olony+ 'C0 on a slide Slide 11 15 :   mulsion 'C0 is created by mixing oil and water. &he result are little dro*lets 3micro reactors5. &his is also a random reaction so not all dro*lets contain all needed material : one bead one 7N1 strand *rimers and 'C0 mix. &he bead contains the com*lementary sequence of the ada*ter site on the 7N1 strand. Slide 16 : -nly 9; < of the dro*lets are ideal for emulsion 'C0. 6eft two columns : before 'C0 right two columns : after 'C0. 1fter 'C0 some dro*lets will only contain a bead 3to* left5 some will be ideal : one bead one 7N1 strand 3to* right5 some will only contain 7N1 3middle left5 some will contain two beads and one 7N1 strand : these will result in the exact same sequence 3middle right5 some are em*ty 3bottom left5 some will contain two or more 7N1 strands and one bead : these are *olyclonal and will result in double signal when sequencing 3bottom right5. Slide 18 19 : =ridge am*lification for Illumina. % &he slide contains the com*lementary sequence of the *rimer and ada*ter.% ss7N1 is attached to the surface 3I didn,t find out how this ha**ens : ligation >5% &he other end of the 7N1 anneals to the com*lementary *rimer on the surface.% xtension starts and a double stranded bridge is formed. % 1fter denaturation : each strand is attached to the surface 3one strand with ada*ter side other strand with *rimer side5.% 1m*lification goes on until clusters or *olonies 3'C0 colonies are formed5 Slide 20 : Second form of )*olony+ 'C0 for S-6i7 Slide 21 : !ildfire am*lification for S-6i7 3not commercial at this time S-6i7 still uses mulsion 'C05% ss7N1 anneals to the com*lementary *rimer site on the surface.% xtension of the tem*late.% 'artial denaturation : strand )walks+ to nearby *rimersite.% xtension and strand dis*lacement : two strands become four strands... Slide 22 :   Next Generation Sequencing for UMMIES Andy Vierstraete (2012) 9(  Something about ?uality Scores in sequencing : e.g. ?@ means that there is 9 error in 9@@ bases. &his means that you will ha#e 9@.@@@.@@@ errors in a 9Gb genome that is sequenced with one time co#erage. Since the sequencing errors are randomly the chance that a base in a strand of 7N1 is sequenced incorrectly twice is #ery low. So if you sequence with a co#erage of 9; times 3a *articular base is read 9; times in different sequences5 errors will disa**ear in the background. Slide 2! : Start ex*laining the different systems. Start with (;( sequencing. Slide 26 : ach well contains maximum one bead. &he bead contains single stranded 7N1. 1 *rimer is annealed and *olymerase am*lifies the strands. Slide 2 :  'yrosequencing is based on the release of di*hos*hate when a nucleotide is incor*orated 3left *icture5. ach of the four bases are added sequentially. 0ight *icture : when a base is incor*orated a di*hos*hate is released and after a few en8ymatic reactions light is *roduced. &his light is detected by a camera and de*ending on the intensity the number of bases incor*orated is determined. Slide 29 : &he key sequence 3first ( artificial bases Aust after the *rimer5 is used to normali8e the signal. &he instrument knows that only one base is incor*orated so the light intensity corres*onds to one base. If the signal intensity is higher after the first four bases than this means that more bases are incor*orated at the same time. Slide !0 : Next System : S-6i7 Slide !2 :  ;;@@! is for the !ildfire model 3not emulsion 'C05. &hey estimate that the ca*acity will be double of the B6 #ersion because they can attach 7N1 to the bottom and to* of the slide 3emulsion beads only go on the bottom5 and by changing the focus of the camera they can read the bottom of the to* of the slide.Sequencing by ligation : S-6i7 uses *robes based on %base encoding. &his means that  bases ha#e to fit to the tem*late to be attached. 2our colors are used and *er color four dual bases are *ossible. 6eft bottom *icture : e.g. a green color can be C1 39st base C and nd base 15 or 1C or &G or G&.   Slide !!5# :  Sequencing by ligation ste* by ste*. Next Generation Sequencing for UMMIES Andy Vierstraete (2012) $9(  Slide !! :  1ll the *robes are *resent 3( colors x ( combinations5 ligase *rimer and the tem*late attached to the bead. Slide !# : &he *rimer anneals to the ada*ter. &his is the 9st run. Slide !5 :  &he *robe with the com*lementary dual bases anneals and the ligase attach the *robe to the *rimer. Slide !6 :  1 laser excites the fluorescent label and fluorescence is detected by the camera. Slide ! : &here are four different combinations for the first two bases as shown abo#e the two green BB. !e can not know which one it is now. Slide !8 : &he last three bases with the fluorescent label is clea#ed off. Slide !9  #! : &he ligation % excitation % detection and clea#age goes on until /; b* are reached. Slide ## : &he extended sequence is melted off and an new *rimer anneals one *osition to the left in com*arison with the first *rimer. &his is the nd run. Slide #5 : &he ligation % excitation % detection and clea#age starts again. Slide #6 :  &he first of the dual bases 3red BB5 of the *robe anneals to the last base of the '9 ada*ter 3green5 on the bead. Since this first base is known 3e.g. &5 the first 30 75 base is the com*lement : 1. Slide # :  Now we know the first base of the dual base and there is only one *ossibility for the second base : &. If we look at the four green *ossibilities from the first run Aust abo#e we know that the first base from the green *robe was a & 3corres*onds with the last base of the red *robe Aust below5. &here is only one *ossibility for the second green base : G. Next Generation Sequencing for UMMIES Andy Vierstraete (2012) (9(
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