TECHNOLOGY IN THE FOOD SAFETY WORLD: TOOLS SUCH AS WHO GENOME - PowerPoint PPT Presentation

TECHNOLOGY IN THE FOOD SAFETY WORLD: TOOLS SUCH AS WHO GENOME SEQUENCING – FRIEND OR FOE? Room 314 | December 5 2017

CEUs – New Process Certified Crop Advisor (CCA) Pest Control Advisor (PCA), Qualified Applicator (QA), Private Applicator (PA) • Sign in and out of each session you attend. • Pickup scantron at the start of the day at first • Pickup verification sheet at conclusion of each session you attend; complete form. session. • Sign in and out of each session you attend. • Repeat this process for each session, and each day you wish to receive credits. • Pickup verification sheet at conclusion of each session. • Turn in your scantron at the end of the day at the last session you attend. Sign in sheets and verification sheets are located at the back of each session room.

AGENDA • Tim Birmingham , Almond Board of California, moderator • Jesse Miller , NSF International • Maria Hoffmann , FDA Center for Food Safety and applied Nutrition 3

Ne xt Ge ne r ation Se que nc ing – T he T e c hnology and its Applic ations – F r ie nd or F oe ? Je sse D. Mille r , Ph.D. Dir e c tor Applie d Re se ar c h Ce nte r NSF Authe nT e c hnologie s

Ne xt Ge ne r ation Se que nc ing Me thods Applic ations and E xample s Age nda 5

Ne xt Ge ne r a tion Se que nc ing Pr o c e ss o f e xtr ac ting ge ne tic mate r ial and r e ading the “c o de ”. 6

L e ts g o Ba c k in T ime ………….1952.

Ho w do we Ana lyze DNA? Se q ue nc ing Ba c kg ro und  RNA se q ue nc ing wa s first to b e de ve lo pe d (diffe re nt me tho ds)  1965: Ro b e rt W. Ho lle y se q ue nc e d tRNA fro m Sac c haro myc e s  1976: Wa lte r F ie rs’ la b first to c o mple te RNA-b a se d g e no me (MS2 b a c te rio pha g e )  Big b re a kthro ug h wa s DiDe o xy Se q ue nc ing (Sa ng e r Se q ue nc ing )  1977: I nve nte d b y F re dric k Sa ng e r (No b e l Prize 1958, 1980)  1977: F irst to se q ue nc e DNA-b a se d g e no me (PhiX b a c te rio pha g e )  T e rme d the “Cha in T e rmina tio n Me tho d”  Di-De o xy Nuc le o tide s a re la b e le d with fluo ro pho re s Use d to b e ra dio la b e le d. Pro b a b ly mo uth pipe tte d to o !  T he se Di-De o xy NT Ps (ddNT Ps) te rmina te the e xte nsio n re a c tio n whe n inc o rpo ra te d via PCR  T he e nd o f e a c h fra g me nt ha s a fluo re sc e nt sig na l  Curre nt me tho d is to run thro ug h a c a pilla ry g e l to size a nd o rde r Use d to b e a po lya c ryla mide g e l  Ca pture the sig na l se q ue nc e a nd tra nsla te to nuc le o tide b a se s

Wha t is Ne xt-Ge n Se q ue nc ing ?  T e rm use d fo r se q ue nc ing tha t ha s a hig he r thro ug hput tha n tra ditio na l Sa ng e r se q ue nc ing  No w E nc o mpa sse s ma ny pla tfo rms – T he rmo F ishe r, I llumina , Pa c ific Bio sc ie nc e s, Oxfo rd Na no po re  Ca n be Whole Ge nome Se que nc ing , 16S rRNA Me ta g e nomic s, Shotg un Me ta g e nomic s, T a rg e te d Ge ne Se que nc ing , RNA- SE Q  1 st Ge n – Sa ng e r, ABI (3130xl)  2 nd Ge n – 454, I llumina (So le xa ) a nd T he rmo F ishe r (Ma ssive ly Pa ra le ll Se q ue nc e rs – Sho rt Re a d)  3 rd Ge n – Pa c ific Bio sc ie nc e s, Oxfo rd (L o ng Re a d Se q ue nc e rs) BASE S T O BYT E S

Ho w is NGS Diffe re nt tha n T ra ditio na l Se q ue nc ing ?  Se q ue nc ing do ne o n flo wc e lls/ c hips no w. No 2D g e ls o r c a pilla rie s re q uire d  MUCH mo re da ta g e ne ra te d (T e ra b a se s no w, kilo b a se s the n)  So phistic a te d Bio I nfo rma tic pro g ra ms e xist to pa rse o ut the da ta - I n so me insta nc e s, c a n se q ue nc e a sa mple fo r a ro und $40  MUCH c he a pe r tha n histo ric a l  Ope n so urc e da ta sha ring fo r ma ssive da ta se ts  Clo ud c o mputing c a pa b ility MORE DAT A CHE APE R PE R BASE INT E RNE T MAKE S COMMS AND ANAL YSIS E ASY

Me thods Cho o se the Right “F it fo r Pur po se ” T o o l fo r the Jo b 11

Pulse -F ie ld Ge l E le c tro pho re sis (PF GE )  “Go ld Sta nda rd” o f b a c te ria l DNA fing e rprinting  Re stric tio n e nzyme s c ut b a c te ria l DNA in spe c ific lo c a tio ns  Multi-dire c tio na l g e l e le c tro pho re sis pro duc e s uniq ue pa tte rn b a se d o n the fra g me nt size s  Allo ws Co mpa riso ns b e twe e n o rg a nisms fo r I D  No t q ua ntita tive  $100-260

Po lyme ra se Cha in Re a c tio n Se mi Qua ntita tive (With a sta nda rd c urve ) T a rg e ts a re g io n o f g e no me fo r a mplific a tio n  Po sitive re a c tio n = g e ne is pre se nt Ca n de te c t se ve ra l ta rg e t g e ne s a t o nc e (Multiple x) Che a p! $5-10/ re a c tio n Se ve ra l ho urs to run

I mmuno lo g ic a l Me tho ds E L I SA L a te ra l F lo w Che a p ($5-10) F a st Ye s/ No a nswe rs  E L ISA c a n b e q ua ntita tive

Wha t is Who le Ge no me Se q ue nc ing ?  Who le Ge no me Se q ue nc ing is the te rm use d fo r e xtra c tio n o f DNA fro m a n o rg a nism a nd the sub se q ue nt ma pping o f its g e no me .  T he g e ne tic c o de (AGCT ) is re a d o n a n instrume nt a nd writte n into a dig ita l file .  T ha t dig ita l file c a n b e a sse mb le d (like a line a r puzzle ) to de te rmine the o rde r o f the c o de in the o rg a nism.  Onc e yo u ha ve the o rde re d c o de , yo u c a n a na lyze the da ta a nd ma ke c o mpa riso ns a nd YF B da ta -drive n de c isio ns a b o ut the o rg a nism.  No t q ua ntita tive  $50/ se q ue nc e – Up to $500 fo r a sse mb ly/ c lo sure 1 (Ba c te ria ) 5

Wha t is 16S/ Sho tg un Me ta g e no mic s?  16S Se q ue nc ing o n Ne xt-Ge n pla tfo rms fo llo ws a simila r wo rkflo w, e xc e pt tha t it ta rg e ts a spe c ific g e ne (16S Rib o so ma l RNA) use d to ide ntify b a c te ria  Me ta g e no mic s a pplie s this c o nc e pt to mixe d c o nso rtia , re sulting in a pro file o f b a c te ria a b unda nc e (e .g ., yo ur mic ro b io me ) 1. Se q ue nc e DNA 2. Alig nme nt to re fe re nc e da ta b a se s. Cla ssifying unkno wn b a c te ria in to ta xo no mic g ro ups 3. Visua lize in phylo g e ne tic tre e s, pie c ha rts, o r o the r a na lyse s b a se d o n q ue stio n to b e a ske d POPUL AT IONS

NGS Be ne fits Ove r Othe r Me tho ds Mo ving F o rwa rd?  NGS diffe re ntia tio n (re so lutio n) is unma tc he d  With hig h-thro ug hput e ffic ie nc ie s, NGS is c he a pe r a nd fa ste r  NGS e na b le s muc h mo re in-de pth da ta a na lysis, suc h a s func tio na l g e ne s a nd he re dity  Co st will c o ntinue to de c re a se  Da ta b a se a va ila b ility a nd po we r will c o ntinue to inc re a se  Glo b a l a do ptio n a nd da ta sha ring will inc re a se va lue

Applic a tions a nd E xa mple s 18

Who le Ge no me Se q ue nc ing  Wha t c a n I use it fo r?  E pide mio lo g y  Re sista nc e  Stra in le ve l I D YF B  Authe ntic ity  MUCH de e pe r lo o k into g e no me tha n Pulse F ie ld Ge l E le c tro pho re sis o r RF L P  L o o king a t e ve ry b a se , no t just whe re e nzyme s c ut

Stra in L e ve l I D  Is the re va lue in kno wing who yo ur re sid e nt stra ins a re ?  Ca n yo u e ra dic a te the m mo re e a sily?  Ca n yo u mo dify pro c e sse s a nd c le a ning re g ime ns?  Pro a c tivity?  Va lue in tra nspa re nc y a nd o wne rship?  Wo rking to wa rd a po sitive so lutio n  T hird Pa rty se q ue nc ing  Me ta da ta ho use d b y third pa rty

Spe c ia tio n o f Campylo b ac te r Co mme nsa l b a c te ria o n Chic ke n a nd o the r fo wl I nte rve ntio ns c a n kno c k do wn numb e rs, b ut ha rd to c o mple te ly e ra dic a te T hre e stra ins unde r sc rutiny  Je juni  Co li  L a ri

F o o d Pa tho g e n I D – Off the She lf I so la te g e rms fro m o ff the she lf me a ts Assa y fo r pa tho g e ns Who le Ge no me Se q ue nc e fo r spe c ie s  Co mmo n tre nd s?  F o o d type  Ge o g ra phy  Inte rve ntio n me tho d  Pre se rva tio n me tho d

Wha t is 16S/ Sho tg un Me ta g e no mic s?  16S Se q ue nc ing o n Ne xt-Ge n pla tfo rms fo llo ws a simila r wo rkflo w, e xc e pt tha t it ta rg e ts a spe c ific g e ne (16S Rib o so ma l RNA) use d to ide ntify b a c te ria  Me ta g e no mic s a pplie s this c o nc e pt to mixe d c o nso rtia , re sulting in a pro file o f b a c te ria a b unda nc e (e .g ., yo ur mic ro b io me ) 1. Se q ue nc e DNA 2. Alig nme nt to re fe re nc e da ta b a se s. Cla ssifying unkno wn b a c te ria in to ta xo no mic g ro ups 3. Visua lize in phylo g e ne tic tre e s, pie c ha rts, o r o the r a na lyse s b a se d o n q ue stio n to b e a ske d POPUL AT IONS

I rrig a tio n Wa te r Mic ro b io me L o o king a t c ha ng e s in wa te r mic ro b io me whe n E . c o li pre se nt  Se a rc hing fo r ma rke rs o f c o nta mina tio n Almo nd Ha rve st  Sha king tre e s to re le a se fruit  Drying fo r a fe w d a ys  Ha rve ste r  Hulling  She lling www.pinte re st.c o m