Tech n ology a n d societ y Com p u t er Scien ce

In vest iga t ion of Pa t h wa y An a lysis Tools for m a ppin g om ics da t a t o pa t h wa ys -Focu s on lip id om ics a n d gen om ics d a t a Un der sök n in g a v a n a lysver kt yg för a t t ka r t lä gga om ik da t a t ill r ela t ion svä ga r – F ok u s p å d a t a a v t y p en l i p i d om i k och g en om i k

A u t h or : A t t i l a K on r á d

Degree: Bachelor of Computer Science 180hp Major: Information Systems Programme: Information Systems Date of exam: 2012-09-20

Supervisor(s): Céline Fernandez, Annabella Loconsole Examiner: Bengt J. Nilsson

An ed u cation isn 't h ow m u ch you h ave com m itted to m em ory, or even h ow m u ch you k n ow . It's bein g able to d ifferen tiate betw een w h at you k n ow an d w h at you d on 't. /An a t ole F r a n ce

Ac k n o w le d g e m e n t s I wou ld like sa y t ha n k you t o ever yon e who h elped m e wit h m y t h esis. To m y su per visor s I t h a n k you for you r pa t ien ce, gu ida n ce a n d a ll t h e good feedbacks.

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Abst r a ct Th is t h esis exa m in es P ATs fr om a m u lt idisciplin a r y view. Th er e ar e a lot of P AT's exist in g t oda y a n a lyzing specific t ype of om ics da t a , t h er efor e we in vest iga t e t h em a n d wh a t t hey ca n do. By defin in g som e specific r equ ir em ent s su ch as h ow m a n y om ics dat a t ypes it ca n h a n dle, t he a ccu r a cy of t h e P AT ca n be obt a in ed t o get t h e m ost su it a ble P AT wh en it com es t o m a ppin g om ics da t a t o pa t hwa ys . Result s sh ow t h at n o P ATs fou n d t oda y fu lfills t h e specific set of r equ ir em en t s or t he m a in goal t h ou gh soft wa r e t est in g. Th e In gen u it y P AT is t h e closest t o fu lfill t h e r equ ir em en t s. Requ est ed by t he en d u ser , t wo P ATs a r e t est ed in com bin a t ion t o see if t h ese ca n fu lfill t h e r equ ir em en t s of t h e en d u ser . Un ipr ot ba t ch con ver t er was t est ed wit h F E vE R a n d r esu lt s did n ot t u r n ou t su ccessfu lly sin ce t h e com bin at ion of t h e t wo P ATs is n o bet t er t h a n t h e In gen u it y P AT. F ocu s t h en t u r ned t o a n a lt er n at ive com bin a t ion , a h om epa ge ca lled NCBI t h at h a ve sea r ch en gin es con n ect ed t o sever a l fr ee P ATs a va ila ble t h u s fulfillin g t he r equ ir em en t s. Thr ou gh t h e sea r ch en gin e “om ics” da t a ca n be com bined a n d m or e t h a n on e in pu t ca n be t a ken a t a t im e. Sin ce t ech nology is r a pidly m ovin g for wa r d , t h e n eed for new t ools for dat a in t er pr et a t ion a lso gr ows. It m ea n s t h a t in a nea r fut u r e we m ay be a ble t o find a P AT t ha t fu lfills t h e r equ ir em en t s of t h e en d u ser s. Ke y w o rd s : Bioch em ist r y, Ca r diovascu lar Lipidom ics, Met a bolom ics, P AT, Tech n ology

disease,

Da t a base,

Gen om ics,

Lipids,

Sa m m a n fa t t n in g Det t a exa m ensa r bet e gr a n ska r a n a lysver kt yg u r et t t vä r vet enska pligt per spekt iv. Det fin n s en h el del olika a n a lysver kt yg ida g som a n a lyser a r specifika t yper a v om ik dat a och dä r för u n der söker vi h u r m å n ga det fin ns sa m t va d de ka n gör a. Gen om a t t defin ier a et t a nt a l specifika kr a v så som h u r m å n ga t yper a v om ik da t a den ka n h a n t er a, n oggr a n n h et a v ver kt yget s a n a lys så ka n m a n se vilka som ä r m est lä m pliga a n a lysver kt ygen n ä r det gä ller ka r t lä ggn in g a v om ik da t a . Resu lt a t en visa r a t t det ida g in t e fin n s a n a lysver kt yg som u ppfyller de specifik t a n givn a kr a ven eller h u vu dsyft et gen om t est n in g a v pr ogr a m va r a n . In gen u it y a n a lysver kt yget ä r det n ä r m a st e vi ka n kom m a för de kr a v som vi söker . P å begä r a n a v slu t a n vä n dar en t est ades t vå a n a lysver kt yg för a t t se om en kom bin a t ion a v dessa ka n u ppfylla slu t a n vä n dar ens kr a v. An a lysver kt yget Un ipr ot ba t ch con ver t er t est as m ed F E vE R m en r esult a t är in t e fr a m gå n gsr ikt , då kom bin a t ion en a v dessa ver kt yg in t e ä r bä t t r e ä n In gen u it y a n a lysver kt yget . F oku s vä n ds m ot en a lt er n at iv kom bin a t ion som är en h em sida och h et er NCBI. H em sida n h a r en sökm ot or koppla d t ill fler a olika a n a lysver kt yg som är gr a t is a t t a n vä n da . Gen om sökm ot or n ka n ”om ik” dat a kom bin er a s och m er ä n et t in m a t a t vä r de k a n h a nt er a s i t a get . E ft er som t ekn iken sn a bbt gå r fr a m å t in n ebär det dä r em ot a t t n ya a n a lysver kt yg beh övs för da t a h a n t er in g och in om en sn a r fr a m t id så h a r vi ka n ske et t a n a lysver kt yg som u ppfyller kr a ven a v slut a n vä n da r n a . N y c k e lo rd : Biokem i, Ka r diova sku lä r sjukdom , Da t aba s, Gen om ik, L ipider , Lipidom ik, Met a bolom ik, An a lysver kt yg, Tekn ik

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Co n te n ts Abst r a ct ................................................................................................................................................. 3 Sa m m a nfa t t nin g ................................................................................................................................. 3 1. In t r odu ct ion .................................................................................................................................. 5 1.1. P u r pose ...................................................................................................................................... 5 1.2. P r oblem definit ions a n d Aim s .............................................................................................. 6 1.3. P r oblem discu ssion .................................................................................................................. 6 1.4. Rela t ed wor k wit h P AT .......................................................................................................... 8 2. Met h ods ......................................................................................................................................... 8 2.1. Model in use .............................................................................................................................. 8 2.1.1. R equ irem en t collection , d ocu m en tation an d valid ation ...................................... 8 2.1.2. R equ irem en t processin g an d test case creation ..................................................... 9 2.1.3. Objective ...................................................................................................................... 12 2.1.4. Un d erlyin g objectives ................................................................................................ 12 2.2. Alt er n at ive r esear ch m et h ods ............................................................................................ 13 3. Biom edica l backgr ou nd ............................................................................................................ 13 3.1. Genet ics ................................................................................................................................... 13 3.1.1. Gen e .............................................................................................................................. 14 3.1.2. S N P .............................................................................................................................. 15 3.2. Bioch em ist r y of Lipids .......................................................................................................... 16 3.2.1. L ipid d efin ition .......................................................................................................... 17 3.2.2. Classes of L ipid s ........................................................................................................ 17 3.2.3. E n zym es in volved in th e syn th esis of lipid s ......................................................... 18 3.2.4. L ipoproteins ................................................................................................................ 21 3.3. Genom ics ................................................................................................................................. 21 3.4. Met a bolom ics .......................................................................................................................... 22 3.5. Lipidom ics ............................................................................................................................... 22 3.6. Ca r diovascu lar disea ses ....................................................................................................... 22 4. Com pu t er Scien ce ba ckgr ou n d ............................................................................................... 23 4.1. Dat a ba ses, Da t a m in in g a n d Kn owledge discover y ....................................................... 23 4.2. P AT ........................................................................................................................................... 23 5. Requ ir em ent s a n d Test elicit at ion ........................................................................................ 24 5.1. Requ ir em en t s ......................................................................................................................... 24 5.2. Test in g ..................................................................................................................................... 25 5.3. Test ca ses ................................................................................................................................ 26 6. Resu lt ........................................................................................................................................... 26 6.1. F in din g t h e P ATs .................................................................................................................. 26 6.2. Sor t in g t he P ATs ................................................................................................................... 27 6.3. Test in g t h e P ATs ................................................................................................................... 27 6.4. E va lu a t in g t he P ATs ............................................................................................................ 28 6.5. F in a l eva lu at ion of t h e P ATs .............................................................................................. 28 6.6. Th e best P AT fr om t h e r a nked list .................................................................................... 29 6.7. Com bin in g P ATs .................................................................................................................... 31 6.8. F u n ct ion alit ies ....................................................................................................................... 34 6.9. Qu a lit y ..................................................................................................................................... 35 7. Discu ssion ................................................................................................................................... 36 7.1. Is it possible t o fin d a P AT t h at pr ocesses m et a bolom ics a n d lipidom ics r aw da t a a s inpu t a nd com bine t h em wit h gen et ic infor m a t ion ? ........................................................ 36 7.2. Wh a t a r e t h e fu n ct ion a lit ies offer ed by t he a va ila ble a n a lysis t ools? ....................... 36 7.3. Wh a t a r e t h e qu a lit ies of t h ese t ool's a nd how t o eva lu a t e t h em ? ............................. 37 Page | 4

7.4. Wh y n ot In gen u it y a n d wh y Un ipr ot wit h F E vE R? ...................................................... 38 8. F u t u r e Va lu e .............................................................................................................................. 38 9. Refer en ces ................................................................................................................................... 38 Appen dix 1 – Test Ca ses .................................................................................................................. 42 Appen dix 2 – Lipid, MI SNP a n d Met a bo SNP da t a sheet ...................................................... 46 Appen dix 3 – Requ ir em en t s Ma t r ixes .......................................................................................... 50 Appen dix 4 – Respon s Tim es .......................................................................................................... 53

1. In t r odu ct ion Va st a m ou n t s of r esea r ch is don e in lipidom ics a n d gen om ics, m a king com pu t er s, In t er n et a n d va r iou s a n a lysis t ool's ver y com m on t oda y bot h in sim ple a n d a dva n ced for m s. As a n exa m ple a sim ple ca lcula t ion ca n be per for m ed on on e com pu t er a n d t r a n sfer r ed or copied t o a n ot h er if n eeded. Mor e a dva n ced per for m a n ces som et im es r equ ir e a soft wa r e t ool t h a t ca n per for m a cer t a in t a sk on a given set of da t a in or der t o give a cer t a in r esu lt . Th e r esu lt is in t u r n u su a lly n ot logica lly or der ed a n d a visu a l pr esen t a t ion is n eeded. Th is is wh er e a pa t h wa y a n a lysis t ool (P AT) is n eeded. A pa t h wa y a n a lysis t ool (P AT) is a n a dva n ced t ool t h a t pr ocesses given da t a , com pa r es t h e given da t a wit h st or ed da t a in a da t a ba se a n d pr esen t s t h e r esu lt s obt a in ed visu a lly. A com pa n y t en ds t o h ir e a pr ogr a m m er t o develop a pa t h wa y a na lysis t ool (P AT) in or der t o in t egr a t e it wit h in t he or ga n iza t ion [34]. On e of t h e m a in gr ou ps of scien t ific u ser s is t h e gr ou p of r esea r ch er s in fields of bioin for m a t ics, gen et ics, gen om ics a n d m et a b olom ics. Resea r ch er s a r e depen den t of t h ese pa t h wa y a n a lysis t ools in t h eir scien t ific wor k. In som e scien t ific fields su ch a s gen om ics a n d m et a bolom ics, t h er e a r e t oo m a n y a n a lysis t ools (P AT), doin g a ll kin ds of differ en t t a sks. Too m a n y pa t h wa y a n a lysis t ools in a specific field ca n con fu se r esea r ch er s wh o do n ot h a ve en ou gh kn owledge in t ech n ology [5]. Th is m a kes it difficu lt t o decide wh a t pa t h wa y a n a lysis t ools a r e su it ed for cer t a in da t a a n d wit hin wh a t scien t ific field. Sin ce t ech n ology is a lso m oving for wa r d ext r em ely fa st , people wit h m u lt idisciplin a r y kn owledge a r e n eeded m or e a n d m or e [20]. F or r esea r ch er s wh o wor k wit h in t h e biom edica l field of m et a bolom ics a n d gen om ics t h er e a r e specific a n a lysis t ools. Th e pu r poses of t h ese pa t h wa y a n a lysis t ools (P AT) a r e t o h elp t h e u ser s in t h eir wor k, wh er e t h ey ca n visu a lize da t a t ha t m a y lea d t o n ew scien t ific discover y. Tech n ology a n d infor m a t ion sh a r in g h a s t a ken a big st ep for wa r d a n d h a s h elped su bst a n t ia lly in differ en t a r ea s a r ou n d t h e wor ld su ch a s in h ea lt h ca r e a n d m edicin e. 1.1.P u rp o s e F in din g r elia ble pat h wa y a n a lysis t ools (will be r efer r ed t o a s P AT fr om n ow on) t h at ca n do a ll t h e n ecessa r y da t a com pu t at ions a n d ca n visu a lly pr esent t h e r esu lt s is r equ est ed by Célin e F er n a n dez fr om Clin ica l Resea r ch Cen t er (CRC) in Ma lm ö (will be r efer r ed t o as t h e en d u ser ). CRC wor k s in discover in g n ew m edicin e, dia gn ost ic t ools a n d im pr oved t r eat m en t s in or der t o im pr ove h ea lt h wor ldwide.

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1.2.P ro ble m d e fin itio n s a n d Aim s Sin ce t h er e a r e m a n y P AT a va ila ble wit h lot s of in for m a t ion , t h e following r esea r ch qu est ion s a r e defin ed in t h is t h esis:  Is it possible t o fin d a P AT t h a t pr ocesses m et a bolom ics a n d lipidom ics r a w da t a a s in pu t a n d com bin es t h em wit h gen et ic in for m a t ion ?  Wh a t a r e t h e fu n ct ion a lit ies offer ed by t h e a va ila ble a n a lysis t ools?  Wh a t a r e t h e qua lit ies of t h ese t ool's a n d h ow t o eva lu a t e t h em ?

Th e object ives a r e defin ed in or der t o h elp a n swer t h e t h r ee r esea r ch qu est ion s. Th e m a in a im of t h is t h esis is t h e followin g: To fin d a P AT t h a t ca n pr ocess a com bin a t ion of da t a in pu t s wit h t h e t ype of “om ics” da t a , i.e. lipidom ics/m et a bolom ics, gen om ics da t a . In or der t o r ea ch t h e m a in pu r pose, sever a l u n der lyin g object ives a r e n eeded. Th ese a r e t h e followin g: 1) F in d P ATs t h a t a r e a ble t o m a p pa t h wa ys of t h e following t ype of da t a : a ) Over a ll m et a bolom ics da t a b) Lipidom ics da t a c) Gen om ics da t a 2) E va lu a t e t h e select ed P AT a n d t h eir fu n ct ion s. Test t h e cu r r en t a ccu r a cy of t h e exist in g P AT in or der t o a n swer if t h e ou t put fr om t h ese t ools sh ows t h e “cor r ect ” r esu lt s. 3) E va lu a t e t h e select ed P AT a ccor din g t o specific r equ ir em en t s given by t h e en d u ser ; see sect ion 1.3 for t h e specific r equ ir em en t s. Aft er t h e eva lu a t ion of t h e P AT a ccor din g t o r equ ir em en t s, t wo opt ion s a r e possible: Opt ion 1: On e or m or e P AT pa sses st eps 2 a n d 3 a n d is deliver ed t o t h e en d u ser . Opt ion 2: If n o P AT fu lfillin g t h e r equir em en t s is fou n d. Alt er n a t ive solu t ion s will be t o see if it is possible t o a da pt a n y of t h e eva lu a t ed a n a lysis t ools, com bin e m or e t h a n on e or m a ke a n in h ou se developm en t of a P AT m eet in g t h e r equir em en t s of t h e en d u ser . 1.3.P ro ble m d is c u s s io n In or der t o solve t h e pr oblem we m u st con sider wh a t P ATs a r e, h ow com plex t h ey a r e a n d wh a t t h ey ca n do. Th e fu n ct ion a lit ies of t h e P AT n eed t o be t est ed [28] t o see if t hey fu lfill t h e specific r equ ir em en t s (S ee T able 1).

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Ta ble 1. 8 specific r equ ir em en t s list ed t h a t n eeds t o be fu lfilled by a P AT. Re qu ire m e n t Re qu ire m e n t d e s c rip tio n ID User is a ble t o see a n d select on t h e P AT 1 wh a t t ype of da t a it m u st pr ocess (if t h e in pu t field is for m et a bolom ic, lipidom ic or gen om ic) User m u st be a ble t o con t r ol if obt a in ed 2 r esu lt is va lid fr om t h e P AT a ccor din g t o lit er a t u r e, In t er n et or la bor a t or y r esu lt s Th e u ser m u st r eceive r esu lt s by t h e P AT 3 wit h in a cer t a in t im e Th e u ser ca n n a viga t e bet ween st a r t of 4 sea r ch (in pu t da t a ) t o t h e en d of sea r ch (r esu lt s obt a in ed). Th e u ser ca n get a visu a l pr esen t a t ion of 5 m et a bolom ics, lipidom ics a n d gen om ics da t a fr om t h e P AT Th e u ser ca n zoom in a n d ou t expa n din g 6 t h e view t o n eigh bor in g possible r esu lt s t o see con n ect ed pa t h wa ys on t h e r eceived r esu lt s fr om t h e P AT. Th e u ser ca n in pu t a specific t ype of da t a 7 in t o t h e P AT (m et a bolom ic, lipidom ic or gen om ic) Th e u ser ca n in pu t com bin ed om ics da t a 8 a n d t h en m a p t h em t o pa t h wa ys

Acqu ir in g kn owledge fr om lit er a t u r e gives u s in for m a t ion a bou t t h e com plexit y of a P AT [27]. Th e fu n ct ion a lit ies fr om a P AT ca n be obt a in ed wit h h elp of soft wa r e t est in g of da t a in pu t s [9] a n d t h is wa y we ca n ch eck if t h e P AT sa t isfy t h e r equ ir em en t s of t h e pot en t ia l u ser s. Th e defin it ion of qu a lit y is of a bigger sca le a n d h a r der t o defin e sin ce qu a lit y h a s differ en t m ea n in gs t o differ en t people [36]. Th e qua lit ies of t h e P AT a r e a ccept a ble if t h ey a r e fu lfillin g a ll t h e r equ ir em en t s [36] a ccor din g t o a set of r equ ir em en t specifica t ion s. We will be u sin g t h e r equ ir em en t specifica t ion s a ccor din g t o t a ble 1. H om epa ges a ssocia t ed wit h P AT a lso n eed t o be qu a lit y ch ecked a n d five select ed a spect s a r e u sed: Accu racy an d Correctn ess (h ow t r u st wor t h y is t h e in for m a t ion pr ovided on t h e h om epa ges), Com pleten ess (a r e t h e h om epa ges com plet e or u n der con st r u ct ion ), R elevan ce (h ow r eleva n t is con t en t or in for m a t ion on a h om epa ge t o t h e P AT), T im e an d Pu n ctu ality (h ow fa st ca n a h om epa ge be fou n d wh en sea r ch in g), T raceability (is t h e in for m a t ion pr ovided on t h e h om epa ges t r a cea ble t o t h eir or igin a l sou r ce).

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1.4.Re la te d w o rk w ith P AT Most P AT t oda y is m a de specifica lly wit h focu s on m et a bolom ics a n d gen om ics. Th is is du e t o t h e r esea r ch wor k in m et a bolic en gin eer in g, cellula r m et a bolism a n d in t oxic gen om ics [16, 25]. Com pa n ies spen d va st a m ou n t s of m on ey developin g a P AT wh ile t r yin g t o com pet e wit h ea ch ot h er [8, 15]. Th e com pet it ion for t h e com pa n ies in volves bu ilding, a da pt in g a n d eva lu a t ing ea ch ot h er 's P AT, t ellin g wh y t h eir P AT is bet t er t h a n t h e ot her [8, 15, 33]. Sin ce t h e P AT is specifica lly developed for a biom edica l field [41], t h er e exist s n o fu ll-sca le a n a lysis on t h e en t ir e P AT yet . Ou r st u dy is a fir st a t t em pt a t su ch a n a n a lysis of a com plet e set of a ll P AT.

2. Met h ods Th is sect ion descr ibes t h e scien t ific m et h ods u sed t o eva lu a t e t h e differ en t P AT. St a r t in g wit h t h e select ed m et h od in u se, h ow t h e in for m a t ion is ga t h er ed a n d det a ils on t h e object ives a n d u n der lyin g object ives. 2.1.Mo d e l in u s e Th e m a in pu r pose (t o fin d a P AT t h a t ca n pr ocess a com bin a t ion of da t a in pu t s wit h t h e t ype of “om ics” da t a , i.e. lipidom ics/m et a bolom ics, gen om ics da t a ) of t h e pr oject wa s divided in t o fou r u n der lyin g object ive , ea ch wit h it s specific object ive . Met h ods t h a t will be per for m ed a r e ba sed on a n em pir ica l m odel wit h a st u dy on P AT in or der t o t est a n d a n a lyze ea ch of t h e P AT a n d t h eir h om epa ges. Test ca ses a r e design ed ba sed on t h e r equ ir em en t s fr om t h e en d u ser a t t h e fir st in t er view. Th e r equ irem en t s a r e r ech ecked a few weeks la t er wit h t h e en d u ser in a secon d in t er view. On ce a ckn owledged, t h e soft wa r e t est in g begin s wit h r equ ir em en t s a n d t est ca ses, in or der t o see if in goin g da t a m a t ch es t h e ou t com in g da t a of t h e P AT. Da t a is ba sed on a gen e n a m e (e.g. NP P A), r efer en ce SNP a ccession ID (r s n u m ber su ch a s r s5068) or a lipid cla ss n a m e (su ch a s lipopr ot ein s). Ver ifica t ion (fr om t h e P AT) of t h e ou t com in g da t a t o see if it s r eleva n t is per for m ed by com pa r in g t h e r eceived r esu lt s wit h in for m a t ion fou n d in lit er a t ur e. A r a n ked list is m a de r a n kin g t h e best P AT fir st , ba sed on h ow m a n y r equ ir em en t s a r e m et . If n o P AT m eet s a ll t h e r equir em en t s, t h e en d u ser h a ve a r equ est t o a da pt or com bin e 2 specifica lly select ed t ools, wh ich en d u ser is a lr ea dy fa m ilia r ized wit h , wh ile t h e r a n ked list get s disca r ded .

2.1.1. R equ irem en t collection , d ocu m en tation an d valid ation F ive m eet in gs a r e booked a t t h e Clin ica l Resea r ch Cen t er (CRC) in or der t o m a ke in t er views. All pa r t icipa n t s (r esea r ch er s in clu din g t h e en d u ser ) a r e goin g t o discu ss a bou t t h e pr oblem t h a t n eeds t o be solved. Discu ssion will focu s on P AT in gen er a l a n d specific fu n ct ion s a r e goin g t o be desir ed by t h e r esea r ch er s t h a t h a ve t o be on a P AT. Requir em en t s a r e m a de con n ect ed t o t h ese fu n ct ion s on a P AT a n d a n ew m eet in g is booked. Du r in g ea ch m eet in g ever yt h in g is wr it t en down a n d docu m en t ed. Aft er ea ch m eet in g, Page | 8

r equ ir em en t s a r e collect ed t o be sor t ed a n d pr ocessed in or der t o m a ke t est ca ses. La t er a ch ecku p t a kes pla ce a t sa m e pla ce, t o see if ever yt h in g is on t h e r igh t t r a ck .

2.1.2. R equ irem en t processin g an d test case creation Th e r equ ir em en t s a re pr ocessed a n d for m u la t ed. Th ey a r e a lso sh or t en ed down fr om 15 t o eight r equ ir em en t s wit h t h e m ost im por t a n t t hin gs t h a t a P AT m u st do. E a ch of t h e r equ ir em en t s is given a n iden t ifica t ion n u m ber . Test ca se t em pla t es a r e sou gh t a n d on e t em pla t e is select ed, down loa ded a n d t h en cu st om ized (Fig 1). Specific t est ca ses a r e design ed t o su it t h e r equ ir em en t s a n d linkin g t h em t o t h eir r espect ive r equ ir em en t (S ee T able 2). Th e design s of t h e specific t est ca ses a r e m a de by a ddin g t h e goa l of t h e t est a lon g wit h t h e even t s t o a ch ieve t h e goa l. La st ly t h e expect ed r espon se is wr it t en , descr ibin g wh a t r esu lt s we sh ou ld expect by followin g t he even t s. Th e wh ole pr ocess st a r t s by ca r efu lly ch eckin g a r equ ir em en t fr om t h e list a n d t r yin g t o see if t h ey ca n be m a de a s a sin gle t est ca se in on e go. If t h a t is n ot possible sever a l t est ca ses a r e n eed ed. If we look a t fir st r equ ir em en t in t a ble 1 a bove, we see t h a t 3 differ en t da t a t ypes n eed t o be t est ed. So we h a ve t o split t h e r equ ir em en t in t o m or e t h a n 1 t est ca se sin ce a ll P AT m a y n ot be a ble t o pr ocess a ll 3 da t a t ypes. We decide t o t a ke t h e fir st da t a t ype wh ich is for m et a bolom ic in pu t da t a . We a lso select a da t a in pu t t h a t we kn ow sh ou ld give a r espon se a n d pr esen t som e r esu lt s. F r om t h is we ca n wr it e down ou r even t s in t h e t est ca se by h a vin g a n in pu t a n d t h en get t in g a r espon se. So we ca n t h en a lso st a t e t h e expect ed r espon se. In ou r ca se it is t h a t t h e m et a bolom ic da t a t ype gives da t a in for m a t ion r ela t ed t o ou r da t a in pu t t ha t we m a de. Next 2 t est ca ses will be sim ila r wit h t he sm a ll differ en ce of h a vin g a differ en t in pu t da t a t ype. Sa m e a ppr oa ch m et h od is a pplied t o t h e r est of t h e t est ca ses. Requ ir em en t s a r e goin g t o be ch ecked, eva lu a t ed if it ca n be m a de a s on e t est or split t in g t h em in t o m or e t est ca ses for sa m e r equ ir em en t , wr it in g t h e even t s a n d t h e expect ed r espon se.

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F ig u re 1. A t est ca se t em pla t e u sed in t h is st u dy.

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Ta ble 2. A t a ble sh owin g r equ ir em en t ID wit h descr ipt ion lin ked t o specific Test Ca se ID ID Re qu ire m e n t Ty p e Lin k e d w ith Te s t d e s c rip tio n Ca s e ID User is a ble t o see an d F u n c tio n a l 1 1, 2 a n d 3

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select on t h e P AT wh a t t ype of dat a it m u st pr ocess (if t h e inpu t field is for m et a bolom ic, lipidom ic or gen om ic) User m u st be a ble t o ch eck if t h e r esult s obt a in ed is va lid fr om t h e P AT a ccor din g to lit er a t ur e or la bor at or y r esu lt s Th e u ser m u st r eceive r esu lt s by t h e P AT wit h in a cer t a in t im e Th e u ser sh ould n a viga t e bet ween st ar t of sear ch (in pu t dat a ) t o t h e en d of sea r ch (r esu lt s obt ain ed). Th e u ser sh ou ld get a visu a l pr esent a t ion of m et a bolom ics, lipidom ics a n d gen om ics da t a fr om t h e P AT Th e u ser m u st be a ble t o zoom in and ou t expa n din g t h e view t o n eigh bor in g possible r esu lt s t o see con n ect ed pa t h wa ys on t h e r eceived r esu lt s fr om t h e P AT. Th e u ser m u st in put a specific t ype of dat a in t o t h e P AT (m et a bolom ic, lipidom ic or gen om ic)

Non fu n c tio n a l

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Th e u ser m u st be a ble t o F u n c tio n a l in pu t com bin ed om ics da t a a n d t h en m a p t hem t o pat h wa ys

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2.1.3. Objective Th e m a in pu r pose is a ch ieved by a cquir in g kn owledge fr om lit er a t u r e su ch a s books a n d a r t icles a n d by doin g soft wa r e t est in g. Th e r esu lt s obt a in ed fr om t h e t est s a r e t h a n com pa r ed wit h r equ ir em en t s m a de by t h e pot en t ia l u ser s of t h e P AT.

2.1.4. Un d erlyin g objectives Object ive 1: Ga t h er in g of in for m a t ion by sea r ch in g books a n d a r t icles , fin din g lot s of P AT a n d obt a in wh a t da t a it ca n pr ocess. Down loa d P AT if possible t o a n a lyze t h em . Object ive 2: E va lu a t e t h e select ed P AT wit h t h eir fu n ct ion s a n d m et h ods by going t h r ou gh ea ch t ool, clickin g a r ou n d a n d in pu t t in g da t a . Test ca ses a r e design ed fr om t h e given r equ ir em en t s. Test s on t h e P AT a r e ba sed u pon : a ) F r om t h e lit er a t u r e kn own m et a bolom ics, lipidom ics, a n d gen et ic pa t h wa ys a n d cor r ela t ion s b) Com pa r ison bet ween r esu lt s obt a in ed fr om t h e lit er a t u r e a n d fr om t h e P AT c) Com pa r ison bet ween exist in g la bor a t or y r esu lt s a n d t h e P AT d) H ow lon g it t a kes t o pr ocess da t a by t h e a n a lysis t ool Cor r ect r esu lt s a r e con sider ed t o be t h ose t h a t com e fr om scien t ific a r t icles, books or la bor a t or y r esu lt s ver ified by scien t ist s. P a t h wa ys a n d cor r ela t ion s wit h m et a bolom ics, lipidom ics, a n d gen et ics a r e t est ed a ga in st lit er a t u r e kn own r esu lt s. Com pa r ison bet ween r esu lt s obt a in ed fr om P AT a ga in st a r t icle a n d book r esu lt s a r e goin g t o be don e fir st , a ft er wa r ds t h e exist ing la bor a t or y r esu lt s. Accu r a cy of t h e P AT a r e a cqu ir ed by t h e ou t pu t da t a a n d r esu lt s will eit h er a ccu r a t ely m a t ch a ll da t a or n ot . A sim ple t im er is u sed t o r ecor d t h e pr ocessin g t im e of a P AT. F in a lly a list of P AT will sh ow wh ich P AT pa ssed, fa iled a n d wh y t h ey fa iled ou r exa m in a t ion . Object ive 3: In or der t o h a ve a sa t isfied en d u ser , specific set of r equ ir em en t s a r e n eeded t h a t m u st be fu lfilled wit h a fin a l eva lu a t ion . Requ ir em en t s a r e collect ed a t a n ea r ly st a ge wit h in t er views fr om r esea r ch er s a n d t h e en d u ser wh o a lso r epr esen t ot h er pot en t ia l u ser s. Th e m ost desir ed a n d im por t a n t r equ ir em en t s wer e discu ssed a n d iden t ified t o be t h e followin g: Select ed a n a lysis t ool m u st be a ble t o: a ) Na viga t e bet ween da t a a n d r esu lt s b) Ma ke visu a l pr esen t a t ion of obt a in ed m et a bolom ics, lipidom ics or gen om ics da t a c) H a ve zoom in a n d zoom ou t fu n ct ion s expa n din g t h e view t o n eigh bor in g possible r esu lt s con n ect ed t o pa t h wa ys on t h e r esu lt s obt a in ed

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d) Th e P AT sh ou ld be a ble t o pr ocess m or e t h a n on e t ype of da t a (m et a bolom ic, lipidom ic or gen om ic) e) Be a ble t o com bin e om ics da t a a n d t h en m a p t h eir pa t h wa ys Na viga t ion will be t est ed by lookin g a t t h e ou t pu t da t a (r esu lt s obt a in ed) t o t h e in goin g da t a (t h e begin n ing of wh er e da t a is in ser t ed). In ser t ion s of da t a a r e m a de in t h e r equ ir ed fields wh ile t r a cea bilit y or clicka ble t r a ckin g views a r e sou gh t wh en obt a in in g r esu lt s. An y visu a l pr esen t a t ion s on obt a in ed r esu lt s a r e a ccept ed bu t det a iled view of pa t h wa y com bin a t ions a n d cor r ela t ion s a r e pr efer r ed. On ou t pu t da t a zoom fu n ct ion s a r e sou gh t t h a t is a sm a ll m a gn ifyin g gla ss wit h a plu s or m in u s sign in t h e P AT. To t est h ow m a n y t ype of da t a (m et a bolom ic, lipidom ic or gen om ic) t h e P AT ca n pr ocess, on e of ea ch da t a t ype will be select ed. Th r ee da t a t ypes t oget h er (m et a bolom ic, lipidom ic a n d genom ic t oget h er ) a r e goin g t o be t est ed fir st , t wo da t a t ypes (m et a bolom ic wit h lipidom ic or gen om ic, lipidom ic wit h gen om ic or m et a bolom ic) a r e t est ed secon dly a n d la st ly one by on e in pu t s of ea ch (m et a bolom ic, lipidom ic, gen om ic). If a P AT pa sses a ll a im s a ft er eva lua t ion , a ll r esu lt s a n d t est m a t er ia l a r e in t en ded t o be t u r n ed over t o t h e en d u ser . F u r t h er su ppor t will be pr ovided in for m of a n swer in g qu est ion s on specific P AT. Test s on t h e P AT, Un ipr ot a n d F E vE R a r e goin g t o be don e if n o P AT will be fou n d t h a t fu lfill t h e r equ ir em en t s. 2.2.Alte rn a tiv e re s e a rc h m e th o d s Th er e a r e a lt er n a t ive m et h ods t o con du ct t h is st u dy bu t it wou ld in volve wor kin g in a bioch em ist r y la bor a t or y t o obser ve, in t er view a n d obt a in r esu lt s fr om exper im en t s a n d a ft er wa r ds design in g wh ile a lso buildin g a com plet e P AT. An ot h er m et h od is t o m a ke a h om epa ge con n ect in g it t owa r ds a P AT t h a t is bein g u sed in t h e la bor a t or y. Met h od select ed in sect ion 3.1 a n d descr ibed m or e in sect ion 4 is bein g don e by r ea son s of get t ing good qua lit y r esu lt s, t im e sa vin g a n d efficien cy.

3. Biom edica l ba ck gr ou n d Th is sect ion con t a in s ba ckgr ou n d in for m a t ion n eeded in or der t o u n der st a n d t h e biom edica l pa r t . Ga t h er ed in for m a t ion is a bou t gen et ics, lipids a n d t h eir bioch em ist r y, m et a bolom ics, gen om ics a n d ca r d iova scu la r disea se. 3.1.Ge n e tic s Gen et ics is t h e st u dy of gen es wit h t h eir st r u ct u r es, sequ en ces a n d t h eir r ole in h er edit y. It is a wa y t o t r y a n d expla in h ow t h ey wor k, wh a t t h ey a r e a n d wh a t t h ey ca n do [32]. Gen et ics in volve scien t ific st u dies of gen es a n d t h eir effect s lea din g t o va r ia t ion in living or ga n ism s [32]. Mea n in g h ow cer t a in t r a it is or con dit ion s a r e bein g pa ssed down fr om on e gen er a t ion t o t h e n ext . Also h ow gen es a r e u n it is of h er edit y t h a t ca r r y in st r u ct ion s for m a kin g pr ot ein s Page | 13

t h a t dir ect a ct ivit ies in cells a n d fu n ct ion s of ou r bodies. An exa m ple of fu n ct ion is in h er it ed disor der s lea din g t o disea ses [32]. Disor der s h a ve been det ect ed du e t o t h e la r ge a m ou n t of la bora t or y exper im en t s a n d t ech n ology a dva n cem en t s, da t a st or in g pr ovide u se of P ATs, t h u s givin g fu n ct ion s t o sea r ch a n d m a t ch gen es wit h ea ch ot h er .

3.1.1. Gen e Gen es a r e sm a ll m olecu la r u n it is t ha t ca r r y t h e h er edit y of living or ga n ism s. Th e gene h olds t h e in for m a t ion t o bu ild a n d m a in t a in a n or ga n ism . E u ka r yot ic cells h a ve a n u cleu s, wh ich con t a in s t igh t ly pa cked DNA a n d a r e well pr ot ect ed [5]. Th e m a in bu ildin g blocks of a gen e con sist of cova len t ly lin ked n it r ogen ba ses A, T, C a n d G. Th e st r u ct u r es a r e t h en st r en gt h en ed by ca r bon a n d h ydr ogen bonds. Th is m a kes a sequen ce t h a t in t h e en d for m s a lon g dou ble h elix DNA ch a in . Th e DNA ch a in is t igh t ly pa cked t oget h er wit h h ist on es, wh ich a r e pr ot ein s, t o for m a n or ga n ized st r u ct u r e. Th e or ga n ized st r u ct u r e is ca lled ch r om osom es [11]. All t h e ch r om osom es a r e well pr ot ect ed wit h in t h e n u cleu s (Fig 2). Th e DNA ch a in in t u r n codes for m a n y fu n ct ion s of livin g or ga nism s [5]. Gen et ic in for m a t ion a n d t r a it is a lso get s pa ssed on t o t h e offspr in g wh en m a t in g. In ou r gen om e t h er e a r e som e st r u ct u r a l gen es wh ich u pon r ea din g, t ell u s wh a t m a t er ia ls a r e n eeded in or der t o bu ild u p a cell or a n or ga n ism . Th is is ou r gen ot ype. Th e st r u ct u r a l gen es we a r e goin g t o u se a r e det er m in ed in com bin a t ion wit h t h e en vir on m en t a n d t his is ca lled ou r ph en ot ype. Th e ph en ot ype is a lso a ffect ed by t h e en vir on m en t of ea r lier gen er a t ion s a n d t h is is ca lled epigen et ic [5]. Th ose ph en ot ypes a r e e.g. eye color a n d blood t ype. Th e gen ot ypes a r e iden t ica l in a ll hu m a n in dividu a ls u p t o a bou t 99 per cen t . Rem a in in g 1 per cen t va r y fr om per son t o per son cr ea t in g t he fea t u r es t h a t m a kes u s a ll u niqu e. Tin y differ en ces in t h e gen om e sequ en ces dist in gu ish a n in dividu a l fr om a n ot h er [5]. Th e t iny differ en ce on t h e ch a n ges of sin gle ba ses in volves r epr odu ct ion fr om t wo in dividu a ls cr ea t in g a n offspr in g a n d ch a n ges by Sin gle Nu cleot ide P olym or ph ism (SNP ) a s m en t ion ed m or e in t ext below. Keepin g t r a ck of t in y differ en ces is h a r d a n d som e of t h ese t in y gen et ic va r ia t ion s a r e im por t a n t du e t o su scept ibilit y t o cer t a in disea ses (like a st h m a , dia bet es, scler osis a n d ca n cer ), u n less you h a ve a n a na lysis t ool a t you r disposa l [5].

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F ig u re 2. A sch em a t ic pr esen t a t ion of h u m a n DNA a ssem bled in t o a ch r om osom e.

3.1.2. S N P SNP is sh or t for Sin gle Nu cleot ide P olym or ph ism a n d it is a sequ en ce va r ia t ion in DNA. Th is m ea n s t h a t a n it r ogen ba se is differ en t in a gene sequ en ce for on e in dividu a l while t h e r est of t h e gen e sequ ence is st ill sim ila r t o a n ot h er in dividu a l [5]. F or a n exa m ple t h e gen e sequ en ce ATAGGC is a lm ost t h e sa m e a s t h e gen e sequ en ce AT CGGC, h owever , we h a ve a ch a n ge on t h e secon d A t o h a vin g a C in st ea d. Ch a n ges of on e n u cleot ide in t h e sequ en ce of ou r gen es a r e n a m ed Sin gle Nu cleot ide P olym or ph ism (SNP ) a n d occu r t h r ou gh ou t t h e wh ole gen om e [3]. Sin gle Nu cleot ide P olym or ph ism (SNP ) va r ia t ions occu r in a ll species, lea ding t o gen et ic va r ia t ion s a nd m a y r esu lt in differ en t ph en ot ype of t h e or ga n ism . In [4] r esea r ch r esu lt s sh ow h ow differ en t ia t ion h a s occu r r ed. Th e gen et ic ch a n ges a r e ba sed on n a t u r a l select ion t o su it t h e m ost fa vor a ble a da pt ion of t h e gen es [3]. Som e of t h ese Sin gle Nu cleot ide P olym or ph ism (SNP ) sequ en ces a r e even specific t o a n et h n ic gr ou p wh ile it m a y be m issin g in a n ot h er gr ou p. Accor din g t o [32] bot h t h e codin g a n d t h e n on codin g r egion s of t h e DNA ca n be a ffect ed. Sin gle Nu cleot ide P olym or ph ism (SNP ) sequ en ces in volve su scept ibilit y t o disea ses a s m en t ion ed in t h e en d of sect ion 2.1.1. A scen a r io given will descr ibe wh y Single Nu cleot ide P olym or ph ism s (SNP : s) a r e im por t a n t [32]. Cou ples r egist er s for a h ea lt h ch eck a n d gives blood t o be a n a lyzed in or der t o det ect h ow h ea lt h y t h ey a r e. Th e blood goes t h r ou gh t r ea t m en t s so on ly sm a ll sequ en ces of n u cleot ides a r e left . Th e Sin gle Nu cleot ide P olym or ph ism (SNP ) sequ en ce of on e in dividu a l is the followin g: “GCCAGTATTGTCGATTTCACAAGTGCCTTTCTGTCGGGATGTCACACA Page | 15

ACGG”. Ot h er per son has the followin g of “GCCAGTATTGTCGATTTCACAAGTGCGTTTCTGTCGGGATGTCACACA ACGG”. Th e sequ en ces fr om bot h in dividu a l’s a r e codes for a pr ot ein , coding t h e u pt a ke of fa t a n d su ga r in t h e h u m a n body. Th e sm a ll va r ia t ion s bet ween t h ese t wo in dividu a ls a r e m a r ked wit h a color . On e of t h em h a s h igh r isk of get t in g dia bet es. Wit h t h e h elp of t oda y’s t ech n ology, SNP a n a lyses a r e u sed t o det er m in a t e disea se su scept ibilit y [32]. An a lysis r evea ls t er r ible n ews for t h e cou ple, wer e t h e in dividu a l wit h t h e sin gle ba se ch a n ged t o G ha s t o st a r t u sin g in su lin wit h a syr in ge, u n less food h a bit ch a n ge wit hin a yea r or t wo. Th e scen a r io descr ibed a bove a r e ver y com m on in h ea lt h ca r e t oda y a n d a lso n ot t h e on ly wor k a r ea exploit ing gen et ic va r ia t ion s. In for en sic scien ce t h e gen et ic va r ia t ion s a r e exploit ed du r in g DNA fin ger pr in t in g [32]. 3.2.B io c h e m is try o f Lip id s Bioch em ist r y is a lso ca lled biologica l ch em ist r y wh ich is t h e st u dy of ch em ica l pr ocesses in living or ga n ism s. Bioch em ist r y r egu la t es a n d gover n s over a ll livin g pr ocesses wit h in a ll living or ga n ism s [5]. Th is occu r s by bioch em ica l sign a lin g. Th e signa lin g is sor t of a n in form a t ion flow a s in sen din g a m essa ge fr om on e pla ce t o a n ot h er . Sign a ls flow t h r ou gh ever y pa r t in a n or ga n ism r egu la t in g t h e m et a bolism . Met a bolism st a n ds for t h e m ea n in g of livin g or ga n ism s t o su st a in life a n d r epr odu ce t h em s elf. On e im por t a n t pa r t in bioch em ist r y is t h e lipids. Lipids a r e im por t a n t com pon en t s in a cell a n d for m cell m em br a n e, vit a l t issu es a n d ser ve a s a n en er gy sou r ce for t h e or ga n ism [1]. Lipids a r e st or ed a s en er gy r eser ves wit h in t h e or ga n ism a nd u sed wh e n n eeded. Lipids h elp keepin g t h e elect r och em ica l ba la n ce of a cell, cell sign a lin g a n d t r a ffickin g r ega r din g wh a t is goin g in or ou t t o t h e cell [1 1]. Th e lipids u su a lly con sist of a pola r h ea d a n d a h ydr oph obic t a il. Th e lipids bin d t o ea ch ot h er du e t o t h e hydr oph obic pa r t wa n t s t o st a y in con t a ct wit h ot h er h ydr oph obic m olecu les [3]. Th e dist r ibu t ion bet ween t h e h ydr oph obic a n d pola r pa r t s of t h e lipids dir ect s t h e 3 -dim en sion a l st r u ct u r e of t h e m olecu les [7] a n d wit h a r ela t ively la r ge pola r pa r t , t h e lipids for m m icelles wh ile m or e equ a l dist r ibu t ion , lea ds t o t h e for m a t ion of dou ble la yer s kn own a s m em br a n es (Fig 3).

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F ig u re 3. P ict u r e of lipids wit h h ydr oph obic t a ils bou n d t oget h er a n d wit h ot h er com pon en t s for m in g t h e m em br a n e. (Modified pict u r e t a ken fr om H u m a n Cell Biology r ef. [43]).

3.2.1. L ipid d efin ition Ch em ist s, bioch em ist s a n d ot h er a n a lyst s t h a t wor k wit h lipids h a ve a gr ea t a n d fir m u n der st a n din g of t h e t er m ca lled lipid a ccor din g t o [19]. But t h er e is n o widely a ccept ed defin it ion t oda y a n d t h ey a r e sa id t o be a gr ou p of n a t u r a lly occu r r in g com pou n ds. In a n or ga n ism , [44] a n d [53] st a t e t h a t t h ou sa n ds of va r iou s for m s of lipid m olecu les ca n be fou n d a n d lipids ca n be ca t egor ized in t o six m a in ca t egor ies (Fig 4). Th ey a ll h a ve a low solu bilit y in wa t er a n d h igh solu bilit y in or ga nic solven t s.

3.2.2. Classes of L ipid s Recen t ly a n ew n om en cla t u r e syst em wa s pr oposed by [26] du e t o t h e diver sit y of lipids in h u m a n pla sm a , sepa r a t in g lipids in t o eight cla sses or ca t egor y wh er e six of t h em a r e con sider ed m a in cla sses. E a ch cla ss ca n be fu r t h er divided in t o su b cla sses a n d in dividu a l m olecu la r species (Fig 3).  Th e fir st ca t egor y is t h e fa t t y a cyls a n d is a lso ca lled fa t t y a cids. Th e fa t t y a cids ca n h a ve t h r ee for m s su ch a s fa t t y a cids, oct a deca n oids a n d eicosa n oids. Th ey a r e t h e m ost com m on bu ildin g block for m or e st r u ct u r a l com plex lipids a n d ca n be sa t u r a t ed or u n sa t u r a t ed. Cells u se t h ese lipids t o for m t h e va r iou s m em br a n es fou n d in a cell, t o st or e en er gy a n d t o a dju st t h e m em br a n e flu idit y in m a ny ce lls. [43, 53]  Secon d ca t egor y is t h e Glycer olipids a n d h a s t h r ee for m s a s m on o-, di- a n d t r ia cylglycerolipids. Th eir fu n ct ion s a r e m a in ly a s en er gy st or a ge a n d a r e bu lked u p in t h e t issu e a s fa t in a n im a ls. [43, 53]

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 Th ir d ca t egor y is ca lled Glycer oph ospolipids bu t t h ey a r e u su a lly ca lled ph osph olipids. Th e m a in for m s a r e P h osph a t idylch olin e (P C), P h osph a t idylch et h a nola m in e (P E ) a n d P h osph a t idic a cid (P A). Th e glycer oph ospolipid cla sses a r e t h e on ly on es t h a t h a ve a ph osph or bin din g a n d t h ey a r e t h e key com p on en t in or der t o for m bila yer s. [43, 53]  Th e fou r t h ca t egor y con sist s of Sph in golipids. Th e m a in for m s a r e Sph in gom yelin a n d Cer a m ides. Th e Sph in golipids h a ve a pola r h ea d a n d t wo n on pola r t a ils. Sph in gom yelin a ct a s a pr ot ect ion for m in g a m yelin sh ea t h t o pr ot ect n er ves. [43, 53]  Th e fift h ca t egor y is t h e St er ol lipids a n d t h ey a r e of va r iou s a lcoh ol for m s. St er ol lipids a r e a n im por t a n t com pon en t for biologica l r oles. St er ols a ct a s r egu la t in g h or m on es a n d a s sign a ling m olecu les. [43, 53]  Th e la st ca t egor y is t h e P r en ols t h a t for m t er pen es a n d a ct a s a pr e cu r sor m olecu les of vit a m in s a s vit a m in A, E a n d K. [43, 53]

3.2.3. E n zym es in volved in th e syn th esis of lipid s A deeper in sigh t is pr esen t ed in t h is sect ion wit h focu s on lipids a n d it is syn t h esis, for a m or e u n der st a n din g on t h e a m ou n t of in for m a t ion a P AT m u st be a ble t o pr ocess. St a r t in g fr om t h e st a r t of da t a in pu t s (a lipid n a m e con n ect ed t o glycer olipids) t o r esu lt s obt a ined. Som e lipid ch a in s a r e ver y lon g or com plex wh ile ot h er s a r e sh or t . It wou ld t a ke a lon g t im e t o ch em ica lly syn t h esize t h e lipids, h owever , wit h t h e h elp of en zym es it is m u ch fa st er a s [37] pr esen t s. Nu m er ou s for m s of lipids occu r a n d sever a l en zym es a r e n eeded. In [46] a syst em biology view pr esen t s n eeded en zym es by u se of a P AT. E .g. t h e syn t h esis of fa t t y a cids occu r s in t h e cyt opla sm a n d key en zym es in volved a r e t h e a cet yl -CoA ca r boxyla se (ACC) a n d m a lon yl-CoA ca r boxyla se (MCC) st a t ed in [51]. Wh ile a n ot h er gr ou p of coen zym e ca lled Acyl-CoA, ch or est er ol a cylt r a n sfer a se (ACAT) wor ks on ch olest er ol [51]. This is st r engt h en ed in [45] sh owin g a clea r view by pict u r es. Th e fa t t y a cids a r e so m a n y a n d ca n be sa t u r a t ed or u n sa t u r a t ed a n d for t h is pu r pose design a t ed sym bols a r e given [31] in or der t o keep t r a ck of t h e ca r bon a t om s a n d t h eir bin dings. Th e sym bols con sist of t wo n u m ber s bet ween a colon (:) [31]. Th e fir st n u m ber t ells u s t h e ca r bon len gt h of t h e fa t t y a cid a n d t h e secon d n u m ber t h e st a t e of sa t u r a t ion . A fa t t y a cid wit h sever a l u n sa t u r a t ed bou n ds sh ows a h igh er n u m ber a t it is secon d va lu e (S ee T able 3). Syn t h esis of fa t t y a cids beyon d 16 ca r bon s len gt h goes t h r ou gh a t wo-ca r bon elonga t ion pr ocess, a ccor din g t o [31] by en zym es in t h e en dopla sm ic r et icu lu m (E R). Not only elon ga t ion occu r s bu t a lso desa t u r a t ion by en zym es in t h e en dopla sm ic r et icu lu m (E R) u sin g fou r en zym es n a m ed desa t u r a se delt a fou r , delt a five, delt a six a n d delt a nin e. Th e design a t ed delt a n a m es wit h a n u m ber a r e given a ccor din g t o wh ich posit ion in t h e fa t t y a cid ca r bon ch a in t h e desa t u r a t ion occu r s [31]. Th e m a in den a t u r a se is delt a nin e a n d is ca lled St ea r oyl-CoA desa t u r a se-1. Th e desa t u r a t ion r equir es oxygen (O2), a coen zym e ca lled Nicot in a m ide a den in e dinu cleot ide h ydr ogen (NADH ) a n d Page | 18

a n elect r on t r a n spor t in g h em opr ot ein ca lled Cyt och r om e b5 [47]. In fa t t y a cid desa t u r a t ion t wo h ydr ogen a t om s a r e r em oved fr om t h e fa t t y a cid m a kin g a n oxida t ion on bot h t h e fa t t y a cid a n d NADH . Th is cr ea t es a dou ble bon d bet ween ca r bon s in t h e fa t t y a cid ch a in .

Ta ble 3. Th e m a in fa t t y a cids in or ga n ism s (Modified t a ble t a ken fr om Cyber lipid cen t er r ef. [31] a n d Vir gin ia web edu ca t ion r ef. [10]) Main fatty acids Number of Name Systematic name Symbol Structure carbons Saturated fatty acids 12

Lauric acid

Dodecanoid acid

12:0

CH3( CH2)10COOH

14

Myristic acid

Tetradecanoic acid

14:0

CH3( CH2)12COOH

16

Palmitic acid

Hexadecanoic acid

16:0

CH3( CH2)14COOH

18

Stearic acid

Octadecanoic acid

18:0

CH3( CH2)16COOH

20

Archidic acid

Eicosanoic acid

20:0

CH3( CH2)18COOH

22

Behenic acid

Docosanoic acid

22:0

CH3( CH2)20COOH

24

Lignoceric acid

Tetracosanoic acid

24:0

CH3( CH2)22COOH

Unsaturated fatty acids 16

Palmitoleic acid

9-Hexadecanoic acid

16:1

CH3( CH2)5CH=CH(CH2)7COOH

18

Oleic acid

9-Octadecanoic acid

18:1

CH3( CH2)7CH=CH(CH2)7COOH

18

Linoleic acid

18:2

CH3(CH2)4(CH=CHCH2)2(CH2)6COOH

18

a-Linolenic acid

18:3

CH3CH2(CH=CHCH2)3(CH2)6COOH

18

g-Linolenic acid

18:3

CH3(CH2)4(CH=CHCH2)3(CH2)3COOH

20

Arachidonic acid

20:4

CH3(CH2)4(CH=CHCH2)4(CH2)2COOH

24

Nervonic acid

9,12-Octadecanoic acid 9,12,15-Octadecanoic acid 6,9,12-Octadecanoic acid 5,8,11,14Eicosatetraenoic acid 15-Tetracosanoic acid

24:1

CH3(CH2)7CH=CH(CH2)13COOH

Com plex lipids h a ve a lon ger biosyn t h et ic pa t h wa y a n d t wo m a in pa t h wa ys a r e kn own a ccor din g t o [47], t h e sn -glycer ol-3-ph osph a t e pa t h wa y (a lso kn own a s t h e Ken n edy pa t h wa y) a n d t h e m on oa cylglycer ol pa t h wa y (Fig. 5 an d 6). Syn t h esis by t h e Ken n edy pa t h wa y occu r s in t h e liver a n d a dipose t issu es wh ile t h e m on oa cylglycer ol pa t h wa y t a kes pla ce in in t est in e con fir m ed in [42]. Bot h st a r t s by ca t a bolism of glu cose (glycolysis) r esu lt in g in t h e bio-syn t h esis of glycer ol, h owever , n ew eviden ce in [47] in dica t es som e glycer ol is syn t h esized a n ew (de n ovo) fr om sin gle m olecu les by a pr ocess ca lled glycer on eogen esis. Th e followin g r ea ct ion s occu r in t h e Page | 19

en dopla sm ic r et icu lu m (E R) of m a m m a lia n or ga n ism s [47]; sn -glycer ol-3ph osph a t eis est er ified by a fa t t y a cid coen zym e in a ca t a lyt ic r ea ct ion by t h e en zym e glycer ol-3-ph osph a t e a cylt r a n sfer a se (GP AT) a t t h e sn - posit ion in or der t o for m lysoph osph a t idic a cid. Lysoph osph a t idic a cid t h en becom es a cyla t ed for m in g ph osph a t idic a cid, a n in t er m edia t e pr odu ct in t h e syn t h esis of a ll glycer olipids [47]. Du r in g syn t h esis of t r ia cyl-sn -glycer ol t he ph osph a t e gr ou p is r em oved by a fa m ily of en zym es ca lled lipid ph osph a t e ph osph a t a se (P AP ), st a t ed by [47], for m in g 1,2-dia cyl-sn -glycer ols a n d fu r t h er a cyla t ed by dia cylglycer ol a cylt r a n sfer a se (DGAT) in t o t r ia cyl -sn glycer ol (Fig 5). Du r in g syn t h esis of glycer oph ospolipids, ph osph a t idylch olin e (P C), ph osph a t idylch et h a n ola m in e (P E ) and ph osph a t idylser in , t h e ph osph a t e gr ou p is n ot r em oved st a t ed by [6] fr om ph osph a t ic a cid. In st ea d ph osph a t ic a cid a r e u sed a s pr e -cu r sor m olecu les in t h e syn t h esis of glycer olipids (Fig 6). Th e syn t h esis by t h e m on oa cylglycer ol pa t h wa y is less com plex a n d in volves on ly a few en zym es belon gin g t o a n a cylglycer ol a cylt r a n sfer a se fa m ily t o for m t h e t r ia cylglycer ols in t h e in t est in e [47].

F ig u re 4. Seven lipid cla sses a n d h ow t h ey in t er a ct bio-syn t h et ica lly (Modified pict u r e t a ken fr om Molecu la r bioch em ist r y r ef. [45]).

F ig u re 5. Th e Ken n edy pa t h wa y syn t h esis in m a m m a ls (Modified pict u r e t a ken fr om Th e AOCS Lipid Libr a r y – Tr ia cylglycer ols r ef. [47]). Page | 20

F ig u re 6. Syn t h esis of a cylglyer ides a n d glycer oph osph olipids sh owin g a lin k bet ween t h e t wo pa t h wa ys (Modified pict u r e t a ken fr om Lipid Libr a r y r ef. [35]).

3.2.4. L ipoprotein s Lipids a r e a lm ost in solu ble, h owever , t h er e a r e wa ys for t h em t o be t r a n spor t ed or pa ss t h r ou gh t h e blood cir cu la t ion [5]. Lipopr ot ein s a llow t h e lipids t o be t r a n spor t ed t h r ou gh t h e blood cir cu la t ion in or der t o r ea ch differ en t t issu es [5]. Th e lipopr ot ein s a re a ssem bled in a wa y t ha t it con t a in s bot h pr ot ein s a n d lipids. The pr ot ein pa r t ser ves a s a n em u lsifica t ion for t h e lipids [11] a n d t h er e a r e five m a jor cla sses, t wo being ver y im por t a n t cla sses of t h e lipopr ot ein s [1], h igh den sit y lipopr ot ein s (H DL) a n d low den sit y lipopr ot ein s (LDL). Rem a in in g lipopr ot ein s a r e In t er m edia t e den sit y lipopr ot ein (IDL), ver y low den sit y lipopr ot ein s (VLDL) a n d ch ylom icr on s [11]. Bot h HDL a n d LDL ca r r y lipids a s ch olest er ol a n d LDL is som et im es r efer r ed t o a s t h e ba d ch olest er ol wh ile H DL is t h e good ch olest er ol. P r oblem s ca n occu r du r in g t h e oxida t ion of t h e LDL a ccor din g t o [11], lea ding t o a lm ost u nst oppa ble ch a in r ea ct ion s. Ch a in r ea ct ion effect r esu lt s in a t h er oscler osis m a n y yea r s la t er [1 1]. 3.3.Ge n o m ic s Gen om ics is a disciplin e wit h in gen et ics t h a t focu s on t h e st u dy of t h e gen om e of a ll or ga n ism s [32]. Wit h in t h is field of r esea r ch t h e pu r pose is t o det er m in e t h e en t ir e DNA sequ en ce of a ll or ga n ism s a n d m a kin g a sca led m a ppin g of a ll t h e gen es [32]. Th is in clu des a lso m a ppin g of wh a t a gen e does a n d t h e a ssocia t ion it h a s t o pr ocesses wit h in a n or ga n ism e.g. m et a bolom ics or lipidom ics [7]. Du r in g t h e pr ocess of m a ppin g a n d a ssocia t ion ea ch gen e get s a design a t ed n a m e a n d n u m ber (a s a n ID t a g) wit h a ll t h e n ecessa r y in for m a t ion pr ovided a bou t t h a t specific gen e in a da t a ba se [32]. In for m a t ion ca n be r et r ieved wit h a P AT fr om t h ese da t a ba ses wh en n eeded. Page | 21

3.4.Me ta bo lo m ic s Met a bolom ics is t h e st u dy of ch em ica l pr ocesses in volving m et a bolit es a n d soph ist ica t ed a n a lyt ica l t ech n ologies a r e u sed t o m a ke syst em a t ic st u dies [8, 18]. A syst em a t ic st udy con sist s of t a r get a n a lysis, m et a bolit e pr ofilin g a n d m et a bolic fin ger pr in t in g. Th e m et a bolit es a r e fou n d in a ll biologica l cells a n d a ll h a ve u niqu e ch em ica l sign a t u r es, like a fin ger pr in t [46]. Th e u n iqu e fin ger pr in t s a r e a n en d pr odu ct a ft er a cellu la r pr ocess a n d ca n be u sed t o see h ow specific ch em ica l pr ocesses h a ve occu r r ed [8, 46]. Th e ch em ica l pr ocesses t h a t a r e exa m in ed ca n be fr om a livin g or ga n ism , cells, t issu es a n d even fr om a n or ga n . Resea r ch field of m et a bolom ics con sist s of m a n y su b pa r t s a n d t h e pa r t s we a r e focu sin g on a r e t h e st u dy of lipidom ics (lipids/fa t t y a cids) [40]. 3.5.Lip id o m ic s Lipidom ics is u sed for descr ibin g t h e com plet e pr ofile of lipids in cells, t issu es or or ga n ism s [47]. Lipidom ics a r e on e su bpa r t of m et a bolom ics a n d a n ewly em er ged r esea r ch field t h a t h a s been dr iven fa st for wa r d by r a pid a dva n ces in t ech n ology [53]. Such t ech n ologies a r e e.g. m a ss spect r om et r y (MS), flu or escen ce spect r oscopy (F S) [24], a n d Nu clea r Ma gn et ic Reson a n ce (NMR) [39]. Th ese t ech n ologies sa ve la r ge a m ou n t s of da t a in da t a ba ses, givin g P AT's possibilit y of a n ew m et h od for da t a a n a lysis [18]. 3.6.Ca rd io v a s c u la r d ise a s e s Th er e a r e m a n y disea ses a r ou n d t h e wor ld. On e of t h em is a cla ss of disea ses t h a t in volve h ea r t or vessels t h a t t r a n spor t s blood (a r t er ies a n d vein s) a n d a r e ca lled ca r diova scu la r disea ses [46]. Ca r diova scu la r disea ses in clu de t h e following: An eu r ysm (Abn or m a l bu lge in a n a r t er y), An gin a (Ch est pa in du e t o la ck of blood t o t h e h ea r t m u scle), At h er oscler osis (pla qu e bu ilds u p in side t h e a r t er ies), Cer ebr ova scu la r Acciden t (St r oke), Con gest ive H ea r t F a ilu r e, Cor on a r y Ar t er y Disea se a n d Myoca r dia l In fa r ct ion (H ea r t At t a ck). Sever a l r esea r ch er s in [12] cla im som e kn own fa ct or s a s lipid or fa t con t en t ca n a ffect t h e ca r diova scu la r syst em , poin t in g t h a t high den sit y lipopr ot ein s (H DL) a n d low den sit y lipopr ot ein s (LDL) a r e r ega r ded t o be a fa ct or beh in d t h e ca r diova scu la r disea ses. On ce t h e disea se is det ect ed it h a s u su a lly pr ogr essed for yea r s a n d lea ds t o t h e n ecessit y of opera t ion or even dea t h . An ot h er gr ou p of r esea r ch er s in [29] h a ve sh own t h a t t h er e a r e sever a l SNP :s wh ich is a ssocia t ed wit h pla sm a level of h igh den sit y lipopr ot ein s (H DL) a n d low den sit y lipopr ot ein s (LDL) wh ich a r e a ssocia t ed wit h m yoca r dia l in fa r ct ion. Aft er t est in g in dividua ls in [2], fr om five differ en t et h n ic gr ou ps wit h r espect of eigh t SNP fr om t h r ee gen es a ssocia t ed wit h ch olest er ol a n d lipopr ot ein syn t h esis, a clea r cor r ela t ion wit h m yoca r dia l in fa r ct ion s wa s obt a in ed. A st u dy on r isks for m yoca r dia l in fa r ct ion in [48] st r en gt h en s t h is t h eor y.

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4. Com pu t er Scien ce ba ck gr ou n d Th is sect ion con t a in s ba ckgr ou n d in for m a t ion n eeded in or der t o u n der st a n d t h e IT pa r t a n d h ow a P AT wor ks. 4.1.D a ta ba s e s , D a ta m in in g an d Kn o w le d ge d is c o v e ry A da t a ba se con sist s of t a bles wit h m a n y colu m n s a n d r ows wit h a collect ion of va st a m ou n t of da t a in for m of in for m a t ion . Th e in for m a t ion is st or ed a t a specific pla ce a n d is oft en bein g well or ga n ized. Wit hin t h e da t a ba se depen din g on wh a t is in ser t ed in t o it , t he in for m a t ion ca n be of gen et ics, lipidom ics or a bou t som et h in g else en t irely. Da t a ba ses oft en r equ ir e som e for m of t ool in or der t o r ea d a n d r et r ieve specific in for m a t ion fa st a m on g t he va st a m ou n t of da t a . Th is is wh er e da t a m inin g com es in , wh en specific in for m a t ion is sou gh t a n d ga t h er ed by a t ool, t h en pr esen t ed a s r esu lt s. F r om t h e pr esen t ed r esu lt s, kn owledge ca n be ga in ed . Th e ga in ed kn owledge h a ve m a n y for m s bu t a few of t h ese ca n per h a ps be t o m a ke im pr ovem en t in exper im en t s, con fir m in g exper im en t r esu lt s or per h a ps ch a n ge a ppr oa ch m et h ods t o solve a scien t ific pr oblem [13]. 4.2.P AT As m en t ion ed ea r lier a ll kin ds of IT-ba sed t ools h a ve been developed in va r iou s biom edica l fields [21]. This h a s been don e in or der t o keep t r a ck of a ll t h e n ecessa r y scien t ific in for m a t ion obt a ined du r in g t h e pa st yea r s [20]. P AT t h a t we a r e wor kin g wit h pr ocess in for m a t ion a bou t gen es, SNP :s a n d lipid m et a bolism . Th er efor e t h e P AT pla y a n im por t a n t r ole in lipidom ics a n d gen om ics r esea r ch . Th e t ools ca n be eit h er web ba sed soft wa r e or down loa da ble pr ogr a m s t h a t t a ke da t a in for m s of a gen e n a m e, SNP a ccession ID (r s n u m ber ) or a cla ss n a m e of t h e lipid. Th e P AT t h en pr ocess t h e da t a given , m a kin g sea r ch es in loca l or r em ot e da t a ba ses. Th e da t a ba ses con sist s of m a n y t a bles wit h differ en t in for m a t ion r ela t ed t o genes a n d lipids. Wh a t t h e P AT do is m a kin g m a n y join fu n ct ion s bet ween t h e t a bles a n d pu t t h ese in for m a t ion t oget h er . Wh en a sea r ch occu r s by da t a m in in g, t h e in for m a t ion fr om t h ese t a bles a r e ga t h er ed a n d pr esen t ed a s r esu lt s ba sed on t h e in pu t pa r a m et er s [9]. Th e in pu t pa r a m et er s a r e t h e in ser t ed t ext s in t he sea r ch field. In som e ca ses t h e t ools fa il t o pr ovide r esu lt s on cer t a in in pu t . F in a lly t h e a na lysis t ool sh ows t h e r esu lt s t elling if a n y r esu lt s wer e fou n d, wh er e t h e lipids or gen es a r e u sed in t h e m et a bolism a n d h ow t h ey a r e con n ect ed t o ot h er lipids or gen es in t h e m et a bolism . Th e r esea r ch er s wor kin g wit h t h e P AT ca n fu r t h er a n a lyze t h e r esult s in or der t o ga in n ew kn owledge. Wit h n ew kn owledge, n ew discover ies ca n be m a de in e.g. lipidom ics or gen om ics a n d t h is lea ds t o t h e dem a n d for u pda t es t o t h e da t a ba ses a n d t h e P ATs (Fig 8). Wit h t h e h elp of P AT, n ew discover ies of disea ses like a t h er oscler osis ca n be m a de [24] or n ew pa t h wa y lin ks in t h e m et a bolic syst em r espon ses ca n be fou n d [52].

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F ig u re 8. A view on h ow ever yt h in g is con n ect ed (bot h vir t u a lly a nd ph ysica lly) t o t h e P AT.

5. Requ ir em en t s a n d Test elicit a t ion Th e followin g sect ion con t a in s in for m a t ion a bou t r equir em en t specifica t ion , t est pla n , t est ca se, a n d soft wa r e t est in g. 5.1.Re qu ire m e n ts Ma n y soft wa r e pr ogr a m s or t ools r equ ir e m on t h s of t est in g t o see if t h ey fu n ct ion cor r ect ly a n d n eed ver y t h or ou gh set s of specific r equ ir em en t s in or der t o be con sider ed a s good fu n ct ion a l t ools, a s st a t ed in [39]. Specific set s of r equ ir em en t s ca n be obt a in ed by in t erviews wit h t h e people wh o a r e of im por t a n ce su ch a s st a keh older s or in dividu a ls wit h a key r ole. We u se a n em pir ica l m et h od on soft wa r e t est in g by ga t h er in g r equir em en t s pa r t ia lly ba sed on a r equ ir em en t s ph a se fr om on e of t h e wa t er fa ll m odels u sed by Ia n Som m er ville [50]. In t h e r equ ir em en t s ph a se, som et im es a lso ca lled r equ ir em en t s en gin eer in g ph a se, br a in st or m in g, r esea r ch a n d a n a lysis is bein g con du ct ed on t h e soft wa r e t h a t will eit h er be developed or t est ed. We in t en d t o do t h e t est in g pa r t wit h ou t developin g a n y n ew P AT a n d t h er efor e on ly t h e r equ ir em en t s m et h odology is a da pt ed a n d a pplied in ou r st u dy. Th e br a in st or m in g, r esea r ch a n d a n a lysis is t h e m ost im por t a n t pa r t of r equ ir em en t s ga t h er in g. Ba sic r equ ir em en t s a r e defin ed a n d set for u ses t h a t t h e soft wa r e m u st su ppor t [49]. Du r in g t h is ph a se, in -dept h st u dies of cu r r en t wor kin g pr ocesses a r e don e a n d h ow t h e pr oblem s ca n be a ddr essed or solved. Th u s, wit h ou t u n der st a n din g t h e r equir em en t s given , h opes of deliver in g a Page | 24

su ccessfu l syst em or soft wa r e is u n likely. Requ ir em en t s elicit a t ion s a r e don e in volvin g m a n y in t er views wit h key in dividu a ls. Requ ir em en t s a r e a n a lyzed a n d eva lu a t ed a n d ca n be of t h r ee kin ds. Th ose wit h h igh est pr ior it y a r e t h e m u st h ave, secon d pr ior it y a r e sh ou ld h ave a n d t h e lowest pr ior it y a r e t h e n ice to h ave. Specifica t ions a r e t h en m a de t o t h e r equ ir em en t s a n d va lida t ion s a r e don e wh er e t h e in dividu a ls wit h a key r ole or st a keh older s a ccept t h e r equ ir em en t s. Requ ir em en t s a r e t h er efor e n eeded in or der t o defin e wh a t a soft wa r e pr ogr a m m u st or sh ou ld do, fea t u r es it h a s a n d a cer t a in qu a lit y t h a t it m u st fu lfill [39]. Requ ir em en t s a r e divided in t o fu n ct ion a l a nd n on fu n ct ion a l r equ ir em en t s. F u n ct ion a l r equ ir em en t s a r e a lwa ys defin ed wit h sh a ll or m u st . Non fun ct ion a l r equir em en t s a r e pr oper t ies or cer t a in qua lit ies a pr odu ct m u st h a ve a n d a r e u sed t o descr ibe soft wa r e’s u sa bilit y, r elia bilit y a n d per for m a n ce [17]. Qu a lit y is n ot som et h in g t h a t ca n be m ea su r ed ea sily a s m en t ion ed in sect ion 1.3, bu t on e wa y t o m ea su r e qu a lit y a ccor din g t o [23] is by u sin g m et r ics or st a t ist ics on pr oper t ies t h a t ca n be m ea sur ed a n d t h a t a r e a ssocia t ed wit h qu a lit y. 5.2.Te s tin g In or der t o do good t est in g, t est pla n s a r e developed in t h e for m of a docu m en t so t h a t syst em a t ic a ppr oa ch es ca n be u sed on soft wa r e t est in g. Th e syst em a t ic a ppr oa ch es a r e fa st a n d t im e sa vin g [30]. A t est pla n descr ibes t h e t est in g ph a ses (e.g. u n it t est in g, in t egr a t ion t est in g, a ccept a n ce t est ing), t h e n eeded r equ ir em en t s, a ll a ct ivit ies, t h e n eeded r esou r ces a n d t h e docu m en t a t ion n ot es. In t h is wor k, we u se a ccept a n ce t est in g t o det er m in e if a set of r equ ir em en t s will be m et . Th e a ccept a n ce t est in g is r u n n in g wit h specific da t a . On ce t h e r esu lt s a r e obt a in ed, t h ey a r e com pa r ed wit h kn own expect ed r esu lt s. Upon cor r ect m a t ch of t h e r esu lt , a pa ss or n o pa ss is given . Th er e a r e m a n y wa ys t o m a ke t est s a n d m ost of t h e t im e t h ey differ bet ween com pa n ies [30]. All t est pla n s a re cr ea t ed pr ior t o a n y t est in g. Th e con t en t of a t est pla n u su a lly con sist s of a su it a ble n a m e r ela t ed t o t h e t est s per for m ed. On ce a t est pla n is select ed, it is per for m ed a ccor din g t o a t em pla t e [30] wh er e t h e t est pla n docu m en t is divided in t o t wo pa r t s. Th e fir st pa r t con sist s of gen er a l in for m a t ion a bou t t he t est s t h a t will be per for m ed. Th e gen er a l in for m a t ion con t a in s pa r t icipa n t s, t est st r a t egies, specifica t ion s a n d fu n ct ion s or fea t u r es t h a t will be u sed. Th e secon d pa r t con t a in s t h e pr ocedu r e on scen a r ios a lso ca lled ca ses on h ow t est s will be don e. Th e cr ea t ion of t he t est pla n , r equ ir em en t s a n d t est ca ses will lea d t o t h e a ct u a l soft wa r e t est in g. Th e pu r pose of t h e soft wa r e t est in g is t o in vest iga t e a n d ga in in for m a t ion on h ow t h e select ed soft wa r e pr ogr a m wor ks [39]. Soft wa r e pr oper t ies a r e eva lua t ed du r in g a select ed soft wa r e t est in g. Du r in g t h e soft wa r e t est in g, bu gs a n d er r or s t h a t m igh t occu r a r e a lso elim in a t ed [23]. On ce t h e soft wa r e pr ogr a m or a pplica t ion m eet s t h e r equ ir em en t s, it is con sider ed t o be wor k in g well a n d a lso sa t isfies t h e n eeds of t h ose wh o r equ est ed it .

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5.3.Te s t c a s e s A set of t est con dit ions h a ve t o be wr it t en fr om fu n ct ion a l r equ irem en t s [17] if soft wa r e is goin g t o be t est ed. Th e t est con dit ion s a r e r efer r ed t o a s t est ca ses a n d a r e per for m ed in cer t a in sequ en ces wh ich it m u st follow du r in g t est in g. F ir st t h e goa l of t he t est is descr ibed wit h t h e even t or execu t ion st eps, followed by t h e expect ed r espon se a n d t h e a ct u a l r espon se fr om t h e t est . Ma n y t im es t est ca ses a r e cr ea t ed, wit h a set of con dit ions fr om given r equ ir em en t s a s m en t ion ed a bove, in or der t o elim in a t e t h e a m biguit y t o t h e m in im u m in soft wa r e [21]. Th e a m ou n t of t est ca ses depen ds on t h e a m ou n t of r equ ir em en t s given . To per for m t h e t est in g ph a se, t est er s a r e select ed t o exa m in e, discover a nd det er m in e if t h e soft wa r e is wor kin g cor r ect ly or n ot du r in g t est in g. To keep t r a ck of a t est er 's wor k t r a cea bilit y m a t r ices a r e u sed, lin kin g r equir em en t s t o specific t est ca ses (Fig 8). A t est ca se con sist s of m a n y st eps st a r t in g wit h a n in pu t ba sed on a r equ ir em en t t h a t will be t est ed a n d en din g on ce a ll st eps h a ve been com plet ed. Th e a ct ion or execu t ion even t s a r e t h en goin g t o be m a de on h ow t o do t h e t est wit h descr ipt ion s on expect ed r espon se or ou t com e. Th e a ct u a l r esu lt s obt a in ed will be wr it t en down on ce t h e t est ca se is com plet e.

F ig u re 1. A t est ca se t em pla t e u sed in t h is st u dy.

6. Resu lt 6.1.F in d in g th e P ATs Wit h u se of In t er n et , sea r ch in g for P ATs, m a n y wer e fou n d a n d m ost of t h em h a d t h eir own h om epa ges. Sea r ch es wit h t h e Google sea r ch en gin e wer e m a de a s “P AT”. 46 h om epa ges wer e fou n d, descr ibing a n a lysis t ools t h a t h a d t o be eva lu a t ed. Th e h om epa ges st a t ed wh a t t ype of da t a t h e P ATs cou ld pr ocess, wh ich wa s con fir m ed by down loa din g a n d t est in g t h e P ATs. The down loa ded P ATs wer e eva lu a t ed wit h t est ca ses 1 t o 3 t h a t r equ ir e t h e t ool t o be a ble t o Page | 26

in pu t a n d pr ocess lipidom ics, m et a bolom ics a n d gen om ics da t a a s descr ibed in a ppen dix 1. Used genom ic da t a wit h kn own cor r ela t ion s wit h lipid syn t h esis a n d m et a bolism a r e sh own in a ppen dix 2. Ou t of 46 P ATs, 23 a n a lysis t ools fu lfilled t h e cr it er ia of bein g a ble t o pr ocess m et a bolom ic, lipidom ic a n d gen om ic da t a . 6.2.S o rtin g th e P ATs Sor t in g of t h e P ATs a r e m a de by r a n kin g t h em ba sed on t h e possibilit y of pr ocessin g m or e t h a n on e t ype of da t a . Th e In gen u it y P AT is t h e on ly on e t ha t pr ocesses a ll t ypes of da t a . Th e m a jor it y of t h e select ed P AT's on ly pr ocess gen om ics da t a (13 ou t of 23) wh er ea s 9 of t h e 23 P ATs cou ld pr ocess bot h gen om ic a n d pr ot ein da t a (Fig 9).

F ig u re 9. A r a n kin g list of P ATs ba sed on n u m ber of da t a t hey ca n pr ocess a n d t h e ID n u m ber of t h e r equ ir em en t it pa ssed. 6.3.Te s tin g th e P ATs P AT's goes t h r ou gh t est in g wit h t est ca ses of 4 a n d 5 t h a t a r e lin ked t o r equ ir em en t ID 2 a nd 3, a ccor din g t o su b a im 2 in sect ion 1.2. Ver y lit t le lit er a t u r e in for m a t ion ca n be fou n d wh en sea r ch in g for m et a bolom ics, lipidom ics a n d gen et ic pa t h wa ys or cor r ela t ion s wit h P ATs. Books fou n d descr ibed ba sic in for m a t ion a bou t t h e a ct u a l m et a bolom ics, lipidom ics a n d gen et ics bu t n ot m u ch of t h eir pa t h wa ys or cor r ela t ion s [5, 31 a nd 3]. Sea r ch es on In t er n et give few r esu lt s of a r t icles a nd jou r n a ls (su ch a s [10, 34, 41 a n d 46]). Wh en doin g t h e com pa r ison bet ween obt a in ed r esu lt s fr om t h e P ATs, t h r ee r efer en ces ca n be fou n d in wh ich t wo ou t of t h r ee a r e r ela t ed t o lipids a n d t h eir syn t h esis pa t h wa y [1, 5]. Th e t hir d lit er a t u r e r efer en ce is a bou t cor r ela t ion of gen et ics t o pa t h wa ys [20]. Th ese lit er a t u r e in for m a t ion ’s a ll cor r ela t e wit h r esu lt s obt a in ed by t h e P AT. Sin ce few r esu lt s a r e fou n d in lit er a t u r e a n d on ly a h a n dfu l by t h e Google sea r ch en gin e, wor k focu ses on la bor a t or y r esu lt s obt a in ed by t h e en d u ser

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a n d is kn own t o be va lid r esu lt s. 35 ou t of 45 P ATs a r e com pa r ed a n d sh ow su ccessfu l r esu lt s, cor r ela t in g wit h la bor a t or y r esu lt s. To do a t im e m ea su r em en t du r in g pr ocessin g of da t a on a P AT, a t im er is u sed (r equ ir em en t ID 3). Th e t im er st a r t s fr om zer o a n d cou n t s u pwa r ds u n t il t h e pr ocesses by t h e P AT a r e com plet e. Th r ee t im es a r e r ecor ded for ea ch P AT t o obt a in a m or e a ccu r a t e m ea su r em en t . All P ATs sh ow a r espon se wit h in a m a xim u m of 7 secon ds. 6.4.Ev a lu a tin g th e P ATs E va lu a t ion of t h e P ATs a ccor din g t o su b-a im 2 r esu lt s in a t ot a l of 14 a n a lyzin g t ool's pa ssin g t h e cr it er ia (Fig 10). Th ese t ools fu lfills t he r equ ir em en t s of being fa st in da t a pr ocessin g a n d sh ow s cor r ela t ion wit h r esu lt s r epor t ed in t h e lit er a t u r e a n d obt a in ed a t t h e la bor a t or y.

F ig u re 10. Th e P ATs t h a t pa ssed ba sic eva lu a t ion a ccor din g t o sub -a im 2. 6.5.F in a l e v a lu a tio n o f th e P ATs Th e P ATs a r e fu r t h er eva lu a t ed t owa r ds t h e en d u ser r equ ir em en t s a n d t h e r esu lt sh ows n o a n a lysis t ool's fu lfillin g t h e r equ ir em en t s (Fig 10). Test ca ses 6 t o 10 (lin ked t o r equ ir em en t ID 4 t o 8) a r e u sed for t h e fin a l eva lu a t ion .

F ig u re 10. We see t h e P ATs t h a t pa ssed cer t a in r equ ir em en t s. Th er e wer e n o P ATs t h a t pa ssed su b-a im 3. Th e t ool's fa iled on t h e zoom in a n d ou t fu n ct ion (t o expa n d t h e view t o n eigh bor in g possible r esu lt s) con n ect ed t o pa t h wa ys wh en r et u r n in g resu lt s wer e obt a in ed a n d on t he com bin a t ion of m or e t h a n on e t ype of om ics da t a in pu t . A com plet e view of t h e r esu lt s a n d r ea son for exclu sion a r e sh own in a ppen dix 3.

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6.6.Th e be s t P AT fro m th e ra n k e d lis t Th e In gen u it y P AT is best r a n ked in ou r list con t a in in g a ll of t oda y’s a va ila ble a n a lysis t ools. Th e In gen u it y P AT ca n pr ocess a n d fu lfill a lm ost a ll of t h e r equ ir em en t s given by t h e en d u ser . Th e t ool is even ca pa ble of t a kin g in pu t of m or e t h a n on e om ics da t a a n d du r in g t h e t est in g st a ge of t h e t ool n o in pu t lim it s a r e fou n d wh en com bin in g da t a . The on ly set ba ck is t h e m issin g zoom in a n d ou t fu n ct ion (t o expa n d t h e view) con n ect ed t o pa t h wa ys u pon obt a inin g r esu lt s t h a t t h e pr ogr a m cou ld n ot pr ocess. A gen e n a m e (AP OE ) a n d t wo pr ot ein a ccession n u m ber s (NP _000032 a n d P 02649) is select ed in or der t o t est t h e t ool. Select ed gen e a n d pr ot ein s a r e kn own t o be in volved in t h e syn t h esis a n d for m a t ion of lipopr ot ein s . Th er efor e t h e expect ed r esu lt fr om t h e P AT is t o fin d in for m a t ion r ela t ed t o lipopr ot ein s. Th e gen e n a m e in ser t ed in t h e sea r ch field a n d t h e r et u r n ed r esu lt sh ow s 1 m a t ch fou n d for lipopr ot ein s (Fig 11). F ir st select ed pr ot ein a ccession n u m ber (NP _000032) r et u r n ed 1 m a t ch for lipopr ot ein s lin king it t o t h e gen e AP OE (Fig 12). Secon d select ed pr ot ein a ccession n u m ber (P 02649) r et u r n ed 1 m a t ch for lipopr ot ein s lin kin g it a lso t o t h e gen e AP OE (Fig 13).

F ig u re 11. Ret u r n ed r esu lt fr om t h e In gen u it y P AT a ft er in pu t of t h e gen e AP OE .

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F ig u re 12. Sea r ch r esu lt s a ft er in pu t of a pr ot ein wit h t h e a ccession n u m ber (NP _000032) sh owing t h e pr ot ein t o be lin ked t o lipopr ot ein s a n d t h e gen e AP OE .

F ig u re 13. Sea r ch r esu lt s a ft er in pu t of a pr ot ein wit h t h e a ccession n u m ber (P 02649) sh owin g t h e pr ot ein t o be lin ked t o lipopr ot ein s a n d t h e gen e AP OE .

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6.7.Co m bin in g P ATs No P ATs pa sses exa m in a t ion ou t lin ed in su b-a im 3, sin ce even t h e In gen u it y P AT wit h good scor e fr om ou r t est in g fa iled, du e t o t h e m issin g zoom fu n ct ion s. In st ea d we h a ve t o in vest iga t e if it is possible t o develop a P AT t h a t m eet s t h e r equ ir em en t s by t h e en d u ser or if a com bin a t ion of 2 or 3 P ATs ca n fu lfill t h e r equ ir em ent s of t h e en d u ser (See sect ion 3.1.2). Th e decision a ft er a m eet in g wit h t h e en d u ser is t h a t developin g a P AT by ou r self is n ot a n opt ion du e t o lim it ed t im e a n d in su fficie n t m a n power . Th e pa t h wa y a n a lysis pr ogr a m Unipr ot ba t ch con ver t er is r equ est ed by t h e en d u ser t o be pr im a r ily t est ed a ga in st a pr ogr a m ca lled F E vE R bu t a lso a ga in st a ll ot h er com bina t ions of P ATs t h a t ca n be fou n d. Wit h r ea son t h a t Un ipr ot h a n dle lipidom ics wh ile F EvE R h a n dle m et a bolics, t h e t wo P ATs h a ve fu n ct ion s t h a t in t h is wa y ca n com plem en t ea ch ot h er . Aft er in pu t of gen om ic da t a , t h e Un ipr ot ga ve eit h er a bla n k pa ge sh owin g t h a t n ot hin g is fou n d or a list of possible m a t ches t o t h e pr ot ein en coded b y t h e gen e. Test r esu lt s u sin g Un ipr ot ba t ch con ver t er en ds u p wit h su ccessfu lly con ver t in g som e of t h e gen e n a m es in t o pr ot ein s. In pu t t in g r esu lt s fr om Un ipr ot t o F E vE R wor k s well a n d t h e P AT's st a r t s, h owever , a lwa ys en ds u p wit h 0 r esu lt s. Th e com bin a t ion of t h e P AT's Un ipr ot a nd F E vE R is t h er efor e n ot su ccessfu l. In ou r sea r ch for com bin a t ion bet ween P ATs we t u r n ou r focu s on t h e Na t iona l Cen t er of Biot ech n ology In for m a t ion (NCBI) h om epa ge. At t h e NCBI h om epa ge a com bin a t ion of pa t h wa y a n a lysis pr ogr a m s ca n be fou n d. Th e h om epa ge of NCBI h a ve a ser ies of P AT's a va ila ble wit h ou t n eeds of down loa ds or pa ym en t s a n d con t a in s va st a m ou n t of wor ldwide collect ed in for m a t ion in m olecu la r biology. NCBI’s h om epa ge is a lso a gover n m en t fu n ded h om epa ge by t h e U.S. On t h e NCBI h om epa ge a n on lin e t ool ca n be fou n d wit h a ver y u sefu l sea r ch en gin e, ca pa ble of t a king m or e t h a n on e in pu t in or der t o for wa r d t h e sea r ch t o specific da t a ba ses t o which t h e NCBI h om epa ge is con n ect ed t o. Depen din g on h ow m u ch in pu t is in ser t ed in t h e sea r ch field, r esu lt s a r e r et u r n ed a ccor din gly. Th e gen e n a m ed E NAC wa s select ed wh ich codes for a pr ot ein t h a t a ffect s t h e sodiu m ch a n n els in biologica l or ga n ism s. E xpect a t ion s a r e t o obt a in su bst a n t ia l r esu lt s on r ela t ed in for m a t ion a bou t E NAC a n d t h e sodiu m ch a n n el fr om a ll da t a ba ses con n ect ed t o t h e N CBI h om epa ge. Th e r et u r n ed r esu lt s sh ow s 26 of 37 differ en t da t a ba ses fou n d m a t ch es con t a in in g in for m a t ion a bou t a r t icles, gen es, pr ot ein s, SNP a n d n u cleot ide sequ en ces r ela t ed t o E NAC a n d t h e sodiu m ch a n n el (Fig 14). A secon d gen e n a m e is select ed NP P A codin g for a pr ot ein t h a t m a kes a r ecept or ca lled n a t r iu r et ic pept ide cla ss A, r egu la t in g wa t er a n d sodiu m ba la n ce in biologica l or ga n ism s. Gen e n a m es select ed (E NAC a n d NP P A) a r e pu t in t o t h e sea r ch field a s “E NAC, NP P A”. No com bin a t ion s or r ela t ion s a r e expect ed t o be fou n d bet ween t h ese gen e n a m es (E NAC a n d NP P A) a n d n o r esu lt s a r e eit h er expect ed t o be r et u r n ed fr om t h e da t a ba ses. Resu lt s obt a in ed pr ecedes t h e expect a t ion s wit h m a t ch es for 8 da t a ba ses con t a in ing som e in for m a t ion a bou t a r t icles, gen es, m olecu la r in t er a ct ion s a n d da t a m a ppin g r ela t in g ENAC a n d NP P A t o ea ch ot h er (Fig 15). Wit h t h e possibilit y of m or e da t a ba ses con n ect ed t o ea ch ot h er t h e efficien cy for fin din g com bin ed r esu lt s a r e t h er efor e in cr ea sed. Page | 31

F ig u re 14. A sea r ch on NCBI’s h om epa ge a cr oss a ll da t a ba ses it is con n ect ed wit h . Th e gen e E NAC is u sed a s da t a in or der t o in vest iga t e t h e r et u r n ed sea r ch r esu lt s. Page | 32

F ig u re 15. A sea r ch on NCBI’s h om epa ge a cr oss a ll da t a ba ses it is con n ect ed wit h . Th e gen es E NAC a n d NP P A a r e u sed t oget h er a s da t a in or der t o in vest iga t e see t h e com bin ed sea r ch r esu lt r et u r n ed.

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6.8.F u n c tio n a litie s A va r iet y of fu n ct iona lit ies a r e fou n d t h r ou gh soft wa r e t est in g fr om t h e P AT. Th e m ost com m on fu n ct ion a lit ies a r e t he followin g: t h e sea r ch in pu t field, in pu t t ext a r ea field, dr opdown list s a nd r espon se win dow of h ow m a n y secon ds it t a kes t o r eceive t h e r esu lt s (Fig 16).

F ig u re 16. P ict u r e of t h e sea r ch in pu t field m a r ked blu e, in pu t t ext a r ea field m a r ked yellow a n d pu sh bu t t on m a r ked gr een fr om 2 differ en t websit es. Som e t ools give a n ice visu a l pr esen t a t ion sh owin g h ow con n ect ion s bet ween r esu lt s a r e con n ect ed su ch a s gen es or lipids (Fig 17). Two t ools h a ve 3d view bu t h a lf t h e t im e du r in g t est in g it give on ly a wh it e bla n k pa ge or r esu lt in g in cr a sh in g of t h e pr ogr a m .

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F ig u re 17. A gen e n a m e wa s sea r ch ed a n d t h e P AT visu a lly sh owed t h e r esu lt . Th e sea r ch ed gen e is color ed r ed a n d sh ow h ow it is in r ela t ion t o ot h er gen es, pr ot ein s, sign a lin g r ecept or s a n d biologica l cell pr ocesses. No P AT fu lfill a ll t h e r equ ir em en t s a ccor din g t o su b -a im 3. Of a ll t h e in vest iga t ed P AT's on ly 2 h a ve ea sy n a viga t ion fu n ct ion s a ccor din g t o ou r r equ ir em en t s a n d on ly 5 m a ke visu a l pa t h wa y pr esen t a t ion s. Non e of t h e P ATs h a ve a n y zoom in or ou t fu n ct ion s (t o expa n d t h e view) con n ect ed t o pa t h wa ys on r esu lt s obt a in ed. 6.9.Qu a lity Qu a lit y is decided on t h a t if a ll r equir em en t s a r e fu lfilled on a P AT, t h en t he qu a lit y is good. As t h e r esu lt s sh ows, n o P AT fu lfills t h e r equ ir em en t s. Th er efor e t h e qu a lit y is n ot a ccept a ble on a n y of t h e P AT. Th e following a spect s a r e u sed t o eva lu a t e t h e qu a lit y of t h e h om epa ges: Accu racy an d Correctn ess, Com pleten ess, R elevan ce, T im e an d P u n ctu ality, T raceability. All h om epa ges pr ovides a ccu r a t e in for m a t ion wit h n o er r or s or m islea ding in for m a t ion a lt h ou gh m a n y h om epa ges belon gs t o com pa n ies t h a t con sider t h eir pr odu ct t o be t h e best . Also five h om epa ges st a t es t h a t it is a wor k in Page | 35

pr ogr ess m a kin g it less a ppea lin g by design . Th er e a r e n o com pa n ies st a t ing wh en or wh er e t h eir P ATs a r e developed or h ow lon g it is in exist en ce. Th e Google sea r ch en gin e u ses a pa ge r a n kin g syst em , r a n k in g t h e com pa n ies pa ges h igh . Th is m a kes it fa st t o be fou n d a n d t a kes a bou t 3 secon ds a t m a xim u m t o be fou n d, fu ll r esu lt s a r e sh own in a ppen dix 4. Com pa n ies on t h eir h om epa ges r a r ely u se a n y r efer en ces t o t h e in for m a t ion t ha t t h ey pu t on t h eir h om epa ge m a kin g it h a r d t o t r a ce t h e in for m a t ion t h ey pr ovide a n d few or n on e ca n be fou n d. In t ot a l, on ly on e hom epa ge pr ovides som e r efer en ces a n d in for m a t ion t h a t ca n be t r a ced.

7. Discu ssion 7.1.Is it p o s s ible to fin d a P AT th a t p ro c e s s e s m e ta bo lo m ic s a n d lip id o m ic s ra w d a ta a s in p u t a n d c o m bin e th e m w ith g e n e tic in fo rm a tio n ? Lookin g a t t h e r esu lt s we see t h a t 46 P ATs a r e fou n d bu t on ly 23 fu lfilled cr it er ia ’s for bein g a ble t o pr ocess m et a bolom ic, lipidom ic or gen om ic da t a . Th e P ATs a r e sor t ed a n d r a n ked ba sed on h ow m a n y t ype of da t a t h ey a r e ca pa ble of h a n dlin g a n d fu r t h er eva lu a t ed a ccor din g t o su b -a im 2. A t ot a l of 14 P ATs pa ssed su b-a im 2. Th is lea ds t o on ly 1 a va ila ble P AT t h a t ca n pr ocess a ll t ypes of om ics da t a wh ich is t h e In gen u it y P AT. It looks pr om isin g a s a P AT a n d is n u m ber on e on t h e r a n kin g list , h owever , t h e t ool fa ils t h e en d u ser r equ ir em en t s on t h e zoom in a n d ou t fu n ct ion a lit y (t o expa nd t h e view t o n eigh bor in g possible r esu lt s) con n ect ed t o pa t h wa ys u pon r eceived r esu lt s. Th e In gen u it y P AT it self wa s ea sy t o u se a n d t h e r esu lt s r et u r n ed a r e a lso u n der st a n da ble. If r equ ir em en t s h a ve been sligh t ly differ en t , t h is is a good P AT t o u se. In gen u it y P AT belongs t o a com pa n y so pa ym en t fees a r e r equ ir ed in or der t o u se it a ft er few da ys of t r ia l. 495 U.S dolla r s a r e a h igh pr ice t o pa y for a n in dividu a l per son bu t for a la r ger gr ou p of m in im u m six wor kin g r esea r ch er s t h a t m igh t u se t h e In gen u it y P AT du r in g a per iod of t wo yea r s per iod, t h e pr ice is a ccept a ble. 7.2.Wh a t a re th e fu n c tio n a litie s o ffe re d by th e a v a ila ble a n a ly s is to o ls ? Th r ou gh ou t soft wa r e t est in g, goin g t h r ou gh t est ca ses, a ll P ATs fu n ct ion a lit ies a r e t est ed. Th e P ATs h a ve a va r iet y of fu n ct iona lit ies. As t h e r esu lt s sh ows, m ost com m on ly u sed fu n ct ion s a r e t h e in pu t fields, t ext a r ea in pu t fields a n d a dyn a m ic dr opdown list . Th is a pply on bot h down loa ded a n d web ba sed P AT. Som e P ATs a r e m or e u niqu e a n d offer ext r a fu n ct ion a lit ies su ch a s file u ploa d or lin ks r ela t ed t o t h e in ser t ed sea r ch field. All P ATs ha ve som e for m of visu a l pr esen t a t ion bu t on ly a few gives t h e desir ed effect like t h e KE GG P AT. KE GG h a ve t h e best t ype of visu a l pr esen t a t ion wit h a r r ows a n d differ en t color m a r kin gs. Next in lin e a r e t h e In gen u it y P AT (a t t h e cu r r en t st a t e on 2012-05-20, t h is t ool only h a ve a t est ver sion bu t st ill look s good) sh owin g a visu a l pr esen t a t ion wit h possible cor r ela t ion s. Page | 36

7.3.Wh a t a re th e qu a litie s o f th e s e to o l's an d h o w t o e v a lu a te th e m ? Th e qu a lit y (m en t ioned in sect ion 1.3) is int er pr et ed differ en t ly by people a n d ever yon e h a ve t h eir own poin t of views on wh a t qu a lit ies a r e. Ou r view of good qu a lit y is t h a t if a ll r equ ir em en t s a r e fu lfilled by t h e P ATs, t h ey a r e t h en a lso fu lfillin g t h e n eeds of t h e pot en t ia l u ser s. H owever if qu a lit y is good for som eon e it m a y n ot be good for som eon e else, t h er efor e ch oices of h a vin g a m or e gen er a l view on qu a lit ies a r e m a de. H owever , we m a de t h e ch oice of h a vin g 8 specific r equ ir em en t s t h a t n eeded t o be fu lfilled by a P AT a s seen in sect ion 1.3 a n d is a ccept ed by t h e en d u ser a s good qu a lit y. Qu a lit y defin it ion s a r e h a r d t o m a ke in gen er a l for soft wa r e pr ogr a m s or a pplica t ions , even wh en m a kin g br oa d a n d gen er a l defin it ion s, a s poin t ed ou t in [36] wh ile [38] st r en gt h en ed t h e r ea son s. Accor din g t o [27], h om epa ges ca n be exa m in ed t h or ou gh ly by cer t a in a spect s t o defin e t h a t it is qu a lit y. Th e exa m in ed h om epa ges for r espect ive P ATs h a ve good st a n da r ds fu lfillin g 4 of 5 a spect s. In [38] r equ ir em en t s a r e a lso a wa y t o defin e qu a lit y a n d in [14] a ppr oa ch es a r e sh own m or e t h or ou gh ly. We follow t h e defin it ion of qu a lit y given by [14], sh owin g t h a t a ll r equ ir em en t s n eed t o be fu lfilled in or der t o be con sider ed good qu a lit y, wh ich a r e ea sily seen on a t r a cea bilit y m a t r ix a s [38] st a t es. Lookin g a t t h e m a t r ices a ft er wa r ds a s t r a cea bilit y [22], a ppen dix 3 sh ow t h a t n ot a ll r equ ir em en t s a r e fu lfilled, t h er efor e we ca n n ot sa y t h a t t h e qu a lit ies for t h ese a n a lyzed P ATs a r e good. If we look a t qu a lit y on down loa ded P ATs ver su s t h e h om epa ge of NCBI we m igh t a sk t h e followin g qu est ion : Wh a t a r e t h e pr os a n d con t r a s? Down loa ded P ATs seem t o be m or e cu st om ized by com pa n ies t o a specific gr ou p of biom edica l field u ser s, t h er e by fu lfillin g t h e qu a lit y for t h a t specific gr ou p. Mon t h ly fees a r e r equ ir ed or t h e t ool ca n be bou gh t a n d in or der t o get su ppor t a ddit ion a l pa ym en t s a r e n eeded. Th e h om epa ge of NCBI h a ve fr ee ser vices a n d do n ot cost a n yt h in g t o u se. Resu lt s obt a in ed fr om t h e down loa ded P ATs a r e sa m e a s on t h e h om epa ge of NCBI, h owever , NCBI a r e ba sed on scien t ific r esu lt s dir ect ly lin ked t o scien t ific a r t icles a n d ca n t h er efor e be seen m or e va lid. In for m a t ion ga in ed fr om a ll P ATs a r e m ea n t t o h elp t h e r esea r ch er s in t h eir biom edica l field (wh et h er it m a y be in lipidom ics, gen om ics or a n y ot h er field) for m a kin g n ew discover ies.

F ig u re 18. Sm a ll figur e wit h +/- on down loa ded P ATs ver su s NCBI

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7.4.Wh y n o t In g e n u ity a n d w h y U n ip rot w ith F Ev ER ? As t h e r esu lt s sh ows t h e In gen u it y ha d pr om isin g fea t u r es a n d wer e ver y good a t fu lfillin g a lm ost a ll r equ ir em en t s. Th e en d u ser h owever du r in g discu ssion m a de t h e decision t o disca r d it sin ce it is a n ew t ool a nd wou ld n eed a lot of t im e t o lea r n it . Th e en d u ser wa s m or e fa m ilia r wit h t h e P AT n a m ed Un ipr ot a n d t h e in h ou se developed P AT F E vE R. Sin ce Un ipr ot h a n dle lipidom ics wh ile F E vE R h a n dle m et a bolics, t h e t wo P ATs ha ve fu n ct ion s t h a t in t h is wa y ca n com plem en t ea ch ot h er . By t h is r ea son t h e t wo t ools wer e in st ea d a n a lyzed. As r esu lt s sh ow, t h e t wo t ools wer e n ot wor king so well t oget h er . E it h er t h e con ver sion wen t well bu t n o r esu lt s wer e sh own or it sim ply ga ve a bla n k pa ge, m ea n in g n o pr esen t ed r esu lt s. In t h e en d t h is wa s a lso disca r ded.

8. F u t u r e Va lu e All st a r t ed wit h h ow t o com bin e a n oppor t u n it y for a m u lt idisci plin a r y t h esis a n d t h is t h esis sh ows on e wa y on h ow it ca n be don e. Th e t est ing m et h od u sed on t h e pa t h wa y a n a lysis sh ows t h a t n o pr oblem s a r e en cou n t er ed a n d ot h er u ser s ca n u se t h e sa m e m et h od wit h ea se. Th e h a r d pa r t is wh en n o t ool's a r e fou n d a ccor din g t o r equ ir em en t s. We a lso fou n d ou t t h a t even wit h a sm a ll gr ou p of pr ogr a m m er s, m on t h s a r e n eeded t o develop a P AT, du e t o t h e va st a m ou n t of in for m a t ion r equ ir ed t o be in clu ded in t h e pr ogr a m . Not a ll P ATs ca n com bin e or t a ke m or e t h a n on e in pu t of om ics da t a . Sea r ch es for a n a lt er n a t ive solu t ion lea ds t o a h om epa ge n a m ed NCBI t h a t h a ve sever a l collect ed P ATs fr ee of u se. As discu ssed a bou t pr os a n d con t r a s on down loa ded P AT's ver su s fr ee, in t h e fu t u r e t h er e will pr oba bly be fr ee ba sed r esou r ces. H om epa ges like NCBI gr ow in popu la rit y a t t r a ct in g m a n y u ser s. F r ee a va ila ble P ATs a r e m or e pr efer r ed t o be u sed sin ce t h ey a r e fr ee of u se a n d t h eir qu a lit y is equ a lly good a s t h e down loa ded on es. Toda y we a lr ea dy see t h e glim pse t o t h e begin n in g of t h is pr oces s. Com pa n ies will t r y m a t ch in g t h e dem a n ds of u ser s a n d st a r t s t o r u n eit h er lon ger fr ee t r ia ls or even m a kin g t h eir t ool fr ee of u se wh ile spon sor ed by a dver t isem en t s.

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22. J or da n W. K, Nor den st a m J , La u wer s Y. G, Rot h en ber ger A. D, Ala vi K, Ga r wood M, Ch en g L. L, (2009), M etabolom ic Ch aracterization of H u m an R ectal Ad en ocarcin om a w ith In tact T issu e M agn etic R eson an ce S pectroscopy, Disea ses of t h e Colon a n d Rect u m Volu m e 52 Issu e 3 23. Ka n n en ber g A, Sa iedia n H , (2009), Wh y S oftw are R equ irem en ts T raceability R em ain s a Ch allen ge, Cr ossTa lk: Th e J ou r n a l of Defen se Soft wa r e E n gin eer in g Volu m e 22 Nu m ber 5. 24. Kin g Y. J , F er r a r a R, Ta bibia za r R, Spin M. J , Ch en M. M, Ku ch in s ky A, Va ila ya A, Kin ca id R, Tsa len ko A, Den g X-F . D, Con n olly A, Zh a n g P , Ya n g E , Wa t t C, Ya kh in i Z, Ben -Dor A, Adler A, Br u h n L, Tsa o P , Qu er t er m ou s T, Ash ley A. E , (2005), Path w ay an alysis of coron ary ath erosclerosis, Resea r ch Ar t icle P h ysiologica l Gen om ics Volu m e 23 Nu m ber 1 25. Kla m t S, St ellin g J , (2002), T w o approach es for m etabolic path w ay an alysis, Tr en ds in biot ech n ology Volu m e 21 Issu e 2. 26. LipidMa ps Na t u r e – Lipidom icsga t ewa y, (2012) WL ipid classification system > h t t p://www.lipidm a ps.or g/da t a /cla ssifica t ion /LM_cla ssifica t ion _exp.ph p < 2012-06-14 27. Lu n dh D, (2011), In for m a t ion Qu a lit y a n d Secu r it y, Skövde Un iver sit y 28. Lu o L, (2001), S oftw are T estin g T ech n iqu es– T ech n ology M atu ration an d R esearch S trategy, Ca r n egie Mellon Un iversit y 29. Meer G, (2005), Cellu lar L ipid om ics, Th e E MBO J ou r n a ls m em ber s r eview Volu m e 24 30. Mogyor odi G.E , (2005), R equ irem en ts-B ased T estin g – Am bigu ity R eview s, Soft wa r e Test in g Services Nu m ber 1. 31. Molecu la r bioch em ist r y, (2012) Fatty acid syn th esis > h t t p://www.r pi.edu /dept /bcbp/m olbioch em /MBWeb/m b2/pa r t 1/fa syn t h esis.h t m < 2012-06-14 32. Na t iona l H u m a n Gen om e Resea r ch In st it u t e: Gen et ic a n d Gen om ic Scien ce, (2012) Gen etic an d gen om ic scien ce > h t t p://www.gen om e.gov/19016904 < 2012-04-20 33. Net wor k Scien ce – Net Sci, (2012) Welcom e to N etS ci’s L ists of S oftw are for B ioin form atics: PAT s > h t t p://www.n et sci.or g/Resou r ces/Soft wa r e/Bioin for m /pa t h wa ya n a lysis.h t m l < 2012-02-27 34. Olson L. D, Kesh a r wa n i S, (2010), E n t er pr ise In for m a t ion Syst em s: Con t em por a r y Tr en ds a n d Issu es, Sin ga por e: Wor ld Scien t ific P u blish in g Co. P t e. Lt d, 7-23. 35. P h osph a t idic a cid, lysoph osph a t idic a cid a n d r ela t ed lipids: stru ctu re, occu rren ce, bioch em istry an d an alysis, (2012) Ph osph atid ic acid – Occu rren ce an d B iosyn th esis > h t t p://lipidlibr a r y.a ocs.or g/Lipids/pa /in dex.h t m < 2012-07-11 36. Qu a lit y, (2012) Qu ality > h t t p://www.qua lit ydigest .com /h t m l/qu a lit ydef.h t m l < 2012-04-09 37. Qu eh en ber ger O, Ar m a n do M. A, Br own H . A, Miln e B. S, Myer s S. D, Mer r ill H . A, Ba n dyopa dh ya y S, J on es N. K, Kelly S, Sh a n er L. R, Su lla r ds M. C, Wa n g E , Mu r ph y C. R, Ba r kley M. R, Leiker J . T, Ra et z H . R. C, Page | 40

Gu a n Z, La ir d M. G, Six A. D, Ru ssell W. D, McDon a ld G. J , Subr a m a n ia m S, F a h y E , Den n is A. E , (2010), L ipid om ics reveals a rem ark able d iversity of lipid s in h u m an plasm a, J ou r n a l of Lipid Resea r ch Volu m e 51. 38. Reeves A. C, Bedn a r A. D, (1994), Defin in g Qu ality: Altern atives an d Im plication s, Aca dem y of Ma n a gem en t Review Volu m e 19 Nu m ber 3. 39. Rosen ber g L.H , H a m m er F . T, H u ffm a n L. L, (1998), R equ irem en ts, T estin g an d M etrics, Cit eSeer 15:t h An n u a l P a cific Nor t h west Soft wa r e Qu a lit y Con fer en ce. 40. Sch illing H . C, Let sch er D, P a lsson ∅. B, (2000), T h eory for th e S ystem ic Defin ition of M etabolic Path w ays an d th eir u se in In terpretin g M etabolic Fu n ction from a Path w ay-Orien ted Perspective, J ou r n a l of Th eor et ica l Biology Volu m e 203 Issu e 3. 41. Sch u st er S, Da n deka r T, F ell A.D, (1999), Detection of elem en tary flu x m od es in bioch em ical n etw ork s: a prom isin g tool for path w ay an alysis an d m etabolic en gin eerin g, Tr en ds in biot ech n ology Volu m e 17 Issu e 2. 42. Th e AOCS Lipid Libr a r y – Tr ia cylglycer ols, (2012) B iosyn th esis an d m etabolism > h t t p://lipidlibr a r y.a ocs.or g/lipids/t a g2/in dex.h t m < 2012-0614 43. Th e lipid ch r on icles, (2012) L ipid om ics > h t t p://www.sa m u elfu r se.com /2011/12/lipidom ics/ < 2012-03-19 44. Th e Lipid Libr a r y, (2012) L ipid syn th esis > h t t p://lipidlibr a r y.a ocs.or g/in dex.h t m l < 2012-04-15 45. Th e Medica l Bioch em ist r y P a ge, (2012) L ipid syn th esis > h t t p://t h em edica lbioch em ist r ypa ge.or g/lipid -syn t h esis.ph p < 2012-03-09 46. Va n ce E . D, Va n ce E.J , (2008), B ioch em istry of L ipid s, L ipoprotein es an d M em bran es 5 th ed ition , Am st er da m : E lsevier , 278-279; 583-588. 47. Vir gin ia web edu ca t ion , (2012) L ipid s > h t t p://web.vir ginia .edu /H eidi/ch a pt er 8/ch p8.h t m < 2012-06-26 48. Voigh t et a l, (2012), Plasm a H DL ch olesterol an d risk of m yocard ial in farction : a m en d elian ran d om ization st u d y, Th e La n cet Volu m e 380 Issu e 9841 49. Wa t er fa ll Model, (2012), All abou t th e w aterfall m od el > h t t p://www.wa t er fa ll-m odel.com / < 2012-08-18 50. Som m er ville Ia n , (1996), S oftw are process m od els, J ou r n a l of ACM Com pu t in g su r veys Volu m e 28 Issu e 1 , p269-271 51. Wa t son D. A, (2006), L ipid om ics: A global approach to lipid an alysis in biological system s, J ou r n a l of Lipid Resea rch Volu m e 47. 52. Wa t son D. A, (2006), T h em atic review series: S ystem s B iology Approach es to M etabolic an d Card iovascu lar Disord ers, J ou r n a l of Lipid Resea r ch Volu m e 47. 53. Wen k MR, (2005), T he em ergin g field of lipid om ics, Na t u r e Reviews Dr u g Discover y Volu m e 46. 54. Willia m W. C, Xia nlin H , (2010), L ipid An alysis: Isolation , S eparation , Id en tification an d L ipid om ic An alysis, Br idgwa t er : Th e Oily P r ess

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Appen dix 1 – Test Ca ses Te s t Ca s e Test ca se: is a docu m en t wh ich descr ibes INP UT, ACTION, E VE NT a n d E XP E CTE D RE SP ONSE t o det er m in e if fea t u r e of a n a pplica t ion is wor kin g cor r ect ly or n ot . A set of in pu t s, execu t ion pr econ dit ion s, a n d expect ed ou t com es developed for a pa r t icu la r object ive, su ch a s t o exer cise a pa r t icu la r pr ogr a m pa t h or t o ver ify com plia n ce wit h a specific r equ ir em en t . (Com p Soft wa r e t est in g, (2010), Test Ca se for m a t s > h t t p://www.fa qs.or g/qa /qa -4044.h t m l < 2010-12-18) Te s t Ca s e 1 “Me ta bo lo m ic ty p e o f d a ta in p u t” (Re qu ire m e n t ID 1): Go a l: See if t h e P AT ca n process m et a bolom ic da t a . Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy r u n n in g). 1. In pu t m et a bolom ic da t a t ype (su ch a s “NP P A”). 2. Get r esu lt r ela t ed t o t h e m et a bolism fr om t h e P AT. Ex p e c te d re s p o n s e : Th e u ser ca n in pu t m et a bolom ic da t a t ype on select ed P AT a ch ievin g da t a in for m a t ion r ela t ed t o “NP P A”. Te s t Ca s e 2 “Lip id o m ic ty p e o f d ata in p u t” (Re qu ire m e n t ID 1): Go a l: See if t h e P AT ca n process lipidom ic da t a . Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy r u n n in g). 1. In pu t lipidom ic da t a t ype (su ch a s “r s 4420638”). 2. Get r esu lt r ela t ed t o lipids fr om t h e P AT. Ex p e c te d re s p o n s e : Th e P AT pr ocess lipidom ic da t a t ype get t ing da t a in for m a t ion r ela t ed t o “r s4420638”. Te s t Ca s e 3 “Ge n o m ic ty p e o f d ata in p u t” (Re qu ire m e n t ID 1): Go a l: See if t h e P AT ca n process gen om ic da t a . Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy st a r t ed). 1. In pu t gen om ic da t a t ype (su ch a s “AP OE ”). Page | 42

2. Get gen et ica lly r ela t ed r esu lt fr om t h e P AT. Ex p e c te d re s p o n s e : Th e P AT pr ocess gen om ic da t a t ype wh er e da t a in for m a t ion is r ela t ed t o “AP OE ”. Te s t Ca s e 4 “Ve rify in g th e re s u lt” (Re qu ire m e n t ID 2): Go a l: Get cor r ect a n d va lid r esu lt s r et u r n ed by t h e P AT. Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy st a r t ed). 1. In pu t om ics (m et a bolom ics, lipidom ics a nd gen om ics) da t a . 2. Get r esu lt fr om t h e P AT. 3. Ch eck r esu lt s obt a in ed wit h va lid r esu lt s fr om books, a r t icles or la bor a t or y r esu lt s. 4. Con fir m t h a t t h e r eceived r esu lt is va lid. Ex p e c te d re s p o n s e : Th e r esu lt r et u r n ed fr om t h e P AT is va lid t o lit er a t u r e or la bor a t or y r esu lt . Te s t Ca s e 5 “Tim e e ffe c tiv e n e s s ” (Re qu ire m e n t ID 3): Go a l: In for m a t ion a bou t h ow fa st t h e P AT r et u r n s a r esu lt . Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy st a r t ed). 1. In pu t om ics (m et a bolom ics, lipidom ics a nd gen om ics) da t a . 2. St a r t t h e t im er . 3. Get r esu lt fr om t h e P AT. 4. St op t h e t im er . 5. Recor d t im e. Ex p e c te d re s p o n s e : Th e r esu lt r et u r n ed fr om t h e P AT t ook less t h a n 5 secon ds a n d is displa yed. Te s t Ca s e 6 “N a v ig a tio n ” (Re qu ire m e n t ID 4): Go a l: See t h e n a viga t ion ca pa bilit ies bet ween t h e da t a in ser t ed a n d t h e r esu lt given by t h e select ed P AT. Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy st a r t ed). 1. In pu t om ics (m et a bolom ics, lipidom ics a nd gen om ics) da t a . 2. Get r esu lt fr om t h e P AT. Page | 43

3. Tr y n a viga t in g bet ween t h e st a r t of in pu t t ed da t a a n d t h e r esu lt s obt a in ed by scr ollin g on t h e r esu lt win dow. 4. Seein g a pa t h lea din g fr om st a r t (in pu t da t a ) t o en d (r esu lt ). Ex p e c te d re s p o n s e : It wa s possible t o n a viga t e bet ween t h e in ser t ed da t a a n d t h e r esu lt obt a in ed. Te s t Ca s e 7 “Vis u a l p re s e n ta tio n ” (Re qu ire m e n t ID 5): Go a l: See if t h e r esu lt ca n be visu a lly pr esen t ed/displa yed by u sin g t h e P AT. Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy st a r t ed). 1. In pu t om ics (m et a bolom ics, lipidom ics a nd gen om ics) da t a . 2. Get r esu lt fr om t h e P AT. 3. Get a visu a l pr esen t a t ion wh er e you ca n m a p r esu lt s t o ea ch ot h er . Ex p e c te d re s p o n s e : Th er e wa s a visu a l pr esen t a t ion wh en u sing t h e P AT. Te s t Ca s e 8 “Zo o m in g ” (Re qu ire m e n t ID 6): Go a l: See if t h e P AT displa ys a n y zoom fu n ct ion s. Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy st a r t ed). 1. In pu t om ics (m et a bolom ics, lipidom ics a nd gen om ics) da t a . 2. Get r esu lt fr om t h e P AT t h a t is con n ect ed t o pa t h wa ys. 3. Sea r ch for a sm a ll m a gn ifyin g gla ss wit h a plu s or m in u s sign . 4. Sea r ch for specific fu n ct ion wit h a r r ows for zoom in g. 5. Tr y zoom in g in t o na r r ow t h e view. 6. Tr y zoom in g ou t t o expa n d t h e view. Ex p e c te d re s p o n s e : Th e P AT h a s zoom fun ct ion s. Te s t Ca s e 9 “S p e c ific d a ta in p u t ty p e ” (Re qu ire m e n t ID 7): Go a l: See if t h e P AT ca n t a ke specific t ype of da t a a s a n in pu t . Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy st a r t ed). 1. In pu t specific t ype of da t a (su ch a ccession n u m ber s: AC_0088966, r s n u m ber : r s896530). Page | 44

2. Get r esu lt fr om t h e P AT. Ex p e c te d re s p o n s e : Th e P AT cou ld pr ocess t h e specific t ype of da t a . Te s t Ca s e 10 “Ma p p in g c o m bin e d d a ta ty p e s ” (Re qu ire m e n t ID 8): Go a l: See if t h e P AT ca n com bin e om ics da t a a n d m a p t h em t o r ela t ed pa t h wa ys. Ev e n t: (P r esu m pt ion m a de t h a t t h e P AT is a lr ea dy st a r t ed). 1. In pu t m or e t h a n one t ype of om ics (m et a bolom ics, lipidom ics a n d gen om ics) da t a com bin ed. 2. Get r esu lt fr om t h e P AT. 3. See if r esu lt com bin ed t o ot h er da t a t ypes sh owin g a m a pped view wit h t h e ot h er da t a t ypes. Ex p e c te d re s p o n s e : Th e P AT cou ld com bin e a n d m a p t h e da t a for t h e u ser .

Page | 45

Appen dix 2 – Lipid, MI SNP a n d Met a bo SNP da t a sh eet Gen e Na m e F ADS123

Lipid a n d rs (n u m ber ) 174546

UBE 2L3

181362

LILRA3

386000

AP OE

439401

KLH L8

442177

TTC39B

581080

CITE D2

605066

SORT1

629301

MSL2L1

645040

LOC55908

737337

SCARB1

838880

AP OA1

964184

AP OB

1042034

LDLR

1122608

GCKR

1260326

NAT2

1495741

LIP C

1532085

LP A

1564348

ZNF 648

1689800

H NF 4A

1800961

ABCA1

1883025

MI SNP SNP fu n ct ion Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid

Met a bo SNP Gen e Na m e r s SNP (n u m ber ) fu n ct ion E NAC Met a bo SNP E NAC Met a bo SNP NP P A 5068 Met a bo SNP BDNF 6265 Met a bo SNP SLC35F 1 89107 Met a bo SNP NP P A 198358 Met a bo SNP BCL11A 243021 Met a bo SNP MDS1 419076 Met a bo SNP 579459 Met a bo SNP NP P B 632793 Met a bo SNP TME M133 633185 Met a bo SNP P NP LA3 738409 Met a bo SNP P LCE 1 932764 Met a bo SNP TF AP 2B 987237 Met a bo SNP CH RNA3 1051730 Met a bo SNP SGK1 1057293 Met a bo SNP C5or f174 1173771 Met a bo SNP Gla sgow 1230297 Met a bo SNP J AG1 1327235 Met a bo SNP LOC10018 1329650 Met a bo SNP CYP 1A1 1378942 Met a bo Page | 46

CYP 26A1

2068888

ANGP TL3

2131925

TRP S1

2293889

CAP N3

2412710

P CSK9

2479409

LACTB

2652834

C6or f106

2814944

AMP D3

2923084

CMIP

2925979

F RMD5

2929282

TRIB1

2954029

IRS1

2972146

LRP 4

3136441

MYLIP

3757354

CE TP

3764261

P GS1

4129767

ABCA8

4148008

ABCG58

4299376

AP OE

4420638

P ABP C4

4660293

KLF 14

4731702

ZNF 664

4765127

1LNT2

4846914

SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP

C10or f10

1530440

Gla sgow2

1703492

STC1

1731274

SGK1

1743966

HFE

1799945

MDS1

1918974

E NAC

2228576

E SR1

2234693

H NF 1a

2259816

NE DD4L

2288774

H CCA2

2334499

FES

2521501

LYP LAL1

2605100

MOV10

2932538 2987983

DBH

3025343

SLC22A2

3127573

E GLN2

3733829 3741913

ULK4

3774372

CACNB2

4373814

ZBE D3

4457053

GCK

4607517

SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Page | 47

TOP 1

6029526

P LTP

6065906

LDLR

6511720

P DE 3A

7134375

MVK

7134594

LIP G

7241918

ANGP TL4

7255436

NYNRIN

8017377

ABO

9411489

MAP 3K1

9686661

COBLL1

10195252

P LE C1

11136341

LRP 1

11613352

P INX1

11776767

STARD3

11869286

COBLL1

12328675

LP L

12678919

MC4R

12967135

SLC39A8

13107325

TYW1B

13238203

LCAT

16942887

MLXIP L

17145738

AP OB

1367117

H LA

2247056

Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid SNP Lipid

SLC7A9

4805834

J AG1

6040055 6983267

GLIS3

7034200

ADM

7129220

KIAA1486

7578326

RBMS1ITG 7593730 MSRA

7826222 7931342

ME IS2

8031633

TBX2

8068318

IL6

10242595 10993994

C6or f204

11153768

NP P A

11191548

E BF 1

11953630

ZNF 652

12940887

P LCD3

12946454

CST3CST9

13038305

SLC4A7

13082711

GUCY1A3

13139571

ZNF 652

16948048

F GF 5

16998073

ATP 2B1

17249754

Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo Page | 48

SNP P LA2G6 5756931 Lipid SNP OSBP L7 7206971 Lipid SNP LRP P P 1R3 9987289 Lipid SNP J MJ D1C 10761731 Lipid SNP SMG6 216172 MI SNP SORT1 SORT1 AP OA1 LDLR LP A CXCL12 KIAA1822 LP A ADAMTS7 LDLR P H ACTR ABO MRAS KCNE 2 P CSK9 ZC3H C1 TCF 21 CNNM2 P P AP 2B MIA3 ANKS1A SH 2B3 CDKN2A WDR12 RALI

629301 646776 964184 1122608 1564348 1746048 2895811 3798220 3825807 6511720 9349379 9411489 9818870 9982601 11206510 11556924 12190287 12413409 17114036 17465637 17609940 3184504 4977574 6725887 12936587

MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI MI

VP S13C

17271305

SH ROOM3

17319721

MTH F R

17367504

GOSR2

17608766

STK39

35929607

SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP Met a bo SNP

SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP

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Appen dix 3 – Requ ir em en t s Ma t r ixes

Page | 50

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Appen dix 4 – Respon s Tim es

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