PIMMS-Dash: Accessible analysis, interrogation, and visualisation of high-throughput transposon insertion sequencing (TIS) data

Motivation C urre nt me t hods f or v i s u alising an d int errog a tin g hig h-t hr oug h put trans p o so n in sertion mu ta g e nes i s sequ enci ng ( TIS ) d a t a re q ui res a si g n ifica nt t ime i n v e st me n t in l e ar ni n g bi o i nf ormatics , o fte n pr o d uci n g st ati c fi g ures t hat d o n ot f a c i li tat e re al ti me a n al y s is . Summary We ha v e c reate d an a c cessib le we b - bas ed bro w ser to ol for v i s u alisat ion a nd dow ns t re am a n a ly s i s o f hig h- t h r o u g h p u t T I S d a t a . T h i s i n c l u d e s m u l t i p l e i n t e r a c t i v e a n d s o r t a b l e t a b l e s t o a i d t h e u s e r t o i d e n t i f y g e n e s o f i nt erest , e n a b li n g t he u ser to g ai n a greater und ersta n d i n g of t h e ge nes con trib ut in g to fi tness i n t heir e x pe ri men tal w ork. PIM MS - Da s h pe r mits re s e arch ers, w i t h a ny l evel of bio infor mat i c s know l e d g e, t o i n te r r og a te d at a s e ts a n d g en e r at e pub l i ca t ion qu a l i ty f ig u re s. Availability P IMM S - Da s h is free ly a v ail a ble a nd is a c cessible on l ine at h t t p s ://pimms- da s hb o ar d- uon. az ure w e b sit es. net

T ra ns p os on / ins e rtion a l m u t ag e n esis is a p o werful t ool freq uen tly use d in mic robiolog y t o eluc ida te ge n e s t ha t contri but e to fi t ness ( grow t h/s urviv al ). Di sru pti ng gen e s by r an dom i n s e r t i o nal m ut agenes is, p r ovi d es a re s ourc e to achieve a g r eater und e r sta ndin g of how bact erial gen ot y p e s co ntrib u te t o t heir o bser v e d phe not y pe s . A p pr oach e s i nv olve t he co ns tructi on of m uta nt li b r a rie s c o n ta inin g a h i g h-de ns it y of i ns erti ons b ef ore t h e l ib rar y i s e x pos ed t o t he s electi ve con d it io n. Fre que ncy of m u ta t io ns ca n t h e n be co mp a re d before a n d a ft er ex p os ure t o t he sele c tive en vi ro n ment or b e tw een s ele c t ive and n o n-s ele c t ive en vi r on ment s . T his is a c h ie ved b y hig h t hro u gh pu t s equ enc i n g, focu ss ed on t h e gen ome/ins ertio n junc tions, a llowin g f or t h e qua n t ific a ti o n of co nditi o na l l y e sse nt ia l se qu en e c s wi t hin t h e s e lecti ve co n diti on. The ma in laborat or y met h ods f or high-t hro u ghp ut mu ta g e n e s i s ma pping are Ins e rt io n Se q uencin g (INS eq) [ 1], T ra ns p os on I nser tio n Se que ncin g ( TN-S eq) [2], Hig h-t hro ug h put In serti o n Tracki ng b y d eep Seq uenc i n g (HI TS) [3 ], Tran sp os o n- Di r e c ted Ins e r tio n sit e Seq uenc ing ( Tra DIS) [4] a nd Prag matic Insert i on Mut ant M app ing S y ste m (PIM MS) [ 5 , 6]. E ach a ppr oa c h is si milar, ho we ve r th ere are s ubt le diff erenc e s i n ap pli c able tr ans p os o n s o r in sertional eleme n t s , m u tant li brar y prep a ration, se que ncing ap proa c h es and s of tware t ools us e d f or b ioin f orma tics a na lys is. There a re n umero u s bioinf o r ma tic t ools for ana ly sin g t h e raw s eq uenc e da ta f or th e mu lti p l e diff e re nt exp eri m e n t al a ppro ache s whic h are s u mm arised els ew here ( C a i n et a l 202 0) bu t curre n t ly .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 14, 2024. ; https://doi.org/10.1101/2024.04.10.588854doi: bioRxiv preprint there a r e no to ol s w hich an alyse t h e ra w dat a and s e a mless ly offe r s t a ti st i c al anal y s is a n d vi s ualis a t io n t hat can b e fi n el y t u ned f or each dat ase t. To co m pl e men t t h e curre ntly a v a i la ble to ol s we ha ve d e v elope d PIM MS - Da sh, a c o mp leme nt a ry colle c tion o f tools w o rk in g fro m a si m p le b r o w ser base d int erface a ll o w i ng for q u ick an d e as y anal ysis , stat istica l eva l uat io n a n d v is ua l isa ti o n of ins erti o na l mu tat ion m a p pin g s equ e n c e da ta . Implementation The PI M MS- Da s hbo a r d w as dev e lo p e d in Py th o n v ersio n 3.9 using t he IDE P ych a rm v e r sion 2 0 20.2. The ap p ma k es use o f f ollow in g ext ernal pyt h on p a ckages; nu mp y ( v 1 . 2 0.1), pa nda s ( v1. 2 . 3), pl o tl y ( v 4 .1 4.3), das h (v1. 19.0 ). A dditi o nall y , DE S eq process i ng is do ne u si n g R vers ion 4.0. 4 a n d t he pa c kage DE S eq2 ( v3.14) . I nt eractio n be twe en p yt hon a n d R s cript s is han dled w it h t he pa c kage R p y2 ( v 3. 4 . 4). The w eb a pp is ho ste d on the Az ure A pp S er vice Pl a n a nd ca n be a cc e s sed he re [ h t t ps ://pi mms- d as h b o ard- uo n.a z ure w ebsit es. net /]. Do c k er ( v20.10.7 ) w as u se d t o co ntaine r i s e th e a pplicat ion an d pu s h t he co nt a iner image to t h e Az ure c o n ta in e r re g i st ry . There are t wo set s o f ex am p le da t a pro v ided o n t he w eb app “PI MM S _ Exa mple_ A”. Th e res pective “ con trol” a n d “te st” fi le s c a n be s electe d in t he l e ft co ntro l pan el al on gsi de t h eir “coord inat e-gff files” a n d ru n t hro ug h the das hb oard. Us e r s c a n als o u pl o a d t h eir own cs v a nd g ff fi l es w hic h c a n be ge nerated u s i ng PIM MS2 (h t t p s ://git hu b.co m/S t rept ococ cal - Re sea rc h- Group/PIM MS2) via t he upl o a d b ox, w hi ch w i ll m ake t h e m a v a ila b le to r un in t h e dro pd ow n li s t s. A ny u ploa ded d a ta is o n ly ava ilable t o t h e curre nt brow ser sess io n an d does not b eco me p ublicl y ava il a b le. It is r ecom me n d e d t hat t he d as h b o ard is ex plor ed wit h t he exa m p le d a tas ets prior t o u s e w it h n ew dat a to pr o v ide a n o verv iew of its us e. The g e n e r a l das hb oard o pt io ns can b e foun d in t he op ti on s t a b o n t he left co ntr ol p a nel. T h es e i ncl ude plot c o n fi gu r ati o n s f or th e vis ualis atio n ta b s a n d t he a bilit y t o t ogg l e DE S e q p r o c e s s in g . We re c omme nd t ha t t he defaul t o ut l ier re mov a l i n D ESe q2 is left en a b le d . The PI M MS- Das h b oar d p r e -loade d d a t ase ts are fro m a hig h-t hrou g h p u t ins ertio n al m ut agenes is s eq ue n c i n g e x pe rimen t c omp aring Streptococus suis (P 1 / 7 ) f o llo wing gr owt h in la b ora tor y m e d iu m ( T o d d H ewitt brot h) a n d p ig ser u m. New da t a ca n be e a sily u ploa ded us ing t he dra g a n d dro p optio n o n th e h ome sc re e n ( F ig 1 A ). A f t e r l o a d i n g d a t a t h e u s e r c a n w o r k t h r o u g h t h e t a b s t o s e e d i f f e r e n t a n a l y s e s a n d r e s u l t s . T h e s i x a v a i l a b l e tabs all o w f or th e user t o vis ualis e t h e u pl o a ded da ta table t o chec k its i ntegrit y. The NIM C o m p a r i s o n t a b a l lo ws us e r s t o v i su al i se th e s atu r a t i on o f in s e r ti o ns a cr o ss the g e no me s , be t we en e a c h co nd it i o n ( Fi g 1 B ) . There are a ls o nu mer ou s met ri c s pro d u c e d from t he DE S e q 2 mo dule w hich de t ermin e s t he rel ative fit ne s s of g e nes bet wee n each co nditio n ( F ig 1 C). On t he V enn ta b , us ers c a n i dentif y ess ential genes w hich are s ha re d o r uniq ue t o t h e c on dit ion s te s ted a n d e x port a s u b s et l is t of g e nes from a cho sen in tersect (Fig 1 D). T h e G e n o m e S c a t t e r t a b p r o d u c e s a n i n t e r a c t i v e f i g u r e w h e r e t h e u s e r c a n z o o m i n o n r e g i o n s o f t h e g e n o m e t o i n ves tig ate larg e r a reas of ess enti a l ge n es a n d se e i f t hey are repres en ted i n bo th c o n d it io ns ( Fig 1 E). The R e pl i c a t e s t ab off e r s a me t hod of q u a lit y c ontr ol t o see if th e re p lica tes cl us ter a s w o ul d be e x pe c ted f or t he two co n d i ti on s (F i g 1 F). Fin a ll y , t h e G eneVie wer ta b e nable s fi n er sc a le a s ses sm ent of th e ins erti o n s det ecte d i n a s pecific gene, w h ic h c an b e se le c ted i n t h e Dat a Table tab (F i g 1 G). .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 14, 2024. ; https://doi.org/10.1101/2024.04.10.588854doi: bioRxiv preprint F i gure O n e: A) La ndi ng p a g e f or u p l oa d i n g d ata and ap plyi ng s et ti ngs B) N o r malis ed dat a t o dire c tl y c o m pa r e betw een tes t a n d c o n t rol m u tant l ibra r ies a n d insert io nal pos iti ons. C) Dat a tabl e wi t h stat ist ical ana l y sis. D) Venn p age to ide ntif y and s u bse t es s enti al genes. E) Ge n o m e S ca tter pag e t o v is u ali s e re g i o ns of ge nom e w hic h la c k s an y m utati o ns or iden tif y areas of h ig h sat u r a t io n. F) Gene View e r p a g e t o in terro gate the l ocal is a ti o n of th e i ns erti ons at a C D S le v e l. G) Scat ter pl ot t o vi su a li se t he (dis )s imi la r i t y of t he p o ol s.

Ex plorati o n a nd an a l ysis of hig h t hrou g hpu t st i ll re m ai ns a da unti n g t ask f or a bi oinfor m a t i c s n o v ice. P IM MS 2 and PIMM S- D a s h offers a n acc e s si ble ap pro ach t o i ns erti on m appi n g dat a anal y s is wi t h t he e m p h a s i s on q ui c k and e as y st at istica l a n a l y s i s an d v is ua l isa ti o n of re s ults t o a p ub l ic a t io n re a dy s ta ndar d. A B C D E F G .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 14, 2024. ; https://doi.org/10.1101/2024.04.10.588854doi: bioRxiv preprint Funding Information T h i s wo r k wa s s uppo r t e d in p a r t by th e In t er n a t ion al Dev el opme n t Re s ea r ch C en t r e ( I D R C ) [ Re f : 1 090 56 ] a nd the Biot ech no l o g y a nd Biol o g ical S c i e nces R es e arch C o u ncil ( B B S RC ) [R ef: B B/T 0 01 9 33/1 ].

1 . G o odma n AL, W u M, Gor d o n JI. Id e n t i f yi ng micr obi al f it ne s s d e term i n a nts b y ins e rt ion s e q ue n c i ng u s in g g e n o m e-wi de tra ns p o s on m u t an t l i brari e s . N at Prot o c . 2 01 1 ; 6: 19 6 9 – 8 0. 2 . v a n Op ijn e n T , B od i K L , C ami l li A. Tn - se q: hi gh - th r o ughp u t pa r a l le l seq uen c i ng f o r fi tn ess an d g e ne t i c i nt eracti o n s tud ies in mi c ro o r g a nis ms . N at M e t ho ds. 20 0 9 ; 6: 76 7–7 2. 3 . Ga wr on sk i JD, Wo n g S MS , Gia nnou ko s G, Wa r d DV , A ke rl ey B J . T r a c k in g i n se r ti on mu tan ts with i n li b r a r i e s b y d e ep s eq uenci n g an d a geno me-w i de sc re en fo r H ae mo p hi l u s g e ne s req uired i n t he lu ng . Proceedi n g s of t he N a ti on a l A c a de my o f S ci en c e s . 2 0 09 ; 106 :164 22 – 7 . 4 . La n g ri d g e G C, Ph an M-D, T urn e r DJ, Pe r kin s TT, Parts L , H aa s e J , et al. Simu ltan eo u s a ss a y of e very S almo n e ll a T yph i ge n e usi ng one millio n tran sp os o n mu ta n t s. G en o me R e s earch. 20 09;1 9 :23 0 8 – 1 6 . 5 . Bla nchar d AM , Leig h J A, E g a n S A, E mes R D. Trans p os o n i n serti o n map pi ng w it h P IM MS – P ra g mat ic I ns e rt io n a l M u t ati o n Ma ppi n g S yst em. Fro nt G e ne t. 2 0 15; 0 6 . 6 . Bl an c h a rd AM, E g a n S A, Em es RD, Wa r r y A, L e ig h JA. P I MMS ( P r ag ma ti c In s e r t ion a l Mu ta t ion Map p in g S ys t em ) La bo r a to ry Me thodo l og y a Re a d i l y Ac c e s si b le To o l fo r Id en ti f i ca ti on o f Es s ent i a l Ge ne s i n S t r ep to c o c cu s. F r ont Mi c ro bi ol . 20 1 6; 7 . .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 14, 2024. ; https://doi.org/10.1101/2024.04.10.588854doi: bioRxiv preprint