Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia

43 SARS-CoV-2, COVID-19 diagnostic testing, Nex t-Ge ner ation S equencing, RT-PCR, G enetic diversi ty 44 HIGHLIGHTS 45 • Colombian SARS-CoV-2 sequences displa yed genetic variabili ty in some targ et r egions used for 46 COVID-19 diagnosis. 47 • Mismatches in critical p rimer r egions cou ld impact thei r performa nce and th e ra te of false-48 negative r esults. 49 50

51 In la te Dec ember 2019 in W uhan ci ty (China), a n ew coron avirus calle d SAR S-CoV-2 (initially nCoV-2019) 52 caused th e ou tbre ak of a respi rat ory disease known as the i nfectious dis ease du e to the new 53 coronavirus (COVID-19 ) ( Zhou et al. , 20 20; Zhu et al ., 2020). Soon afte r lea rning about the po ten tial for 54 transmission of this virus in the con t ext of a glob alized worl d, count ries t ook swift and ex treme 55 measures such as bor der closings, rigor o us follow-up of contacts , and manda tory preventive isola tion. In 56 Colombia, the first case of COVID-19 wa s identified on March 6, 2020, shor tly before th e World He alt h 57 Organiza tion (WH O) declar ed COVID-19 as a pand emic after i t had sp rea d i n 114 countri es in all 58 continen ts and having claimed th e lives of 4,291 people (WHO, 2020a). 59 The first cas e of COVID-19 in Col ombia was import ed from Italy, a co untry tha t had th e mos t ala rming 60 epidemic p eak in Eu rope at tha t time. Sh ortly aft erwards , cases of COVID-19 wer e diagnose d in travel ers 61 from other o rigins, as well as in multipl e of their con tacts . On Ap ril 20, 2020, community transmissio n 62 cases already e xceed ed 10% of the tot al cases register ed in the coun try, which is why the transition to 63 the mitiga tion phas e was declar ed. U ntil May 18, 2020, 4,629,000 cases and 297 ,380 deaths h ave bee n 64 repor ted globally, and in Colombia, 16, 295 cases and 592 deaths have been report ed (Dong, Du, & 65 Gardn er, 2020). 66 SARS-CoV-2 is a bet acoron avirus with a positive pol arity singl e-strand ed R NA g e nome of app roxim ately 67 30 kb. This new cor onavirus sha res a glo bal iden tity of 96 .2% with th e ba t cor on avirus RaTG13 (Zhou et 68 al., 2020), 91 .2% with the Malay Pangoli n coronavirus isola te, Pangolin-CoV (Zhang, Wu, & Zhang, 2020), 69 and even 97.5% with RmYN02 derived from bat when th e ORF1ab gen e is exclu sively analyzed (Wang, 70 Pipes, & Nielsen, 2020). I ts rec ent o rigin is enigmatic since the re is a high similar ity betwe en the amin o 71 acid sequence in th e recep tor-bin ding domain (RBD domain) of subunit 1 (S1) of the Spike prot ein of 72 SARS-CoV-2 and that of Pangolin-CoV , but the lat te r lacks the polybasic furin p rocessing site, exclusiv e 73 to SARS-CoV-2 (Andersen, Rambau t, Lipkin, Holmes, & Garry, 2020). Accumulat ed evidence suggests a 74 zoonotic origin of the virus as a r es ult of r ecombina tion wit h a y et u nid entified c oronavirus or 75 convergent evoluti on drive n by na tura l selecti on to op timize int eracti on with the human ACE2 cell 76 recep tor (Wang e t al., 2020; WHO, 2020 b). After th e publica tion of the compl et e SARS-CoV-2 genome in 77 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint genomic data re posito ries such as NCBI (MN908947.3) and GIS AID in mid-January 2020, health agencie s 78 and r esearch ers fr om differe nt c ount rie s quickly develop ed S ARS-CoV-2 screeni ng tes ts bas ed on r eal-79 time RT-PCR (rRT-P CR) that amplify diffe rent SARS-CoV-2 gene regions. Hundr eds of commercial kits are 80 under d evelopmen t and many of them have b een lic ensed for emergency us e 81 (https://www.finddx.org/covid-19/pipeline/). Various i n-house pro tocols we r e also develo ped and 82 published on the websit e of the World Health O rganiza tion (WHO) for informational pu rposes withou t 83 implying endorsement , prefer ence or v alidation by this en tity (WHO, 2020c). However, most of thes e 84 protocols were publish ed du ring Janua ry 2020 when o nly 230 virus s equenc es were availabl e tha t 85 circulated exclusively in Asia and Euro pe, e xcept fo r a few cases in th e Unit ed Sta tes and Can ad a 86 (Holshue et al ., 2020). 87 Since then , refinemen ts of these p rot o cols are not known in the con te xt of n early 30,000 sequences 88 repor ted on May 18, 2020, worldwide, including Latin America, which pro vide a more complete 89 perspec tive of th e accumula ted ge netic variability and seque nce pa rticula riti es of viruses circulating i n 90 specific regions tha t could affect th e efficiency and sensitivity of the r RT-PCR protocols curr ently sha re d 91 by WHO. The mu ta tion ra te of SARS-CoV-2 as a virus with an RN A genome is higher th an th at of viruse s 92 with a DNA genome (Tang et al., 202 0), with an estimated mean evolutio n ary rate of 2.24 x 10 -3 93 substitu tions/site/year (Li, Li, Cui, & Wu, 2020); therefor e, changes in the seq uence could occur over 94 time t hat compromise the ope rat ional performance of diagnos tic t ests (PAHO, 2020). The objective of 95 this study was to describe t he gene tic variability of Colombian SARS-CoV-2 genomes in hybridizatio n 96 regions of oligonucleo tides of the mai n in-house methods for S ARS-CoV-2 detecti on. 97 98

99 Patients and samples 100 Nasopha ryngeal swab samples from patients with suspected SA RS-CoV-2 infection were received at th e 101 Institu to Nacion al de Salud (I NS) as pa rt of the virological surveill ance of COVID- 19 from 11 Colombian 102 depar tments and the c apital distric t. A ccording to the nati onal law 9/1979, decrees 786/1990 and 103 2323/2006, the INS is t he r eferenc e lab a nd health auth ority of th e nati onal ne tw ork of labora tori es and 104 in cases of public healt h emerg ency or t hose in which scientific rese arch for pu blic health pu rposes a s 105 requir ed, the I NS may use the biological material for res earch purpos es, with out informed consent , 106 which includes th e anonymous disclosu r e of resul ts. This s tudy was perfo rmed i n accordance with t he 107 ethical s tanda rds no ted in th e 1964 Decl arati on of H elsinki and its la te r amend ments. Th e informa tio n 108 used for this study comes from secondary sources of data that were pr eviously anonymized and do not 109 repr esent a risk to th e community. 110 RNA extraction and real-time rRT-PCR 111 Viral RNA was ob tain ed using th e aut o mated M agNA Pure LC nucleic acid ext raction syst em (Roche 112 Diagnostics GmbH, Mannheim, G ermany ) and viral RNA detection was perform e d by rRT- PCR using the 113 SuperScrip t II I Platinum One-S tep Quan tita tive RT- kit. PCR (Thermo Fisher Scientific, Wal tham, MA , 114 USA), following the Chari té-Be rlin pro to col (Victor M. Corman e t al. , 2020) for t he amplification of t he 115 SARS-CoV-2 E and RdRp genes. 116 Next generation sequencing 117 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint The complet e SA RS-CoV-2 genome seq uence of 30 p ati ents was o btain ed thro ugh NGS, t en genom es 118 with Oxford N anopo re (Oxfor d Nanop ore Technologi es, Oxfo rd, UK) and 20 genomes with Illumin a 119 MiSeq (Illumina, S an Diego, CA, USA) te chnologies, following th e ar tic.ne twork "nCoV-2019 sequencing 120 protocol " (Quick, 2020). In bo th str at e gies, SARS-CoV-2 specific oligonucleoti des were us ed for th e 121 genera tion of amplicons by means of a Q5® high fidelity DNA polymerase (Ne w England Biolabs Inc., 122 UK), in order to avoid th e int roduct ion o f artificial muta tions. The g enomes wer e assembled by mapping 123 to the refer ence genome (NC_045512.2 ) using the BWA (Li et al., 2020) and B Bmap (brian-jgi, 2020) 124 software to gen era te a cons ensus genom e by the two assembly to ols. 125 Genetic diversity analysis 126 The Colombian genomes and oligonucleotid es from of the in-house protocols were aligned with th e 127 Muscle to ol (Edgar, 2004) using the M EGA X softwar e (Kumar, St echer , Li, Kn yaz, & Tamura, 2018). 128 Substitu tions ma trices of the Col ombian genomes resp ect to the refer ence ge no me (NC_045512) at th e 129 nucleotid e and amino acid levels we re genera ted fo r th e 13 r RT-PCR protocols published at the WH O 130 website (WHO, 2020c), which several countries have es tablishe d as their pr eferr ed diagnostic prot ocol 131 for SARS-CoV-2. 132 Oligonucleotides analysis 133 Thermodynamic featur es (priming Tm a nd Δ G at t he variabl e sites, mispriming a nd Δ G, hair pin Δ G , and 134 primer dimer Δ G) and the codon positi on at the 3’ end for oligonucle otid es with conflicting sites and 135 optimized oligon ucleoti des were evalua ted int o the Primer Selec t module of th e LaserG ene v8.1 suit e 136 (DNASTAR Inc. Madiso n, WI , USA). 137 138

139 Several target regions for SARS-CoV-2 molecular detection using in-house protocols. 140 A to tal of 39 p rimer and prob e seq uen ces from t he mai n in-hous e rRT-PCR pr otocols for SA RS-CoV-2 141 detec tion publishe d at WHO websi te were aligned to the r efere nce sequenc e derived from the firs t 142 confirmed case at Wuhan, Hu bei pr ovince, China, and nam ed Wuha n-1 strai n (GenBank Accessio n 143 Number : NC_045512.2). The p rotoco ls t arget ed 13 differ ent g enome regions in side th e O rf1ab (Nsp9 , 144 Nsp10, Nsp11, RdRp , ExoN), E (Envelope) and N (Nucleocapsid) genes with 61, 5% (8/1 3) of the assays 145 targe ting the N gene (Figure 1). 146 147 Point mutations in Colombian SARS-CoV-2 genomes at the target hybridization sequences of some in-148 house protocols for SARS-CoV-2 detection. 149 A total 30 whole genome SA RS-CoV-2 sequences from Colombia were included in the previousl y 150 obtain ed alignment for gen etic varia bility analysis. These sequ ences deri ved from cases with 151 confirmation d ates from Ma rch 6 th -24 th . 2020 wer e r emit ted from 11 depa rt ments and the capit al 152 district (Anti oquia, B ogotá D.C., B oliva r, Caldas, Cauca, Magdal ena, Nor te d e Santan der , Quindío, 153 Risaralda , San tand er, Tolima a nd Valle del Cauca) through th e Natio nal Publ ic Healt h Labo rato rie s 154 Netwo rk to th e Colombian Na tional Insti tute of He alth for diagn ostic confirmatio n. 155 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint From the alignm ent of th e 39 oligonucle otides t o th e refe rence a nd Colombian S ARS-CoV-2 sequences, 5 156 showed mismatches with a t leas t one Colombian sequence (Tabl e 1). The conflicting sites in th e 157 primer/prob e sequenc es were due t o 1) a mismatch between the oligonucle otid e and the refe rence an d 158 Colombian seque nces or 2) a mismatc h betwe en t he oligo nucleo tide and on e or mo re Colombia n 159 sequences . 160 The Hong Kong(2020) H KU-NP probe ( Chu et al., 2020) (Figure 2) was extre mely different to all th e 161 refere nce and Colombian SARS-CoV-2 by four nucleotide sit es. This probe also pr esents th e formation o f 162 a highly stabl e h airpin and s elf-dimer s tructu res (Table S1). The Corman Berli n(2020) RdRP SARSr-F2 163 (Victor M. Corman et al., 2020) (Figure 3) region was variabl e in on e Colombia n SARS-CoV-2 sequenc e 164 from the d epar tmen t of Valle d el Cauca (ID: 79943), involving a critical site at th e 3’ pen tame r. This sit e 165 is supposed to pr event th e cor rect hybridizati on of t he 3’ end of the prime r, l eading t o inefficient o r 166 unsuccessful ext ension by the DN A pol ymerase. The R dRP SARSr-R1 prim er (Victor M. Corm an et al. , 167 2020) showed a degen era te site which does no t comp rise the nucle otid e foun d in the refe rence an d 168 Colombian SARS-CoV-2 sequenc es. Howe ver, this misma tch was loca ted at an int e rnal sit e of t he p rimer , 169 only partially affecting the th ermodynam ic profile of the primer-ta rget hybridiz ati on. The Zhu 2020 C DC-170 China Set I Probe(OR F1ab) (Zhu et al., 2020) (Figure 4) was found to be almost comple tely 171 complementa ry with the Colombia n SAR S-CoV-2 sequences, excep t for a viral seq uence obt ained from a 172 human case in the depa rtmen t of A nti oquia (ID: 79253), bea ring a single subs titu tion a t th e seven th 173 probe posi tion, wit hout consi dera ble effect on th e the rmodynamic feat ures for prob e-ta rge t 174 hybridizatio n (Table S1). The Zhu 2020 C DC- China Set II Fw(N) primer (Figur e 5) hybridization was foun d 175 to b e cri tically affect ed by the accumul ated gene tic diversi ty of the Colombian SARS-CoV-2 strains . At 176 the 5’ end of th e primer a t riple-nucle oti de substitu tion G GG /barb2rightAAC in thre e sequ ences from Bogotá an d 177 Valle del Cauca affected th e Tm and Δ G. At th e 3’ r egion, two s equenc es fro m Quindío displayed a 178 substitu tion affecting th e 3’ pen tamer st ability. 179 The primers and prob es sets pr oposed by Pasteur 2020 (France)nCoV IP2 (Fig. S1), Hong Kong (2020) 180 HKU-ORF1b-nsp14 (Fig. S2), Corman Berl in(2020) E Sarbeco (Fig. S3), CDC 2020 (USA) 2019-nCoV N1 (Fig. 181 S4), DMC-MH 2020(Thailand)WH-NIC N (Fig. S4), CDC 2020(USA) 2019-n Co V N3 (Fig. S5), Corman 182 Berlin(2020) N Sa rbeco (Fig. S5), Na o 20 20(Japan)NIID 2019-nCOV N (Suppl. Fig . 6) and CDC 2020(USA) 183 2019-nCoV N2 (Fig. S6) showed corr espondenc e with th e r efer ence s equ ence a nd with all th e 184 accumulated gen etic variabili ty in availab le sequences of Colombian st rains of SAR S-CoV-2. 185 186 The third codon position was found to align with the 3’ end of some primers with intended use for 187 molecular detection of SARS-CoV-2 188 Codon positi ons in co ding r egions ar e differentially susc eptibl e to nucl eotid e substitu tion, being th e 189 third codon p osition associ ate d with a hi gher substit ution rat e. O n the othe r han d, the p erfect ma tching 190 of the l ast n ucleo tide at the 3’ end of every forward and revers e prime r is cri tica l for DNA polymeras e-191 based e xte nsion during PCR amplificati on (Staheli, Ryan, Bruc e, Boyce , & Rose , 2009). Therefor e, th e 192 ration al design of p rimer to b e used w ith rapi dly evolving RNA virus es should have th e re quisite of 193 avoiding third and some times first codon positions. 194 Codon position of th e last nucleo tide at the 3’ end of every analyz ed prime r was identified according to 195 the corr esponding op en-read ing frame. The 3’ end of 11 primers correspon d ed to th e first codon 196 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint position, an othe r 11 primers had 3’ en ds located at th e second codon posi tio n and the 3’ end of 4 197 primers aligned with the third codon p o sition (Table 1). One of th e prime rs (Nao 2020(Japan)NIID 2019-198 nCOV N F2) had the 3’ end aligned with the first codon posi tion of the codon C UA which encodes for 199 alanine . This codon allows a su bstit utio n at the first codo n posi tion (C to U) t o be synonymous an d 200 ther efore may escap e to th e selec tion pr essure a t the p rot ein level. 201 The 3’ end of the prim ers CDC 2020( USA) 2019-nCoV N1-F, CDC 20 20(USA) 2019-nCoV N3-F, CD C 202 2020(USA) 2019-nCoV N3-R and Hong K ong(2020) HKU-NR aligned with the third codon positio n of thei r 203 correspond ing ORFs. Two of these prime rs were the forward and r everse prime rs proposed to amplify a 204 specific targe t (CDC 2020 (USA) 2019-nC oV N3) at t he N gene making this pr oto col very susceptibl e t o 205 false negative r esults as th e viral gene tic variability increas es. 206 207

208 Nex t Gene rati on S equencing (N GS) tech nologies ar e a valuable tool in d ete rmini ng the whol e genom e of 209 microorganisms impacting on public hea lth, they have accel era ted our abili ty to underst and import ant 210 factors in the bi ology of infectious diseas es such as the iden tification of de te rmin ants of virulence, d rug-211 resistanc e associat ed substitu tions, vaccine design and genomic epidemiology, a powerful approach t o 212 integra te epidemi ologic and g ene tic info rmation i n r econstruc ting t ransmission patt erns and infect ious 213 disease dynamics (Gwinn, MacCannell, & Armstrong, 2019). N GS has also enabl ed pathogen discovery in 214 a timely manner compa red to tr aditi ona l methods, b eing essenti al to the virus t axonomic classificatio n 215 during the beginnings of COVID-19 pand emics (Zhou et al., 2020; Zhu et al., 2020). This robust molecular 216 information is als o th e raw mat erial fo r the deve lopmen t and r efinemen t of mo lecular and s erol ogical 217 diagnostic meth ods (Wang et al ., 2020) and all sequenc e-based d esigns are susc eptibl e to improvem ent 218 as the virus disseminat es and its gen etic variability increas es. 219 Muta tion is th e fundamen tal sou rce of g enetic vari atio n and R NA viruses are par ticularly suscepti ble to 220 have high mutati on ra tes duri ng the ge nome replica tion (Sanjuan , Neb ot, Chiri co, Mansky, & Belshaw , 221 2010). Therefore, RN A viruses display high substitution r ates when analyz ed through time . Alth ough 222 sequence id enti ty betwe en th e analyze d genomes and th e Wuh an refe rence s train (NC_045512) was 223 around 99 .9%, th e es timat e subs titu tion rate for S ARS-CoV-2 is in the rang e of 1. 67-4.67 x 10 -3 /site/yea r 224 (Tang et al., 2020). Ther efore , it is evide nt tha t th e genomic da ta of this stu dy allow the refin ement o r 225 even the design of more precise and efficient protoc ols for the molecular d etec tion of the gene tic 226 variants of SARS-CoV-2 circulating in Colombia. 227 An enormous effort of every official and private labo rat ory around t he word has led to the availa bility of 228 hundreds of commercial kits and in- house pro tocols for the molecul ar d etec tion and COVID-19 229 diagnostic confirma tion . Whi le this effort is welcom e and some recomme ndati o ns have b een provide d 230 by regional agencies, th e availa bility of al l thes e me thods imply the n eed for a n ev idence-based crit erio n 231 for decision-making about the best op tio n at country level. In this study, based on NGS data of complet e 232 SARS-CoV-2 genomes from Colombian c ases, timely evidence is provided on th e genetic variabili ty of 233 repr esent ative st rains of the circul atin g viruses in the country t hat shoul d be consider ed for a n 234 evidence-based impr ovement of th e rou t ine diagnostic t ests. 235 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint The majority of the evalua ted in-h ouse molecula r assays displayed ge netic sta bility at th e 236 primers/prob es ta rget regions . However , some t hese oligonucleo tides d isplayed mismatches th at we r e 237 considered of minor o r major import anc e for the t est pe rformance bas ed on th e in silico analysis. Some 238 of the analyzed p rimers/prob es displayed mismatches when compar ed to t arg et sequ ences of some 239 Colombian SARS-CoV-2 strains. Polymorphic sites in the compl ete SARS-CoV-2 genomes ob tain ed in thi s 240 study, were suppo rted by sequ encing depths be tween 123 and 390X sufficient to id entify thes e 241 sequence vari atio ns unequivocally. Th e analyzed g enome seq uences co rres ponded t o th e ea rly 242 introduc tion and dispersio n of the virus in the country. It is exp ected th at some of the identified 243 nucleotid e substitu tions r emain stabl e in subsequent transmission chains of the corresponding clust er s 244 of cases, as demonstr ate d in the p resen t study as characte r sta tes shar ed by two or more sequ ences. 245 Prior to the a rrival of SARS-CoV-2 in C olombia, th e Charité-B erlin pr otocol d e scribed by Corman and 246 collabora tors was establish ed as a rou ti ne in de tec tion (Victor M . Corman et a l., 2020) following the 247 recommenda tion of the Pan Americ an Health O rganiza tion (PAHO). Their thr ee molecular ta rgets (RdRp , 248 E and N genes) wer e a nalyzed and R d Rp displayed s ome conflicting nucleo ti de sit es. Th e an tisense 249 primer RdR p SARS r R1 pr esen ted a de genera te si te a t t he nucle otid e posit io n 15,519 equivalen t t o 250 nucleotid es G o r C (S) while the refer e nce and all Colombian s equences displ a yed T at tha t positi on. 251 However, this mismatch was exp ected not to affect seri ously the over all stabil ity of its hybridization . 252 There was an impor tan t finding in the sense prime r RdRp _SARSr _F1 which dis played a mismatch a t 253 position 15,451 when compar ed to th e hCoV/Colombia/Valle_del _Cauca/79943/2020 genome. Thi s 254 mismatch locat ed a t the second base of the 3’ e nd of the primer is e xpec te d to s everely affect th e 255 primer hybridiza tion and subsequ ent D NA polymeras e-mediat ed ex tensio n. In the pres ent stu dy, we 256 propose to include a degen era te si te (R) as follows (RdRP_ SARSr-F2 M od 5 '-257 GTGA AATG GTCATGTGTG GC R G-3'). Thus, covering the gene tic variabili ty and improving th e 258 thermodynamic s tabili ty with th is gen etic varian t to avoid fals e-negative re sults (Table S2). Th e 259 nucleotid e iden tity of the Colombi an gen omes with respec t to the Corman Berlin ( 2020) E Sarbeco and N 260 Sarbeco designs was 100%. These results suppor ted th e PAHO recommendati on of diagnostic 261 confirmation based on th e single E gene in the conte xt of sustaine d transmissi on at high levels in this 262 region where o ther cor onavirus species of the subgenus Sarbecovirus are e xpec t ed to be absen t (PAHO, 263 2020). 264 Anoth er critical char acte ristic of the pri mers/probes of the curr ent in-house pr otocols listed by WH O 265 and assesse d in the pres ent s tudy was the codon p osition at th e 3’ end of t he sense and antis ense 266 primers in coding r egions. The majo r an d minor susceptibili ty to nucl eotid e sub stituti ons for th e thi rd 267 and second cod on posi tions, r espec tivel y, is widely known. As any change at th e 3’ end can affect the 268 primer hybridiza tion , it is highly recommended th at this posi tion aligns with th e second (prefera ble) o r 269 first codon p osition . Some prime rs and i ndeed p rimer s ets we re found to be coi ncident wit h th e thi r d 270 codon positi on. Despi te not finding subs titu tions a t t hese sites i n th e Colombian strains, tha t posi tion is 271 expec ted t o be unst able t hrough tim e. 272 Genomic da ta available from this st udy allowed the in silico evalua tion/refin ement of the pr otocols fo r 273 molecular de tecti on of SARS-CoV-2 cir culating in Colombia. It is highly reco mmended to est ablish 274 routin e molecular surveillanc e of the virus in order to det ermine th e real impac t of every mutation in 275 the diagnos tic tes t’s perform ance (PAHO , 2020). 276 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint Finally, the implemen tati on of molecular tests at count ry-level should be support ed by the estimatio n of 277 the analytical sensitivity (Limit of det ect ion [LOD] in copies/re action), sp ecificity (Victor M. Corman e t 278 al., 2020), and the accumula ted ge neti c variability should b e tes ted d uring th e implemen tati on of a 279 molecular d et ection pro tocol i ncluding t he clinical s ensitivity in differen t biofluid s (V. M. Corman e t al . , 280 2016), reprod ucibility, rep eat ability, i nt er-oper ato r, in te r-instrume nt, int er-site and in ter-b atch assays 281 (Hu et al., 2019). 282 283

284 Detectio n of SARS-CoV-2 viral RNA using nucleic acid amplificatio n t echniques suc h as rRT-PCR continues 285 to be th e gold s tanda rd for the diagno sis of COVID-19 (WHO, 2020d). Howev er, al l sequ ence-base d 286

ar e susceptible t o nucleoti de substitu tion affecting the oligonucl eotid e hybridizatio n efficiency 287 and resulting in false negatives . Some of the in-house pro tocols analyzed in th e pr esent study req uire an 288 expe riment al evalua tion of th eir pe rfor mance in the con te xt of virus gene tic variability . The genomic 289 data of this study all ow the refin ement o r even the d esign of more precis e and efficient pro tocols for th e 290 molecular d et ection of th e gene tic vari a nts of SAR S-CoV-2 circulating in Colombi a. Howeve r, mor e NGS 291 data fr om Colombian S ARS-CoV-2 will be de termin ant to a b et ter compreh e nsion of th e impac t of 292 genetic variabili ty on specific molecular a ssays of routine use as th e virus evolves. 293 ACKNOWLEDGMENTS 294 For th e joint work with the profession a ls involved in facing th e SA RS-CoV-2 pandemic from differen t 295 aspects and to th e Directo rat es of Publi c Health Resea rch, Public Health N etwo rks and Surveillance of 296 the Na tion al Insti tut e of Healt h. 297 CONFLICTS OF INTERESTS 298 The autho rs declar e tha t th ere is no conf lict of inter est in th e manuscript . 299 FUNDING 300 This study was funded by the Nation al In stitut e of Heal th, in Bogo tá D.C., Colombi a. 301

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint Zhou, P., Yang, X.-L., Wang, X.-G. , Hu, B. , Zhang, L., Zhang, W., . . . Shi, Z.-L. (2020). Discovery of a novel 362 coronavirus associa ted with t he rec ent p neumonia ou tbre ak in humans and its po tenti al bat 363 origin. bioRxiv , 2020.2001.2022.914952 . doi:10.1101/2020.01.22 .914952 364 Zhu, N., Zhang, D., Wa ng, W., Li, X., Yang, B., Song, J., . . . Rese arch, T. (2020). A No vel Coronavirus from 365 Patients with Pneumoni a in China, 2019. N Engl J Med . doi :10.1056/NEJMoa2001 017 366 367 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint Figure Legends 368 369 Figure 1. Target hybridization regions of the primers/probes employed by the principal in-hous e370 protocols for molecular detection of SARS-CoV-2. The pro tocols targe ted 13 different g enome region s371 inside the O rf1ab ( Nsp9, Nsp10, Nsp11 , RdRp, ExoN) , E ( Env elope) y N ( Nucleocapsid) genes. Targe t372 hybridizatio n regions of th e prime rs / probes employed by th e principa l in-house protocols for mol ecula r373 detec tion of SARS-C oV-2. The differen t genes and pro tein p roducts, as well as the coor dinat es i n374 kilobases ( Kb) of the gen es and pro tein p roducts to which t hey ar e dir ecte d wer e estimat ed accor ding t o375 the SARS-C oV-2 refer ence genome av aila ble at G enBa nk ( NC _045512.2) . 376 377 Figure 2. Genetic diversity at the target region of the Hong_Kong (2020)_HKU-NP assay. Hong_Kon g378 ( 2020) _HKU-NP probe displayed misma tches at positio ns 29187-88 and 29197-98 ( highlighted in red) ,379 with all the C olombian seq uences and th e RefSeq displaying C A and C C , respectiv ely. Gen omic position s380 were estima ted according to t he SARS-C oV-2 reference genom e av ailable at G en Bank ( NC _045512.2) . 381 382 383 384 e s t r n o g , s All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint 385 Figure 3 . Genetic diversity at the target region of the Corman_Berlin (2020) _RdRP assay. Th e386 C olombian genome hC oV/C olombia/Valle_del _C auca/79943/2020 displayed a substitu tion ( G to A) a t387 position 15,451 ( highlight ed in r ed) , affecting the p rimer C orm an_B erlin ( 2020) _RdRP _SARSr-F 2388 hybridizatio n r egion. Th e substi tuti on is l ocated in t he p enultim ate positi on a t t h e 3 ’ end of the prome r ,389 genera ting a mismatch tha t could in terfe re with th e 3’ pen tamer s tabili ty. A dege nera te base ( S = C or G )390 in the prime r C orman_ Berli n ( 2020) _Rd RP _SARSr-R1 at p osition 15,519 ( highlighted in red) p roduces a391 mismatch with all th e C olombian gen om es and th e RefSe q as th ey hav e T in th e sense sequ ence, so th e392 degener at e base shoul d be on e tha t inc ludes an A among d egene rat e alt erna ti v es. Gen omic position s393 were estima ted according to t he SARS-C oV-2 reference genom e av ailable at G en Bank ( NC _045512.2) . 394 395 Figure 4. Genetic diversity at the target region of the Zhu_2020_CDC-Set I assay. Th e C olombia n396 genome hC oV/C olombia/Antioquia/792 53/2020 displayed a substitu tion ( C to T) at position 13,38 4397 ( highlighted in red) where the p robe Zhu _2020_C D C -Set I P robe ( ORF1ab) hybridi zes. G enomic positi on s398 were estima ted according to t he SARS-C oV-2 reference genom e av ailable at G en Bank ( NC _045512.2) . 399 400 401 e t 2 , ) a e s n 4 s All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint 402 Figure 5. Genetic diversity at the target region of the Zhu_2020_CDC-China_Set II assay. Thr e e403 C olombian genomes , hC oV/C olombia/Bogota/78390/2020, hC oV/C olombia /404 Valle_del _C auca/81279/2020 and hC oV/ C olombia/Valle_del _C auca/81251/2020 hav e the sam e pat te r n405 of substitu tions ( G GG to A AC ) in primer Zhu_2020_C D C -C hina_Set II Fw ( N) in th e first t hre e nucle otide s406 at th e 5' end ( highlight ed in red) . Furthe rmore , the hC oV/C olombia/Quindio/79911/2020 an d407 hC oV/C olombia/Quindio/80663/2020 g enomes displ ayed a substi tuti on ( G to T) at posi tion 28,89 9408 where this oligonucl eotid e hybridiz es ( hi ghlighted in red) . Gen omic positions we re es timat ed accor din g409 to the S ARS-C oV-2 reference ge nome av ailable a t Ge nBank ( NC _045512.2) . 410 411 Table 1. Target gene/regions and design conflicts of the analyzed primers and probes 412 according to the genetic diversity of the Colombian SARS-CoV-2 strains. 413 Target gene Primer/probe name 1 Coordinate s 2 Codon position at the 3' end Genomes presenting mismatches ORF1ab Pasteur 2020(F rance)nC oV IP2- 126 69Fw 1269 0- 1270 7 1 None Pasteur 2020(F rance)nC oV IP2- 1269 6bPr obe 1271 7- 1273 7 NA None Pasteur 2020(F rance)nC oV IP2- 127 59Rv 1279 7- 1278 0 2 None Zhu 202 0 CDC-China Set I(ORF1a b) fw 1334 2- 1336 2 2 None Zhu 202 0 CDC-Set I Probe(ORF 1ab) 1337 7- 1340 4 NA hCoV/Col ombia /Anti oqu ia/79 253 /20 20 Zhu 202 0 CDC-China Set I(ORF1a b) Rv 1346 0- 1344 2 1 None Corman Ber li n(20 20) RdRP SARS r-F2 1543 1- 1545 2 2 hCoV/Col ombia /Va lle de l Cauca/799 43/ 202 0 e / n s d 9 g All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint Corman Ber li n(20 20) RdRP SARS r-P2 1447 0- 1549 4 NA None Corman Ber li n(20 20) RdRP SARS r-R1 1553 0- 1550 5 1 All Hong Kong(2 020) HKU-ORF 1b- nsp14F 1877 8- 1879 7 2 None Hong Kong(2 020) HKU-ORF 1b- nsp141P 1884 9- 1887 2 NA None Hong Kong(2 020) HKU-ORF 1b- nsp14R 1890 9- 1888 9 1 None Gen E Corman Ber li n(20 20) E Sa rbeco F1 2626 9- 2629 4 2 None Corman Ber li n(20 20) E Sa rbeco P1 2636 2- 2635 7 NA None Corman Ber li n(20 20) E Sa rbeco R1 2638 1- 2636 0 2 None Gen N CDC 2020(USA) 201 9-nCo V N 1-F 2828 7- 2830 6 3 None CDC 2020(USA) 201 9-nCo V N 1-P 2830 9- 2833 2 NA None CDC 2020(USA) 201 9-nCo V N 1-R 2835 6- 2833 5 2 None DMC-MH 2020( Thai land) WH- NIC N-F 2832 0- 2833 8 2 None DMC-MH 2020( Thai land) WH- NIC N-P 2834 1- 2835 6 NA None DMC-MH 2020( Thai land) WH- NIC N-R 2837 6- 2835 8 1 None CDC 2020(USA) 201 9-nCo V N 3-F 2868 1- 2870 2 3 None CDC 2020(USA) 201 9-nCo V N 3-P 287 04- 2872 7 NA None CDC 2020(USA) 201 9-nCo V N 3-R 2875 2- 2873 2 3 None Corman Ber li n(20 20) N Sar beco F 1 2870 6- 2872 4 1 None Corman Ber li n(20 20) N Sar beco P 1 2875 3- 2877 7 NA None Corman Ber li n(20 20) N Sar beco R1 2883 3- 2881 4 1 None Zhu 202 0 CDC-China Set II Fw(N) 2888 1- hCoV/Col ombia /Bogota/ 783 90/ 202 0 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint 2890 2 hCoV/Col ombia /Va lle de l Cauca/812 79/ 202 0 2 hCoV/Col ombia /Va lle de l Cauca/812 51/ 202 0 hCov/Colomb ia/Qui ndi o/7 991 1/2 020 hCoV/Col ombia /Quin dio /80 663 /20 20 Zhu 202 0 CDC-China Set II Probe( N) 2893 4- 2895 3 NA Ninguno Zhu 202 0 CDC-China Set II Rv(N) 2897 9- 2895 8 1 Ninguno Nao 2 020(Ja pan)NIID 201 9-nCOV N F 2 2912 5- 2914 4 1 Ninguno Nao 2 020(Ja pan)NIID 201 9-nCOV N P 2 2922 2- 2924 1 NA Ninguno Nao 2 020(Ja pan)NIID 201 9-nCOV N R 2 2929 9- 2928 0 2 Ninguno Hong Kong(2 020) HKU-NF 2914 5- 2916 6 2 Ninguno Hong Kong(2 020) HKU-NP 2917 9- 2919 8 NA Ninguno Hong Kong(2 020) HKU-N R 2925 4- 2923 6 3 Ninguno CDC 2020(USA) 201 9-nCo V N 2-F 2916 4- 2918 3 1 Ninguno CDC 2020(USA) 201 9-nCo V N 2-P 2918 8- 2921 0 NA All CDC 2020(USA) 201 9-nCo V N 2-R 2923 0- 2921 3 1 Ninguno 414 415 416 417 418 419 420 421 422 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint Supplementary material Figure S1. Alignment of Pasteur 2020 (France)nCoV IP2 primer sets with Colombian SARS-CoV-2 genomes. . Gen omic positions were esti mated acco rding t o th e SAR S-CoV-2 reference gen ome avail abl e at Ge nBank (NC_045512.2). Figure S2. Alignment of Hong Kong (2020) HKU-ORF1b-nsp1 primer sets with Colombian SARS-CoV-2 genomes. Genomic positions we re es ti mated accor ding to the SA RS-CoV-2 reference genom e availabl e at Ge nBank (NC_045512.2). Figure S3. Alignment of Corman Berlin(2020) E Sarbeco primer sets with Colombian SARS-CoV-2 genomes. Genomic positions we re es ti mated accor ding to the SA RS-CoV-2 reference genom e availabl e at Ge nBank (NC_045512.2). Figure S4. Alignment of CDC 2020(USA) 2019-nCoV N1 (Fig. S4) and DMC-MH 2020(Thailand)WH-NIC N primer sets with Colombian SARS-CoV-2 genomes. Genomic posi tions wer e es ti mated acco rding t o th e SARS-CoV-2 reference genom e available at Ge nBank (NC_045512.2). Figure S5. Alignment of CDC 2020(USA) 2019-nCoV N3 and Corman Berlin(2020) N Sarbeco primer sets with Colombian SARS-CoV-2 genomes. Genomic positi ons were estimat ed accor ding to the S ARS-CoV-2 refere nce genome avail able a t Ge nBank (NC_045512.2). Figure S6. Alignment of Nao 2020(Japan)NIID 2019-nCOV N and CDC 2020(USA) 2019-nCoV N2 primer sets with Colombian SARS-CoV-2 genomes. Genomic positions were es timat ed according to the SARS- CoV-2 reference genome avail able a t Ge nBank (NC_045512.2). Table S1. Thermodynamic characteristics of the primers/probes showing mismatches with the

or Colombian SARS-COV-2 sequences. Table S2. Thermodynamic characteristics of RdRP_SARSr-F2 Mod. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprintthis version posted May 26, 2020. ; https://doi.org/10.1101/2020.05.22.20107292doi: medRxiv preprint