Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol.
Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol.
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DOI：
10.3389/fmed.2022.836728
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发表时间：
2022
影响因子：
3.9
通讯作者：
Githinji G
中科院分区：
文献类型：
--
作者：
Lambisia AW;Mohammed KS;Makori TO;Ndwiga L;Mburu MW;Morobe JM;Moraa EO;Musyoki J;Murunga N;Mwangi JN;Nokes DJ;Agoti CN;Ochola-Oyier LI;Githinji G
ARTIC网络基于引物集和扩增子的测序方案是目前应用最广泛的SARS-CoV-2测序方案之一。2021年6月18日发布了V3引物集的更新，以解决在Delta变体中观察到的扩增子下降问题。在这里，我们报告了对ARTIC网络V4协议修改版本的内部优化，该协议在原始V4池策略以扩增子下降为特征的情况下提高了SARS-CoV-2基因组恢复。我们使用了一组匹配的43份临床样本和连续稀释的阳性对照，通过ARTIC V3， V4和优化的V4引物扩增，并使用牛津纳米孔技术公司的GridION测序。我们观察到，与V3引物相比，使用原始V4池策略时，67%的样本的基因组恢复增加了0.5%至46%。所有变异和阳性对照在引物位置23和90处观察到扩增子下降。当使用优化方案时，我们观察到所有样本的基因组回收率提高了60%，扩增子23和90的平均深度增加。因此，在72% （n = 31）的样本中恢复了≥95%的基因组。然而，在ARTIC V3引物基因组覆盖率<28%的样本中，只有60-70%的基因组可以被恢复。Ct值与基因组恢复的相关性无统计学意义（p > 0.05）。利用ARTIC V4引物，在提高α、β、Delta、Eta和非voc /非voi型SARS-CoV-2变异的基因组恢复浓度的同时，增加了下降或平均读取深度较低的扩增子的引物浓度。
The ARTIC Network's primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol. An update to the V3 primer set was released on 18th June 2021 to address amplicon drop-off observed among the Delta variant of concern. Here, we report on an in-house optimization of a modified version of the ARTIC Network V4 protocol that improves SARS-CoV-2 genome recovery in instances where the original V4 pooling strategy was characterized by amplicon drop-offs. We utilized a matched set of 43 clinical samples and serially diluted positive controls that were amplified by ARTIC V3, V4 and optimized V4 primers and sequenced using GridION from the Oxford Nanopore Technologies'. We observed a 0.5% to 46% increase in genome recovery in 67% of the samples when using the original V4 pooling strategy compared to the V3 primers. Amplicon drop-offs at primer positions 23 and 90 were observed for all variants and positive controls. When using the optimized protocol, we observed a 60% improvement in genome recovery across all samples and an increase in the average depth in amplicon 23 and 90. Consequently, ≥95% of the genome was recovered in 72% (n = 31) of the samples. However, only 60–70% of the genomes could be recovered in samples that had <28% genome coverage with the ARTIC V3 primers. There was no statistically significant (p > 0.05) correlation between Ct value and genome recovery. Utilizing the ARTIC V4 primers, while increasing the primer concentrations for amplicons with drop-offs or low average read-depth, greatly improves genome recovery of Alpha, Beta, Delta, Eta and non-VOC/non-VOI SARS-CoV-2 variants.
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DOI：
10.1126/science.abj4336
发表时间：
2021-10-22
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Wilkinson E;Giovanetti M;Tegally H;San JE;Lessells R;Cuadros D;Martin DP;Rasmussen DA;Zekri AN;Sangare AK;Ouedraogo AS;Sesay AK;Priscilla A;Kemi AS;Olubusuyi AM;Oluwapelumi AOO;Hammami A;Amuri AA;Sayed A;Ouma AEO;Elargoubi A;Ajayi NA;Victoria AF;Kazeem A;George A;Trotter AJ;Yahaya AA;Keita AK;Diallo A;Kone A;Souissi A;Chtourou A;Gutierrez AV;Page AJ;Vinze A;Iranzadeh A;Lambisia A;Ismail A;Rosemary A;Sylverken A;Femi A;Ibrahimi A;Marycelin B;Oderinde BS;Bolajoko B;Dhaala B;Herring BL;Njanpop-Lafourcade BM;Kleinhans B;McInnis B;Tegomoh B;Brook C;Pratt CB;Scheepers C;Akoua-Koffi CG;Agoti CN;Peyrefitte C;Daubenberger C;Morang'a CM;Nokes DJ;Amoako DG;Bugembe DL;Park D;Baker D;Doolabh D;Ssemwanga D;Tshiabuila D;Bassirou D;Amuzu DSY;Goedhals D;Omuoyo DO;Maruapula D;Foster-Nyarko E;Lusamaki EK;Simulundu E;Ong'era EM;Ngabana EN;Shumba E;El Fahime E;Lokilo E;Mukantwari E;Philomena E;Belarbi E;Simon-Loriere E;Anoh EA;Leendertz F;Ajili F;Enoch FO;Wasfi F;Abdelmoula F;Mosha FS;Takawira FT;Derrar F;Bouzid F;Onikepe F;Adeola F;Muyembe FM;Tanser F;Dratibi FA;Mbunsu GK;Thilliez G;Kay GL;Githinji G;van Zyl G;Awandare GA;Schubert G;Maphalala GP;Ranaivoson HC;Lemriss H;Anise H;Abe H;Karray HH;Nansumba H;Elgahzaly HA;Gumbo H;Smeti I;Ayed IB;Odia I;Ben Boubaker IB;Gaaloul I;Gazy I;Mudau I;Ssewanyana I;Konstantinus I;Lekana-Douk JB;Makangara JC;Tamfum JM;Heraud JM;Shaffer JG;Giandhari J;Li J;Yasuda J;Mends JQ;Kiconco J;Morobe JM;Gyapong JO;Okolie JC;Kayiwa JT;Edwards JA;Gyamfi J;Farah J;Nakaseegu J;Ngoi JM;Namulondo J;Andeko JC;Lutwama JJ;O'Grady J;Siddle K;Adeyemi KT;Tumedi KA;Said KM;Hae-Young K;Duedu KO;Belyamani L;Fki-Berrajah L;Singh L;Martins LO;Tyers L;Ramuth M;Mastouri M;Aouni M;El Hefnawi M;Matsheka MI;Kebabonye M;Diop M;Turki M;Paye M;Nyaga MM;Mareka M;Damaris MM;Mburu MW;Mpina M;Nwando M;Owusu M;Wiley MR;Youtchou MT;Ayekaba MO;Abouelhoda M;Seadawy MG;Khalifa MK;Sekhele M;Ouadghiri M;Diagne MM;Mwenda M;Allam M;Phan MVT;Abid N;Touil N;Rujeni N;Kharrat N;Ismael N;Dia N;Mabunda N;Hsiao NY;Silochi NB;Nsenga N;Gumede N;Mulder N;Ndodo N;Razanajatovo NH;Iguosadolo N;Judith O;Kingsley OC;Sylvanus O;Peter O;Femi O;Idowu O;Testimony O;Chukwuma OE;Ogah OE;Onwuamah CK;Cyril O;Faye O;Tomori O;Ondoa P;Combe P;Semanda P;Oluniyi PE;Arnaldo P;Quashie PK;Dussart P;Bester PA;Mbala PK;Ayivor-Djanie R;Njouom R;Phillips RO;Gorman R;Kingsley RA;Carr RAA;El Kabbaj S;Gargouri S;Masmoudi S;Sankhe S;Lawal SB;Kassim S;Trabelsi S;Metha S;Kammoun S;Lemriss S;Agwa SHA;Calvignac-Spencer S;Schaffner SF;Doumbia S;Mandanda SM;Aryeetey S;Ahmed SS;Elhamoumi S;Andriamandimby S;Tope S;Lekana-Douki S;Prosolek S;Ouangraoua S;Mundeke SA;Rudder S;Panji S;Pillay S;Engelbrecht S;Nabadda S;Behillil S;Budiaki SL;van der Werf S;Mashe T;Aanniz T;Mohale T;Le-Viet T;Schindler T;Anyaneji UJ;Chinedu U;Ramphal U;Jessica U;George U;Fonseca V;Enouf V;Gorova V;Roshdy WH;Ampofo WK;Preiser W;Choga WT;Bediako Y;Naidoo Y;Butera Y;de Laurent ZR;Sall AA;Rebai A;von Gottberg A;Kouriba B;Williamson C;Bridges DJ;Chikwe I;Bhiman JN;Mine M;Cotten M;Moyo S;Gaseitsiwe S;Saasa N;Sabeti PC;Kaleebu P;Tebeje YK;Tessema SK;Happi C;Nkengasong J;de Oliveira T
通讯作者：
de Oliveira T
DOI：
10.1016/j.ijid.2021.10.050
发表时间：
2022-01
期刊：
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases
影响因子：
--
作者：
Kuchinski KS;Nguyen J;Lee TD;Hickman R;Jassem AN;Hoang LMN;Prystajecky NA;Tyson JR
通讯作者：
Tyson JR
影响因子：
5.3
作者：
Cotten M;Lule Bugembe D;Kaleebu P;V T Phan M
通讯作者：
V T Phan M
影响因子：
3.7
作者：
Itokawa K;Sekizuka T;Hashino M;Tanaka R;Kuroda M
通讯作者：
Kuroda M
影响因子：
4.6
作者：
Park SY;Faraci G;Ward PM;Emerson JF;Lee HY
通讯作者：
Lee HY