ABSTRACT
Reverse transcription-PCRs (RT-PCRs) targeting SARS-CoV-2 variant of concern (VOC) mutations have been developed to simplify their tracking. We evaluated an assay targeting E484K/N501Y to identify B.1.351/P1. Whole-genome sequencing (WGS) confirmed only 72 (59.02%) of 122 consecutive RT-PCR P.1/B.1.351 candidates. Prescreening RT-PCRs must target a wider set of mutations, updated from WGS data from emerging variants.
KEYWORDS: SARS-CoV-2, VOC, reverse transcriptase PCR, whole-genome sequencing, COVID-19, RT-PCR, WGS
OBSERVATION
The recent emergence and successful spread of SARS-CoV-2 variants of concern (VOCs) have triggered alarm (1). Definitive identification of these variants requires whole-genome sequencing (WGS) to identify the constellation of variant-specific mutations (2). In the search for a higher throughput, lower costs, and faster availability of data, alternative reverse transcription-PCR (RT-PCR)-based approaches have been proposed to prescreen these variants. For the first VOC triggering a global alarm, namely, B.1.1.7, an RT-PCR approach (Thermo Fisher TaqPath RT-PCR) took advantage of spike gene (S-gene) impaired detection, caused by the 69/70 deletion, which is a B.1.1.7 genetic marker (3). This RT-PCR demonstrated its usefulness to simplify and accelerate the B.1.1.7 screening in microbiology diagnostic settings. Similarly, other RT-PCR-based assays have been developed by other companies to screen two new worrying VOCs, B.1.351 and P.1, targeting the E484K and N501Y mutations in the S-gene, which are considered markers for these VOCs.
Our group has implemented a two-step procedure to screen VOCs. First, detection of SARS-CoV-2 is performed using routine RT-PCR (Thermo Fisher Taq-Path RT-PCR) on nasopharyngeal specimens. This step allows for identification of case candidates infected by the B.1.1.7 variant, based on the demonstration of S-gene target failure due to the spike 69/70 deletion. Second, cases where S-gene target failure was not identified are further screened using one of the new assays for early detection of B.1.351/P.1 VOCs (it also includes the detection of the B.1.525 variant of interest [VOI] [SARS-CoV-2 variants ELITe MGB kit]). Finally, B.1.351/P.1 and B.1.525 candidates are confirmed by WGS. For WGS, RNA was reverse-transcribed using random hexamers. Whole-genome amplification of the coronavirus was done with the Artic_nCov-2019_V3 panel of primers (artic.network/ncov-2019). Libraries were prepared using the Nextera Flex DNA library preparation kit (Illumina, Inc., California, USA) and sequenced on the Miseq system (Illumina, Inc.) using the v2 kit.
The purpose of this study is to assess the concordance between RT-PCR-based preassignment of P.1/B.1.351 or B.1.525 candidates and final confirmation by WGS.
A total of 142 COVID-19 cases were candidates possibly infected by non-B.1.1.7 variants, identified by our two-step RT-PCR-based approach from 16 April to 1 June 2021. Of these candidates, 121 (85.21%) harbored the E484K and N501Y mutations (B.1.351/P1 candidates as per commercial kit indications), 12 (8.45%) harbored the E484K mutation (B.1.525 candidates), and none of the previous mutations were found in the remaining nine (6.34%) cases (Table 1).
TABLE 1.
Assignment of SARS-CoV-2 VOCs based on RT-PCR and WGS from 16 April to 1 June 2021a
| Sample code | Date (day/mo/yr) | CT valueb | RT-PCR assignment | WGS assignment | Traveler | Country of origin | GISAID code |
|---|---|---|---|---|---|---|---|
| 1 | 4/16/21 | 18 | Non-P.1/B.1.351/B.1.525 | B.1.575.1 | No | EPI_ISL_1914373 | |
| 2 | 4/16/21 | 22 | P.1/B.1.351 | P.1 | No | EPI_ISL_1914365 | |
| 3 | 4/16/21 | 23 | P.1/B.1.351 | P.1 | No | EPI_ISL_1914366 | |
| 4 | 4/16/21 | 27 | P.1/B.1.351 | P.1 | No | EPI_ISL_1914367 | |
| 5 | 4/17/21 | 28 | Non-P.1/B.1.351/B.1.525 | B.1.177 | No | EPI_ISL_1914334 | |
| 6 | 4/17/21 | 18 | B.1.525 | B.1.526 | No | EPI_ISL_1914376 | |
| 7 | 4/17/21 | 16 | B.1.525 | B.1.526 | No | EPI_ISL_1914377 | |
| 8 | 4/17/21 | 22 | P.1/B.1.351 | P.1 | No | EPI_ISL_1914368 | |
| 9 | 4/17/21 | 30 | Non-P.1/B.1.351/B.1.525 | B.1.177.50 | No | EPI_ISL_1914335 | |
| 10 | 4/19/21 | 27 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505904 | |
| 11 | 4/19/21 | 30 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505905 | |
| 12 | 4/19/21 | 24 | P.1/B.1.351 | P.1 | Yes | Mexico | EPI_ISL_3505906 |
| 13 | 4/20/21 | 19 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505907 | |
| 14 | 4/20/21 | 29 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505908 | |
| 15 | 4/21/21 | 21 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505909 | |
| 16 | 4/22/21 | 16 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505910 |
| 17 | 4/22/21 | 28 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505911 | |
| 18 | 4/23/21 | 17 | P.1/B.1.351 | P.1 | No | EPI_ISL_1914327 | |
| 19 | 4/23/21 | 17 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505912 | |
| 20 | 4/23/21 | 22 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505913 | |
| 21 | 4/23/21 | 19 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_1914328 | |
| 22 | 4/23/21 | 18 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505914 | |
| 23 | 4/24/21 | 20 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505915 | |
| 24 | 4/26/21 | 21 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505916 |
| 25 | 4/27/21 | 23 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_1964218 |
| 26 | 4/27/21 | 20 | P.1/B.1.351 | P.1 | No | EPI_ISL_1964180 | |
| 27 | 4/27/21 | 22 | P.1/B.1.351 | P.1 | Yes | Colombia | EPI_ISL_3505917 |
| 28 | 4/27/21 | 17 | P.1/B.1.351 | P.1 | Yes | Colombia | EPI_ISL_1964215 |
| 29 | 4/27/21 | 21 | B.1.525 | B.1.526 | Yes | Dominican Republic | EPI_ISL_3505918 |
| 30 | 4/28/21 | 29 | Non-P.1/B.1.351/B.1.525 | B.1.177 | No | EPI_ISL_3505919 | |
| 31 | 4/28/21 | 18 | P.1/B.1.351 | B.1.111 | Yes | Colombia | EPI_ISL_3505920 |
| 32 | 4/29/21 | 17 | P.1/B.1.351 | P.1 | Yes | Colombia | EPI_ISL_3505921 |
| 33 | 4/29/21 | 29 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505922 | |
| 34 | 4/29/21 | 23 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505923 | |
| 35 | 4/29/21 | 25 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505924 |
| 36 | 4/29/21 | 24 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505925 |
| 37 | 4/29/21 | 21 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505926 |
| 38 | 4/29/21 | 27 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505927 | |
| 39 | 4/30/21 | 18 | P.1/B.1.351 | P.1 | No | EPI_ISL_1964216 | |
| 40 | 4/30/21 | 17 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_3505928 | |
| 41 | 4/30/21 | 13 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505929 | |
| 42 | 5/1/21 | 20 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_3505930 | |
| 43 | 5/1/21 | 23 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505931 |
| 44 | 5/2/21 | 18 | P.1/B.1.351 | P.1 | Yes | Brazil | EPI_ISL_3505932 |
| 45 | 5/2/21 | 18 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505933 |
| 46 | 5/3/21 | 22 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_3505934 | |
| 47 | 5/6/21 | 24 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_2080926 | |
| 48 | 5/6/21 | 13 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_2080927 | |
| 49 | 5/6/21 | 23 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505935 | |
| 50 | 5/6/21 | 25 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_2080930 | |
| 51 | 5/6/21 | 26 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_2080928 |
| 52 | 5/6/21 | 28 | P.1/B.1.351 | P.1 | No | EPI_ISL_2080923 | |
| 53 | 5/7/21 | 29 | P.1/B.1.351 | P.1 | No | EPI_ISL_2080886 | |
| 54 | 5/7/21 | 14 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_3505936 | |
| 55 | 5/7/21 | 21 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505937 | |
| 56 | 5/7/21 | 17 | P.1/B.1.351 | P.1 | No | EPI_ISL_2080922 | |
| 57 | 5/7/21 | 30 | B.1.525 | B.1.280 (E484Q) | No | EPI_ISL_2080885 | |
| 58 | 5/8/21 | 18 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505938 |
| 59 | 5/9/21 | 20 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_3505939 | |
| 60 | 5/10/21 | 21 | B.1.525 | B.1.280 (E484Q) | Yes | Dominican Republic | EPI_ISL_2172424 |
| 61 | 5/11/21 | 16 | P.1/B.1.351 | P.1 | Yes | Colombia | EPI_ISL_3505940 |
| 62 | 5/12/21 | 17 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505941 |
| 63 | 5/12/21 | 28 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505942 | |
| 64 | 5/12/21 | 14 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505943 | |
| 65 | 5/13/21 | 19 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505944 |
| 66 | 5/13/21 | 14 | P.1/B.1.351 | B.1.351 | Yes | Cuba | EPI_ISL_3505945 |
| 67 | 5/13/21 | 29 | P.1/B.1.351 | B.1.621 | Yes | Dominican Republic | EPI_ISL_3505946 |
| 68 | 5/14/21 | 20 | B.1.525 | B.1.1.318 | Yes | Senegal | EPI_ISL_3505947 |
| 69 | 5/14/21 | 30 | P.1/B.1.351 | P.1 | No | EPI_ISL_2178828 | |
| 70 | 5/14/21 | 16 | P.1/B.1.351 | P.1 | Yes | Dominican Republic | EPI_ISL_3505948 |
| 71 | 5/14/21 | 17 | P.1/B.1.351 | P.1 | Yes | Paraguay | EPI_ISL_3505949 |
| 72 | 5/14/21 | 17 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505950 |
| 73 | 5/14/21 | 19 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505951 |
| 74 | 5/14/21 | 17 | Non-P.1/B.1.351/B.1.525 | C.37 | Yes | Colombia | EPI_ISL_2178831 |
| 75 | 5/14/21 | 17 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_2178834 |
| 76 | 5/14/21 | 30 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505952 | |
| 77 | 5/15/21 | 24 | P.1/B.1.351 | P.1 | Yes | Portugal | EPI_ISL_3505953 |
| 78 | 5/15/21 | 15 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505954 |
| 79 | 5/16/21 | 27 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505955 |
| 80 | 5/16/21 | 17 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505956 |
| 81 | 5/16/21 | 17 | P.1/B.1.351 | P.1 | Yes | Colombia | EPI_ISL_3505957 |
| 82 | 5/16/21 | 22 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505958 | |
| 83 | 5/17/21 | 24 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505959 |
| 84 | 5/17/21 | 30 | P.1/B.1.351 | P.1 | Yes | Unknown | EPI_ISL_3505960 |
| 85 | 5/18/21 | 15 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505961 | |
| 86 | 5/19/21 | 22 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505962 | |
| 87 | 5/19/21 | 15 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505963 | |
| 88 | 5/19/21 | 17 | P.1/B.1.351 | B.1.351 | Yes | Cuba | EPI_ISL_3505964 |
| 89 | 5/19/21 | 18 | P.1/B.1.351 | B.1.623 | Yes | Dominican Republic | EPI_ISL_3505965 |
| 90 | 5/19/21 | 18 | P.1/B.1.351 | B.1.623 | Yes | Dominican Republic | EPI_ISL_3505966 |
| 91 | 5/20/21 | 26 | Non-P.1/B.1.351/B.1.525 | B.1 | No | EPI_ISL_2304188 | |
| 92 | 5/20/21 | 28 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505967 | |
| 93 | 5/20/21 | 14 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505968 |
| 94 | 5/20/21 | 31 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505969 | |
| 95 | 5/20/21 | 18 | B.1.525 | B.1 (E484Q) | Yes | Dominican Republic | EPI_ISL_3505970 |
| 96 | 5/20/21 | 22 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_3505971 | |
| 97 | 5/21/21 | 27 | P.1/B.1.351 | B.1.623 | No | EPI_ISL_3505972 | |
| 98 | 5/21/21 | 18 | B.1.525 | B.1.526 | No | EPI_ISL_3505973 | |
| 99 | 5/21/21 | 22 | B.1.525 | B.1.526 | No | EPI_ISL_2304191 | |
| 100 | 5/21/21 | 18 | P.1/B.1.351 | B.1.621 | No | EPI_ISL_3505974 | |
| 101 | 5/21/21 | 30 | B.1.525 | B.1.526 | No | EPI_ISL_2304190 | |
| 102 | 5/21/21 | 30 | Non-P.1/B.1.351/B.1.525 | B.1.617.2 | Yes | India | EPI_ISL_2296706 |
| 103 | 5/21/21 | 21 | P.1/B.1.351 | P.1 | No | EPI_ISL_2304186 | |
| 104 | 5/22/21 | 31 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505976 | |
| 105 | 5/22/21 | 25 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505975 | |
| 106 | 5/22/21 | 17 | B.1.525 | B.1 (E484Q) | No | EPI_ISL_2304153 | |
| 107 | 5/22/21 | 20 | P.1/B.1.351 | B.1.623 | No | EPI_ISL_3505977 | |
| 108 | 5/22/21 | 18 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505978 | |
| 109 | 5/22/21 | 16 | P.1/B.1.351 | B.1.621 | No | EPI_ISL_3505979 | |
| 110 | 5/22/21 | 24 | P.1/B.1.351 | P.1 | Yes | Colombia | EPI_ISL_2304183 |
| 111 | 5/23/21 | 22 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505980 |
| 112 | 5/23/21 | 18 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505981 |
| 113 | 5/24/21 | 20 | P.1/B.1.351 | P.1 | Yes | Brazil | EPI_ISL_3505984 |
| 114 | 5/24/21 | 14 | P.1/B.1.351 | P.1 | Yes | Brazil | EPI_ISL_3505983 |
| 115 | 5/24/21 | 23 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505982 |
| 116 | 5/25/21 | 26 | P.1/B.1.351 | P.1 | No | EPI_ISL_2402499 | |
| 117 | 5/25/21 | 16 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_3505985 | |
| 118 | 5/25/21 | 14 | P.1/B.1.351 | B.1.623 | No | EPI_ISL_2402506 | |
| 119 | 5/26/21 | 18 | P.1/B.1.351 | B.1.623 | No | EPI_ISL_3505986 | |
| 120 | 5/26/21 | 32 | P.1/B.1.351 | B.1.1 | No | EPI_ISL_3505987 | |
| 121 | 5/26/21 | 25 | P.1/B.1.351 | P.1 | No | EPI_ISL_3505988 | |
| 122 | 5/27/21 | 20 | Non-P.1/B.1.351/B.1.525 | B.1.617.2 | No | EPI_ISL_2402478 | |
| 123 | 5/27/21 | 17 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_2402508 |
| 124 | 5/27/21 | 18 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_2402505 |
| 125 | 5/28/21 | 16 | Non-P.1/B.1.351/B.1.525 | B.1.617.2 | No | EPI_ISL_2402477 | |
| 126 | 5/28/21 | 20 | P.1/B.1.351 | B.1.623 | No | EPI_ISL_2402512 | |
| 127 | 5/28/21 | 14 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505989 |
| 128 | 5/28/21 | 16 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505990 |
| 129 | 5/29/21 | 15 | P.1/B.1.351 | P.1 | Yes | Venezuela | EPI_ISL_2402527 |
| 130 | 5/29/21 | 22 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_2402528 |
| 131 | 5/29/21 | 29 | P.1/B.1.351 | B.1.351 | No | EPI_ISL_2402513 | |
| 132 | 5/30/21 | 17 | P.1/B.1.351 | B.1.623 | Yes | Colombia | EPI_ISL_3505991 |
| 133 | 5/30/21 | 21 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505992 |
| 134 | 5/30/21 | 22 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505993 |
| 135 | 5/30/21 | 23 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505994 |
| 136 | 5/30/21 | 19 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505995 |
| 137 | 5/30/21 | 26 | P.1/B.1.351 | B.1.621 | Yes | Colombia | EPI_ISL_3505996 |
| 138 | 5/30/21 | 19 | P.1/B.1.351 | B.1.623 | Yes | Dominican Republic | EPI_ISL_3505997 |
| 139 | 5/31/21 | 22 | B.1.525 | B.1 (E484Q) | No | EPI_ISL_3505998 | |
| 140 | 5/31/21 | 22 | P.1/B.1.351 | B.1.621 | No | EPI_ISL_3505999 | |
| 141 | 5/31/21 | 17 | P.1/B.1.351 | B.1.621 | No | EPI_ISL_3506000 | |
| 142 | 6/1/21 | 24 | P.1/B.1.351 | B.1.621 | No | EPI_ISL_3506001 |
Shading indicates discrepancies between RT-PCR and WGS variant assignations.
CT, threshold cycle.
WGS of all cases confirmed the RT-PCR-based preassignment in 71 of the cases (58.68%; 57 P.1 [80.28%] and 14 B.1.351 [19.72%]) from the 121 P.1/B.1.351 candidates (Table 1). The remaining 50 cases (41.32%) were wrongly assigned, corresponding to B.1.621 (78%) (39 cases), B.1.111 (2%) (1 case), B.1.623 (18%) (9 cases) and B.1.1 (2%) (1 case) (Table 1).
B.1.621 is a VOI, as defined by the World Health Organization (4). It harbors several mutations affecting the spike protein, including E484K and N501Y (5). There are limited data on the global distribution of this variant, which has become predominant in some regions of Colombia, with up to 7% of sequenced cases. In our series, 33 (84.62%) corresponded to travelers from Colombia, 1 (2.56%) from the Dominican Republic, and 5 (12.82%) were unlinked cases.
None of the B.1.525 preassigned cases was confirmed by WGS (Table 1). All of them were wrongly assigned, corresponding to B.1.526 (50%) (6 cases), B.1.1.318 (8.33%) (1 case), B.1 (25%) (3 cases), and B.1.280 (16.67%) (2 cases). In five cases (41.67%), the E484K mutation was wrongly assigned by RT-PCR, corresponding to E484Q (B.1 and B.1.280 variants; Table 1).
For the remaining cases, for which the RT-PCR-based prescreening did not detect the E484K/N501Y mutations, three (33.33%) corresponded to B.1.617.2, a VOC. In the remaining six cases, different non-VOC/VOIs were identified by WGS, specifically, B.1.575.1, B.1.177, B.1.177.50, C.37, and B.1 (Table 1).
Our findings indicate that incorrect B.1.351/P.1 assignment may occur when using a targeted RT-PCR prescreening approach, based on the identification of a limited number of marker single nuclear polymorphisms (SNPs). The emergent presence of the B.1.621 VOI, sharing the two E484K/N501Y mutations with P.1/B.1.351, is mainly responsible for the incorrect assignments in our study. The misassignments found in our study are not associated with specific technical limitations of the test applied; they are the result of assuming that a proper assignment of VOCs can be performed by targeting a low number of marker mutations. Thus, caution is recommended when applying targeted RT-PCR for VOC screening. In the current context of constant emergence of new variants, commercial solutions devoted to prescreen VOCs should rely on a wider set of mutations to ensure specificity. Mutations targeted by these approaches should be constantly evaluated considering updated WGS data on circulating variants.
GENOMIC METHODS
We used 11 μl of RNA as the template for reverse transcription using Invitrogen SuperScript IV reverse transcriptase (Thermo Fisher Scientific, MA, USA) and random hexamers (Thermo Fisher Scientific). Whole-genome amplification of the coronavirus was done with an Artic_nCov-2019_V3 panel of primers (Integrated DNA Technologies, Inc., Coralville, IA, USA) (artic.network/ncov-2019) and the Q5 Hot Start DNA polymerase (New England Biolabs, Ipswich, MA, USA). Libraries were prepared using the Nextera Flex DNA library preparation kit (Illumina, Inc., CA, USA) following the manufacturer’s instructions.
Libraries were quantified with the Quantus fluorometer (Promega, WI, USA) before being pooled at equimolar concentrations (4 nM). Next, they were sequenced in pools of up to 17 libraries on the MiSeq system (Illumina, Inc., CA, USA) and the MiSeq reagent microkit v2 (2 × 151 bp) or in pools of up to 96 libraries with the MiSeq reagent (2 × 201 bp).
FastA files above the GISAID thresholds were deposited at GISAID (Table 1). An in-house analysis pipeline was applied to analyze the sequencing reads. The pipeline can be accessed at https://github.com/pedroscampoy/covid_multianalysis. Briefly, the pipeline goes through the following steps: (i) removal of human reads with Kraken (6), (ii) preprocessing and quality assessment of FASTQ files using fastp v0.20.1 (7) (arguments: –cut tail, –cut-window-size, –cut-mean-quality, -max_len1,-max_len2) and fastQC v0.11.9 (8), (iii) mapping with the Burrows-Wheeler Aligner (BWA) v0.7.17 and variant calling using iVAR v1.2.3 (9) using the Wuhan-1 strain sequence (GenBank accession number NC_045512.2) as the reference, and (iv) recalibration of punctual low-coverage positions using joint variant calling. When necessary, informative noncovered positions were analyzed by standard Sanger sequencing with the corresponding flanking primers from the ARTIC v3 set.
ACKNOWLEDGMENTS
We are grateful to Dainora Jaloveckas for editing and proofreading assistance.
The Gregorio Marañón Microbiology-ID COVID 19 Study Group is Javier Adán-Jiménez, Luis Alcalá, Teresa Aldámiz, Roberto Alonso, Beatriz Álvarez, Ana Álvarez-Uría, Alexi Arias, Luis Antonio Arroyo, Juan Berenguer, Elena Bermúdez, Emilio Bouza, Sergio Buenestado-Serrano, Almudena Burillo, Ana Candela, Raquel Carrillo, Pilar Catalán, Emilia Cercenado, Alejandro Cobos, Víctor Manuel de la Cueva, Cristina Díez, José Egido-Balzategui, Pilar Escribano, Agustín Estévez, Chiara Fanciulli, Alicia Galar, María Dolores García, Darío García de Viedma, Paloma Gijón, Adolfo González, Helmuth Guillén, Jesús Guinea, Laura Vanessa Haces, Marta Herranz, Martha Kestler, Juan Carlos López, Carmen Narcisa Losada, Marina Machado, Mercedes Marín, Pablo Martín, Javier Martín-Escolano, Andrea Molero-Salinas, Pedro Montilla, Patricia Muñoz, María Olmedo, Álvaro Otero-Sobrino, Belén Padilla, María Palomo, Francisco Parras, María Jesús Pérez-Granda, Laura Pérez-Lago, Leire Pérez, Paula Pescador, Sandra R Maus, Elena Reigadas, Carla Margarita Rico-Luna, Cristina Rincón, Belén Rodríguez, Sara Rodríguez, Cristina Rodríguez-Grande, Adriana Rojas, María Jesús Ruiz-Serrano, Carlos Sánchez, Mar Sánchez, Julia Serrano, Pedro J. Sola-Campoy, Francisco Tejerina, Maricela Valerio, María Cristina Veintimilla, Lara Vesperinas, Teresa Vicente, Sofía de la Villa.
Contributor Information
Darío García de Viedma, Email: dgviedma2@gmail.com.
Eleanor A. Powell, University of Cincinnati
REFERENCES
- 1.Boehm E, Kronig I, Neher RA, Eckerle I, Vetter P, Kaiser L. 2021. Novel SARS-CoV-2 variants: the pandemics within the pandemic. Clin Microbiol Infect 27:1109–1117. doi: 10.1016/j.cmi.2021.05.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Zella D, Giovanetti M, Cella E, Borsetti A, Ciotti M, Ceccarelli G, D’Ettorre G, Pezzuto A, Tambone V, Campanozzi L, Magheri M, Unali F, Bianchi M, Benedetti F, Pascarella S, Angeletti S, Ciccozzi M. 2021. The importance of genomic analysis in cracking the coronavirus pandemic. Expert Rev Mol Diagn 21:547–562. doi: 10.1080/14737159.2021.1917998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Brown KA, Gubbay J, Hopkins J, Patel S, Buchan SA, Daneman N, Goneau LW. 2021. S-gene target failure as a marker of variant B.1.1.7 among SARS-CoV-2 isolates in the greater Toronto area, December 2020 to March 2021. JAMA 325:2115–2116. doi: 10.1001/jama.2021.5607. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.World Health Organization. 2021. COVID-19 weekly epidemiological update, special edition: proposed working definitions of SARS-CoV-2 variants of interest and variants of concern. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20210225_weekly_epi_update_voc-special-edition.pdf.
- 5.Laiton-Donato K, Franco-Muñoz C, Álvarez-Díaz DA, Ruiz-Moreno HA, Usme-Ciro JAA, Prada D, Reales-González J, Corchuelo S, Herrera-Sepúlveda MT, Naizaque J, Santamaría G, Rivera J, Rojas P, Hernández Ortiz J, Cardona A, Malo D, Prieto-Alvarado F, Gómez FR, Wiesner M, Martínez MLO, Mercado-Reyesa M. 2021. Characterization of the emerging B.1.621 variant of interest of SARS-CoV-2. medRxiv. doi: 10.1101/2021.05.08.21256619. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Wood DE, Salzberg SL. 2014. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol 15:R46. doi: 10.1186/gb-2014-15-3-r46. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Chen S, Zhou Y, Chen Y, Gu J. 2014. A quality control tool for high throughput sequence data. Bioinformatics 34:i884–i890. doi: 10.1093/bioinformatics/bty560. [DOI] [Google Scholar]
- 8.Andrews S. 2010. FastQC: a quality control tool for high throughput sequence data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
- 9.Grubaugh ND, Gangavarapu K, Quick J, Matteson NL, De Jesus JG, Main BJ, Tan AL, Paul LM, Brackney DE, Grewal S, Gurfield N, Van Rompay KKA, Isern S, Michael SF, Coffey LL, Loman NJ, Andersen KG. 2014. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biol 20:8. doi: 10.1186/s13059-018-1618-7. [DOI] [PMC free article] [PubMed] [Google Scholar]