key: cord-0981368-6a5fl4kb
authors: Cotten, Matthew; Bugembe, Dan Lule; Kaleebu, Pontiano; Phan, My V.T.
title: Alternate primers for whole-genome SARS-CoV-2 sequencing
date: 2020-11-09
journal: bioRxiv
DOI: 10.1101/2020.10.12.335513
sha: ce68fa2cc356bfd5bd01e8bd32bb330cea54cf6e
doc_id: 981368
cord_uid: 6a5fl4kb

As the world is struggling to control the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there is an urgency to develop effective control measures. Essential information is encoded in the virus genome sequence with accurate and complete SARS-CoV-2 sequences essential for tracking the movement and evolution of the virus and for guiding efforts to develop vaccines and antiviral drugs. While there is unprecedented SARS-CoV-2 sequencing efforts globally, approximately 19 to 43% of the genomes generated monthly are gapped, reducing their information content. The current study documents the genome gap frequencies and their positions in the currently available data and provides an alternative primer set and a sequencing scheme to help improve the quality and coverage of the genomes.

Since the first report on 30 th December 2019 in Wuhan China and the WHO declaration 

The phenomenon is unlikely to be due to an isolated set of genomes as we observed 69 similar N200 frequencies in genomes submitted from each month of the pandemic (Table 1) ,

suggesting that gaps in coverage is a more general phenomenon. Of note, genomes 71 generated using Ion Torrent show much lower levels of N200 (Table 1 ). The very low 72 frequency of large gaps in the Ion Torrent data may be due to the use of a dedicated alternative 73 primer set (6). There have been discussions and reports on the SARS-CoV-2 genome 74 changes due to sequencing errors as well as long gaps in the genomes due to missing 75 amplicons from the amplicon-approach sequencing (7) (8). Updates of the ARTIC primers 76 have been presented in late March 2020 to address these issues (9) (10). Additional reports 77 of longer amplicon methods have been published (11) (12) (14) including methods to use a 78 subset of ARTIC primers to generate longer amplicons(13).

However, the percentage of reported complete genomes in GISAID with 1 or more 80 N200s continues, with 10,611 (28%) of the 38,228 genomes deposited in September 2020 81 having 1 or more N200 gaps (Table 1) , indicating the challenges remain largely unsolved. Ion  Torrent  total 8   Ion Torrent  % with  N200 9   Method  unclear 10   Method  unclear %  with N200 11  1Jan20-31Jan20  54  1  2  9  0  2  0  0  0  43  2  1Feb20-29Feb20  126  2  2  44  0  9  0  5  0  65  4  1Mar20-31Mar20  2872  559  19  1518  16  548  32  35  6  771  18  1Apr20-30Apr20  12411  3745  30  4970  38  1286  27  264  0  5424  26  1May20-31May20  19787  8606  43  8634  52  2634  30  529  0  7990  42  1Jun20-30Jun20  21665  8723  40  7043  36  3844  35  629  2  10149  47  1Jul20-31Jul20  17986  4834  27  4965  23  1585  33  471  2  10965  29  1Aug20-31Aug20  17276  4005  23  11074  22  2270  26  486  0  3446  28  1Sep20-30Sep20  38227  10611  28  22740  23  7973  44  580  1 

Alternate primers as a potential solution to avoid gapped genomes.

We explored an alternate set of amplification primers (termed the Entebbe primers) Our experience had suggested an optimum amplicon size of around 1500 bp. The larger amplicons reduced the total primers content of the reactions but still allowed high reverse detailed laboratory methods that we used for reverse transcription, PCR amplification and 

Carefully remove and discard ethanol, being careful not to touch the bead pellet. 

Initial genome release of novel coronavirus (10μM)