Disentangling primer interactions improves SARS-CoV-2 genome sequencing by multiplex tiling PCR

Abstract

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.

Fig 1. Dropout of the amplicon 18 and 76 due to primer interaction.

Examples of depth plot for (A) original ARTIC primer set V1 and (B) V1 with 76_RIGHT replacement for the same clinical sample (previously deposited to GISAID with ID EPI_ISL_416596, Ct = 28.5, 1/25 input per reaction). Regions covered by amplicons with modified primer (76_RIGHT) and the interacting primer (18_LEFT) are highlighted by green and orange colors, respectively. For all data, reads were down-sampled to normalize average coverage to 250X. Horizontal dotted line indicates depth = 30. These two experiments were conducted with the same PCR master mix (except primers) and in the same PCR run in the same thermal cycler.

Fig 2. Predicted primer interactions.

(A) The 14 predicted primer interactions subjected for modification in this study. Primers replaced in the N1 primer set (S1 Table) are shown in green. (B) Violin plots showing the distributions of dimer-scores (the nearest-neighbor ΔG adjusted by empirically determined penalty and bonus scores [10]) at all heterodimers in Pools1 and 2 reported by the PrimerROC algorithm (n = 4,743 for each pool). The scores of interactions depicted in Fig 2A are over plotted as scatter points.

Fig 3. Responses to different annealing/extension temperatures.

Abundance of 98 amplicons at 8 different annealing/extension temperatures with the three different primer sets on a same clinical sample (previously deposited to GISAID with ID EPI_ISL_416584, Ct = 16, 1/300 input per reaction). For all data, reads were down-sampled to normalize average coverage to 500X before analysis. The green lines and points indicate the abundances of amplicons whose primers in V1 primer set were subjected to modification in the N1 primer set. The orange lines and points indicate the abundances of amplicons whose primers were not modified but predicted to be eliminated the adverse primer interactions in the N1 primer set. Other amplicons which were not subjected to the modification are indicated by black lines and points. The plots in the left column shows results of all 98 amplicons while only amplicons targeted by modification are shown in the plots in the right column. Horizontal dotted line indicates fragment abundance = 30. Red vertical lines indicate normal annealing/extension temperature, 65°C. All those experiments were conducted with the same PCR master mix (except primers) and in the same PCR run in the same thermal cycler.

Fig 4. Performance comparison of the three multiplex PCR primer sets.

(A) A depth plot of original (V1) and two modified ARTIC primer sets (V3 and N1) on a same clinical sample (previously deposited to GISAID with ID EPI_ISL_416596, Ct = 28.5, 1/25 input per reaction). Regions covered by amplicons with modified primers and with not modified but interacting primers are highlighted by green and orange colors, respectively. For all data, the reads were down-sampled to normalize average coverage to 250X. Horizontal dotted line indicates depth = 30. These two experiments were conducted with the same PCR master mix (except primers) and in the same PCR run in the same thermal cycler. (B) Volcano plot for coverages of all 98 amplicons in PCR using the primer set N1 compared with PCR using the primer set V1 among three clinical samples (shown in Fig 4A and S1 Fig). Points with green color indicate amplicons whose primer(s) was subject to replacement. Points with orange color indicate amplicons whose primers were not replaced but had been predicted to interact with either of the replaced primers as shown in Fig 2A.