Epidemic trend of SARS-CoV-2 variation
First, we searched the GISAID database (data were from February 4, 2024), the results showed that the top five global SARS-CoV-2 variants changed from JN.1, HV.1, JN.1.4, JN.1.1 and HK.3 in December 2023 to JN.1, HV.1, JN.1.4, JN.1.1 and BA.2.86.1 in January 2024. Nationally, the top five SARS-CoV-2 variants are HK.3.2, HK.3, JN.1, EG.5.1.1, and JN.1.1. In Liaoning, the top five SARS-CoV-2 variants changed from HK.3.2, HK.3, EG.5.1.1, EG.5.1.8 and JN.1.1 in December 2023 to HK.3.2, HK.3, EG.5.1.1, JN.1 and JN.1.1 in January 2024 (Fig. 1A). 59 oral/nasopharyngeal swab samples were sequenced during December 13, 2023 to January 31 2024, that including overseas outpatient and Dalian local outpatient, emergency and inpatient cases. The results showed that JN.1 lineage gradually replaces EG.5 lineage and becomes the dominant strain (Fig. 1B). Further, we analyzed the number of confirmed cases and the proportion of JN.1 lineage in Dalian showed that the inflection point of the growth of local cases in Dalian appeared around January 10, 2024, while the inflection point of the growth of JN.1 lineage appeared on December 25, 2023 after the first detection on December 20, 2023. The second rapid growth occurred on January 22, 2024 (Fig. 1C). Finally, by searching the GISAID database, we further analyzed the effective series of COVID-19 in the world, China and Liaoning Province, showing a consistent trend (Fig. 1D).
Performance comparison of multiple sequencing platforms for SARS-CoV-2 whole genome sequencing
Our experimental strategy was to perform multiple sequencing platforms, including Ion Torrent, optimize amplification primers Ion Torrent V8, Illumina and Nanopore sequencing platforms. The flow chart of multi-sequencing platform was shown in Fig.2.
In order to further compare the performance of different sequencing platforms in detecting the variation sites of SARS-CoV-2, We first analyzed the range difference of Ct values in the samples with valid sequences (coverage rate > 96% according to national CDC requirements), and the results showed that the Ct values of ORF1ab gene on Ion Torrent sequencing platform were 16.91-35.44, and the Ct values of N gene were 15.37-34.49. On the Illumina and Nanopore sequencing platforms, the ORF1ab gene Ct values ranged from 18.35-29 and 18.13-29.03, and the N gene Ct values ranged from 16.96 to 29.82 and 17.43-27.93 (Fig. 3A). In addition, in terms of coverage rate there was almost no difference at 1x, 4x and 10x, but at 30x, the coverage rate of Nanopore sequencing platform results dropped, and it was worth noting that in the 100x, the Nanopore sequencing platform results showed a big difference compared with the other two sequencing platforms (Fig. 3B). The results also showed that Ion Torrent sequencing platform had better coverage rate for Ct value > 30. When the value of 30 < Ct < 35, the short augmented augmented library can be considered for use on Ion Torrent sequencing platform, and the sequence coverage rate was 98.5% - 99.5%, which was not affected by Ct value, and meet the requirements of high-throughput sequencing (Fig. 3C). The results showed that Illumina sequencing platform had the largest total reads, but Nanopore sequencing platform had the lowest total reads at 14497-43584. The read mean length for Ion Torrent sequencing platform was 182-218, while Nanopore sequencing was 1183-1520 (Table 1).
Table 1 Performance comparison of different sequencing platforms
Molecular tracing of SARS-CoV-2 JN.1 lineages
In view of the global public health impact of the SARS-CoV-2 variants, five were currently listed as variants of concern by the World Health Organization (WHO), including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and Omicron (B.1.1.529). Molecular tracking based on viral genomes plays an important role in investigating the chain of transmission of SARS-CoV-2. In order to explore the origin of our JN.1 lineages cases, we performed nucleotide comparisons (e.g. single nucleotide differences) between newly detected JN.1 lineages sequences and GISAID data, including 30 strains that came from Dalian were analyzed, and constructed a phylogeny tree (Figure 4). These results showed that JN.1 lineages were divided into different branches. However, there was coverage rate of 98 that is incorporated into other branches of the JN.1 lineage. There were other unmarked strains of Omicron.
Analysis of SARS-CoV-2 JN.1 lineage variation sites with multiple sequencing platforms
In order to clarify the accuracy of the different sequencing platforms in defecting variation sites of SARS-CoV-2 JN.1 lineage, compared with the SARS-CoV-2 reference genome (NC_045512) in the NCBI database, we first applied Ion Torrent sequencing platform to defect the samples, and found that there were serious missed defection in two regions of 22686-22917 and 23055-23075. After optimizing amplification primer, the defection rate was fully covered. In addition, Illumina sequencing platform combined with MicroFuture capture kit primer 1 was used, the sites of 7842, 13339, 21618-21711 could not be defected completely or partially, when primer 1+2 was used, the defection rates of the same sites were improved to some degrees, but there were still partial deletion. Furthermore, the defection rate of Nanopore sequencing platform combined with BaiYi capture kit was 100%. Further analysis, the results showed that 125 nucleotide variation sites in JN.1 lineage, including 1 MPLF, 9 Del, 116 synonymous/non-synonymous mutations, and 16 sites were not defected in the areas missed by Ion Torrent sequencing platform. Six sites were not defected in Illumina sequencing platform (Table 2).
In non-S protein region: compared with BA-2, BA.2.86 to JN.1.5 had 10 more site-point mutations (A211D, V1056L, N2526S, A2710T, V3593F, T4175I in ORF1a; D3H, T30A, A104V in the M region: contribute to the shape of the virus envelope and promote the completion of the virus assembly; Q229K in region N: participates in viral genome assembly and acts as a viral RNAi inhibitor to antagonize host immune defense system); Compared with BA.2, both BA.2.86 and JN.1 had one less variation site (L3201F in ORF1a); Compared with BA.2 and BA.2.86, JN.1 has three more site-point mutations (K1973R in ORF1a NSP3: promotes cytokines expression and cleavage of viral polyproteins; R3821K in ORF1a NSP6: hypothesized transmembrane protein and potential restrict ion of autophagosome/lysosome expansion; F19L in ORF7b: presumed type I transmembrane protein). JN.1.1 specific site F499L in NSP2: binds to suppressor proteins 1 and 2 (PHB1 and PHB2) and is involved in disrupting the host cell environment. JN.1.4 specific site T170I in NSP1: By promoting cell degradation and blocking host RNA translation, inhibit IFN signal and block host innate immune response; JN.1.5 specific site V1271T in ORF1b NSP13: RNA helicolytic enzyme; JN.1.6 specific site G2387D in ORF1b NSP16:2 '-o-Ribose-methyltransferase.
In S protein region: Compared with BA-2, there were newly 1 ins (16MPLF), 5 del (H69, V70, Y144, N211 and V483) and 24 point mutations (R21T, S50L, V127F, F157S, R158G, L212I, L216F, H245N, A264D, I332V, K356T, R403K, V445H, G446S, N450D, L452W, N460K, N481K, F486P, E554K, A570V, P621S, S939F and P1143L) from BA.2.86 to JN.1.6. Compared with BA.2, BA.2.86 to JN.1.6 had 4 less bit mutations (G339D, E484A, Q493R and P681H). Interestingly, BA.2.86 and JN.1 lineage had the G339H, E484K and P681R mutation, which the same site showed different amino acid variations. Furthermore, compared with BA.2 and BA.2.86, JN.1 lineage had only one more bit mutation (L455S). Most of the new variation sites were located mainly in the N-terminal domain (NTD) and receptor binding domain (RBD) regions, resulting in changes in receptor binding, which leads to immune escape and enhanced transmission (Figure 5).