Accurate Diagnosis of SARS-CoV-2 JN.1 by Sanger Sequencing of Receptor-Binding Domain Is Needed for Clinical Evaluation of Its Immune Evasion

Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used SGTF of TaqPath COVID-19 Combo Kit RT-qPCR as proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥ 6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing instead of proxy indicator to diagnose JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The nested PCR amplicons were sequenced. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed R203K, G204R and Q229K, the 3 mutations associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 of the 26 known amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: Sanger sequencing of a 445-base segment of the SARS-CoV-2 RBD is useful for accurate determination of emerging variants. The CDC may consider using Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.

Sanger Sequencing for Molecular Diagnosis of SARS-CoV-2 Omicron Subvariants and Its Challenges

Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The European Centre for Disease Prevention and Control (eCDC) and the World Health Organization (WHO) recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the polymerase chain reaction (PCR)-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490 - 509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariants when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.2 RBD and an atypical BA.1 NTD, which can only be detected by using specially designed PCR primers. In addition, Sanger sequencing may reveal unclassified subvariants, such as BA.4/BA.5 with L84I mutation in the S gene NTD. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.

A novel CRX mutation by whole-exome sequencing in an autosomal dominant cone-rod dystrophy pedigree

AIMTo identify the disease-causing gene mutation in a Chinese pedigree with autosomal dominant cone-rod dystrophy (adCORD).METHODSA southern Chinese adCORD pedigree including 9 affected individuals was studied. Whole-exome sequencing (WES), coupling the Agilent whole-exome capture system to the Illumina HiSeq 2000 DNA sequencing platform was used to search the specific gene mutation in 3 affected family members and 1 unaffected member. After a suggested variant was found through the data analysis, the putative mutation was validated by Sanger DNA sequencing of samples from all available family members.RESULTSThe results of both WES and Sanger sequencing revealed a novel nonsense mutation c.C766T (p.Q256X) within exon 5 of CRX gene which was pathogenic for adCORD in this family. The mutation could affect photoreceptor-specific gene expression with a dominant-negative effect and resulted in loss of the OTX tail, thus the mutant protein occupies the CRX-binding site in target promoters without establishing an interaction and, consequently, may block transactivation.CONCLUSIONAll modes of Mendelian inheritance in CORD have been observed, and genetic heterogeneity is a hallmark of CORD. Therefore, conventional genetic diagnosis of CORD would be time-consuming and labor-intensive. Our study indicated the robustness and cost-effectiveness of WES in the genetic diagnosis of CORD.

AutoSeqMan:batch assembly of contigs for Sanger sequences

With the wide application of DNA sequencing technology, DNA sequences are still increasingly generated through the Sanger sequencing platform. SeqMan （in the LaserGene package） is an excellent program with an easy-to-use graphical user interface （GUI） employed to assemble Sanger sequences into contigs. However, with increasing data size, larger sample sets and more sequenced loci make contig assemble complicated due to the considerable number of manual operations required to run SeqMan. Here, we present the ＇autoSeqMan＇ software program, which can automatedly assemble contigs using SeqMan scripting language. There are two main modules available, namely, ＇Classification＇ and ＇Assembly＇. Classification first undertakes preprocessing work, whereas Assembly generates a SeqMan script to consecutively assemble contigs for the classified files. Through comparison with manual operation, we showed that autoSeqMan saved substantial time in the preprocessing and assembly of Sanger sequences. We hope this tool will be useful for those with large sample sets to analyze, but with little programming experience. It is freely available at https：//github.com/ Sun-Yanbo/autoSeqMan.

Study of pathogenic genes in a pedigree with familial dilated cardiomyopathy

BACKGROUND Dilated cardiomyopathy(DCM)is a genetically heterogeneous cardiac disorder characterized by left ventricular dilation and contractile dysfunction.The substantial genetic heterogeneity evident in patients with DCM contributes to variable disease severity and complicates overall prognosis,which can be very poor.AIM To identify pathogenic genes in DCM through pedigree analysis.METHODS Our research team identified a patient with DCM in the clinic.Through invest-igation,we found that the family of this patient has a typical DCM pedigree.High-throughput sequencing technology,next-generation sequencing,was used to sequence the whole exomes of seven samples in the pedigree.RESULTS A novel and potentially pathogenic gene mutation-ANK2p.F3067L-was discovered.The mutation was completely consistent with the clinical information for this DCM pedigree.Sanger sequencing was used to further verify the locus of the mutation in pedigree samples.These results were consistent with those of high-throughput sequencing.CONCLUSIONS ANK2p.F3067L is considered a novel and potentially pathogenic gene mutation in DCM.

EGFR Gene Mutation and Methodological Evaluation in 399 Patients with Non-small Cell Lung Cancer

The purpose of the present study was to study the characteristics of epidemic growth factor receptor(EGFR)gene distribution in patients with non-small cell lung cancer(NSCLC),and to detect the mutation rate of EGFR gene by Sanger sequencing and amplification refractory mutation system(ARMS)-PCR.Paraffin-embedded sections of NSCLC tissues from 399 NSCLC patients diagnosed in Renmin Hospital of Wuhan University were collected,103 of them were detected for exons 18-21 mutation of EGFR by Sanger sequencing method,296 cases were detected for exons 18-21 mutation by ARMS-PCR method.DNA extraction of both groups was performed with Qiagen QLAamp DNA FFPE Tissue KIT.Comparisons of detection rates between the two methods were conducted by row X list chi-square test.The total mutation rate of EGFR gene detected by Sanger sequencing was 21.4%,exons 18-21 and combined mutation rates were 1.0%,9.7%,1.0%,7.8%and 2.0%,respectively.And the proportions were 4.7%,45.2%,4.7%,36.3%and 9.4%respectively.The total mutation rate detected by ARMS-PCR was 51.4%,exons 18-21 and combined mutation rates were 2.7%,27%,1.7%,18.2%and 1.7%,respectively.The proportions were 5.3%,52.6%,3.3%,35.5%and 3.3%respectively.Further analysis of mutation rate showed that there was significant difference between the two methods in detecting total mutation of EGFR gene(P<0.001).There were significant differences in mutation detection rates of exons 19 and 21(P<0.001,P<0.05),but there were no significant differences in other exons.And there was no significant difference in mutation detection rates between the two methods.The mutation rate of EGFR gene in NSCLC patients was 50%.And exon 19 deletion was the most common mutation type,followed by exon 21 mutation.Compared with Sanger sequencing method,ARMS method is more sensitive with significant advantages in detecting exon 19 deletions and exon 21 mutations,which can be widely used in clinical detection of EGFR gene mutations.The results of this study will further guide patients with advanced NSCLC to select TKI targeted drugs,and provide clinical diagnostic basis for targeted therapy of NSCLC patients.

PCR Based Detection and Phylogenetic Analysis of Fowl Adenovirus Strains Isolated from 2019 Epidemic from Punjab and Sindh, Pakistan

Hydro-Pericardium Hepatitis (HPH) is an emerging infectious disease of commercial poultry, caused by different serotypes of Fowl Adeno Virus. The vertical transmission of the virus into the progeny may results in devastating damage, causing huge economic losses to its farmers. In present study, molecular typing is performed on basis of partially conserved hexon gene sequences, using a unique set of primers having common reverse oligo for simultaneous detection of FAdV1, FAdV-4 and FAdV-11. A total of 14 fowl adeno virus strains were isolated from 100 suspected adeno virus liver samples, collected from different districts in Pakistan, between 2018 and 2019. FASTA’s sequence alignment and phylogenetic analysis revealed that out of the 14, one isolate which belonged to group A showed 27% similarity with FAdV-1, while three isolates showed 99%, 95% & 45% similarity to FAdV-4 (Group C). Whereas, ten isolates showed more than 99% similarity to FAdV-11 (Group D). The serotypes FAdV1, FAdV-4 and FAdV-11 are prevailing in the breeder and broilers. These results hold great importance in rapid, reliable and simultaneous detection of the three FAdV serotypes. Therefore, fowl adeno virus vaccine production for commercial poultry shall be according to the prevalent field serotypes.

HITAC-seq enables high-throughput cost-effective sequencing of plasmids and DNA fragments with identity

DNA sequencing is vital for many aspects of biological research and diagnostics. Despite the development of second and third generation sequencing technologies, Sanger sequencing has long been the only choice when required to precisely track each sequenced plasmids or DNA fragments. Here, we report a complete set of novel barcoding and assembling system, Highly-parallel Indexed Tagmentation-reads Assembled Consensus sequencing(HITAC-seq), that could massively sequence and track the identities of each individual sequencing sample. With the cost of much less than that of single read of Sanger sequencing,HITAC-seq can generate high-quality contiguous sequences of up to 10 kilobases or longer. The capability of HITAC-seq was confirmed through large-scale sequencing of thousands of plasmid clones and hundreds of amplicon fragments using approximately 100 pg of input DNAs. Due to its long synthetic length, HITACseq was effective in detecting relatively large structural variations, as demonstrated by the identification of a~1.3 kb Copia retrotransposon insertion in the upstream of a likely maize domestication gene. Besides being a practical alternative to traditional Sanger sequencing, HITAC-seq is suitable for many highthroughput sequencing and genotyping applications.

Biased heteroplasmy within the mitogenomic sequences of Gigantometra gigas revealed by sanger and high-throughput methods

Few studies have explored the differences between Sanger and HTS methods in the results of mitogenome sequencing. We used a single individual of insect to study the differences between the sequences given by Sanger and PCR-free HTS methods. Here we provided evidence for biased results of sequencing due to different methods in the mitochondrial genes of atp6, atp8, cox1, cox2, cox3, Cytb, nad2, nad3, nad4, nad5, rrn S, rrnL, trnH, trn I, and control region at various degrees. Especially, in cox1, the differently sequenced nucleotides account for 2.6% of the complete length. Furthermore, the highest value of the intraspecific genetic distance based on K2 P accounts for 2.5% using a barcode fragment size of cox1（651 bp, Sanger）, while the maximum distance of the corresponding cox1 fragment obtained by the two sequencing methods was 5.0%. We revealed that the methods of Sanger and HTS may give different sequencing results of mitochondrial genes, which may reflect the heteroplasmy of mitogenomes within an insect individual. Therefore, researchers should be very cautious in using the mixed data of a gene given by different methods of sequencing.

Laboratory diagnosis of nonpolio enteroviruses:A review of the current literature

Infections by nonpolio enteroviruses(EVs)are highly prevalent,particularly among children and neonates,where they may cause substantial morbidity and mortality.Laboratory diagnosis of these viral infections is important in patient prognosis and guidance of clinical management.Although the laboratory diagnosis of non-polio EVs is mainly based on molecular techniques,classical virus-isolation techniques are still used in refer-ence laboratories.Other techniques,such as antigen detection and serology,are becoming obsolete and rarely used in diagnosis.An important part of diagnosis and surveillance of EV infections is viral typing by VP1 gene sequencing using conventional Sanger technique and more recently,full-genome next-generation sequencing.The latter allows the typing of all EVs,better investigation of EV outbreaks,detection of coinfec-tion,and identification of severity markers in the EV genome.