To Analyze the Sensitivity of RT-PCR Assays Employing S Gene Target Failure with Whole Genome Sequencing Data during Third Wave by SARS-CoV-2 Omicron Variant

Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.

Safety evaluation and whole genome sequencing for revealing the ability of Penicillium oxalicum WX-209 to safely and effectively degrade citrus segments

The microbial potential of Penicillium has received critical attention.The present research aimed to elucidate the efficacy of crude enzyme secreted from Penicillium oxalicum WX-209 in degrading citrus segments and evaluate the safety of the process.Results showed that citrus segment membranes gradually dissolved after treatment with the crude enzyme solution,indicating good degradation capability.No significant differences in body weight,food ingestion rate,hematology,blood biochemistry,and weight changes of different organs were found between the enzyme intake and control groups.Serial experiments showed that the crude enzyme had high biological safety.Moreover,the whole genome of P.oxalicum WX-209 was sequenced by PacBio and Illumina platforms.Twenty-five scaffolds were assembled to generate 36 Mbp size of genome sequence comprising 11369 predicted genes modeled with a GC content of 48.33%.A total of 592 genes were annotated to encode enzymes related to carbohydrates,and some degradation enzyme genes were identified in strain P.oxalicum WX-209.

Genome sequencing provides potential strategies for drug discovery and synthesis

Medicinal plants are renowned for their abundant production of secondary metabolites,which exhibit notable pharmacological activities and great potential for drug development.The biosynthesis of secondary metabolites is highly intricate and influenced by various intrinsic and extrinsic factors,resulting in substantial species diversity and content variation.Consequently,precise regulation of secondary metabolite synthesis is of utmost importance.In recent years,genome sequencing has emerged as a valuable tool for investigating the synthesis and regulation of secondary metabolites in medicinal plants,facilitated by the widespread use of high-throughput sequencing technologies.This review highlights the latest advancements in genome sequencing within this field and presents several strategies for studying secondary metabolites.Specifically,the article elucidates how genome sequencing can unravel the pathways for secondary metabolite synthesis in medicinal plants,offering insights into the functions and regulatory mechanisms of participating enzymes.Comparative analyses of plant genomes allow identification of shared pathways of metabolite synthesis among species,thereby providing novel avenues for obtaining cost-effective biosynthetic intermediates.By examining individual genomic variations,genes or gene clusters associated with the synthesis of specific compounds can be discovered,indicating potential targets and directions for drug development and the exploration of alternative compound sources.Moreover,the advent of gene-editing technology has enabled the precise modifications of medicinal plant genomes.Optimization of specific secondary metabolite synthesis pathways becomes thus feasible,enabling the precise editing of target genes to regulate secondary metabolite production within cells.These findings serve as valuable references and lessons for future drug development endeavors,conservation of rare resources,and the exploration of new resources.

Surveillance of emerging SARS-CoV-2 variants by nanopore technology-based genome sequencing

Objective:To surveill emerging variants by nanopore technology-based genome sequencing in different COVID-19 waves in Sri Lanka and to examine the association with the sample characteristics,and vaccination status.Methods:The study analyzed 207 RNA positive swab samples received to sequence laboratory during different waves.The N gene cut-off threshold of less than 30 was considered as the major inclusion criteria.Viral RNA was extracted,and elutes were subjected to nanopore sequencing.All the sequencing data were uploaded in the publicly accessible database,GISAID.Results:The Omicron,Delta and Alpha variants accounted for 58%,22%and 4%of the variants throughout the period.Less than 1%were Kappa variant and 16%of the study samples remained unassigned.Omicron variant was circulated among all age groups and in all the provinces.Ct value and variants assigned percentage was 100%in Ct values of 10-15 while only 45%assigned Ct value over 25.Conclusions:The present study examined the emergence,prevalence,and distribution of SARS-CoV-2 variants locally and has shown that nanopore technology-based genome sequencing enables whole genome sequencing in a low resource setting country.

Copy number variation sequencing for diagnosis of cytomegalovirus infection based low-depth whole-genome sequencing technology in fetus:Three cases and literature review

Objective:To summarize the application value of copy number variant sequencing(CNV-seq)in the detection of fetal chromosome and cytomegalovirus load.Methods:The study analyzed the clinical basic data,relevant laboratory tests,treatment process,and outcomes of three patients with positive cytomegalovirus load detected by CNV-seq for fetal chromosomes and cytomegalovirus load,and literature review was done simutaneoubly.Results:In all three cases,the amniotic fluid cytomegalovirus load was less than 105 Copies/ml,and there were no significant neurological abnormalities observed during pregnancy or postpartum follow-up.There is no literature review on the application of CNV-seq technology in the detection of cytomegalovirus infection,only literature reports on genome analysis of CMV-DNA in confirmed patients were available.Conclusion:CNV-seq can be used to detect cytomegalovirus load,which may have a certain degree of predictive value for fetal outcome.CNV-seq can simultaneously detect fetal chromosomes and pathogenic microorganisms,which is of great significance for the prevention and control of birth defects.

The bisulfite genomic sequencing protocol

The bisulfite genomic sequencing (BGS) protocol has gained worldwide popularity as the method of choice for analyzing DNA methylation. It is this popular because it is a powerful protocol and it may be coupled with many other applications. However, users often run into a slew ofproblems, including incomplete conversion,overly degraded DNA, sub-optimal PCR amplifications, false positives, uninformative results, or altogether failed experiments. We pinpoint the reasons why these problems arise and carefully explain the critical steps toward accomplishing a successful experiment step-by-step. This protocol has worked successfully (>99.9% conversion) on as little as 100 ng of DNA derived from nearly 10-year-old DNA samples extracted from whole blood stored at -80°C and resulted in enough converted DNA for more than 50 PCRreactions. The aim of this article is to makelearning and usage of BGS easier, more efficient and standardized for all users.

Complete Genome Sequencing and Genetic Variation Analysis of Two H9N2 Subtype Avian Influenza Virus Strains

[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.

Improving the accuracy of genomic prediction for meat quality traits using whole genome sequence data in pigs

Background Pork quality can directly affect customer purchase tendency and meat quality traits have become valu-able in modern pork production.However,genetic improvement has been slow due to high phenotyping costs.In this study,whole genome sequence(WGS)data was used to evaluate the prediction accuracy of genomic best linear unbiased prediction(GBLUP)for meat quality in large-scale crossbred commercial pigs.Results We produced WGS data(18,695,907 SNPs and 2,106,902 INDELs exceed quality control)from 1,469 sequenced Duroc×(Landrace×Yorkshire)pigs and developed a reference panel for meat quality including meat color score,marbling score,L*(lightness),a*(redness),and b*(yellowness)of genomic prediction.The prediction accuracy was defined as the Pearson correlation coefficient between adjusted phenotypes and genomic estimated breeding values in the validation population.Using different marker density panels derived from WGS data,accuracy differed substantially among meat quality traits,varied from 0.08 to 0.47.Results showed that MultiBLUP outperform GBLUP and yielded accuracy increases ranging from 17.39%to 75%.We optimized the marker density and found medium-and high-density marker panels are beneficial for the estimation of heritability for meat quality.Moreover,we conducted genotype imputation from 50K chip to WGS level in the same population and found average concord-ance rate to exceed 95%and r^(2)=0.81.Conclusions Overall,estimation of heritability for meat quality traits can benefit from the use of WGS data.This study showed the superiority of using WGS data to genetically improve pork quality in genomic prediction.

Breed identification using breed‑informative SNPs and machine learning based on whole genome sequence data and SNP chip data

Background Breed identification is useful in a variety of biological contexts.Breed identification usually involves two stages,i.e.,detection of breed-informative SNPs and breed assignment.For both stages,there are several methods proposed.However,what is the optimal combination of these methods remain unclear.In this study,using the whole genome sequence data available for 13 cattle breeds from Run 8 of the 1,000 Bull Genomes Project,we compared the combinations of three methods(Delta,FST,and In)for breed-informative SNP detection and five machine learning methods(KNN,SVM,RF,NB,and ANN)for breed assignment with respect to different reference population sizes and difference numbers of most breed-informative SNPs.In addition,we evaluated the accuracy of breed identification using SNP chip data of different densities.Results We found that all combinations performed quite well with identification accuracies over 95%in all scenarios.However,there was no combination which performed the best and robust across all scenarios.We proposed to inte-grate the three breed-informative detection methods,named DFI,and integrate the three machine learning methods,KNN,SVM,and RF,named KSR.We found that the combination of these two integrated methods outperformed the other combinations with accuracies over 99%in most cases and was very robust in all scenarios.The accuracies from using SNP chip data were only slightly lower than that from using sequence data in most cases.Conclusions The current study showed that the combination of DFI and KSR was the optimal strategy.Using sequence data resulted in higher accuracies than using chip data in most cases.However,the differences were gener-ally small.In view of the cost of genotyping,using chip data is also a good option for breed identification.

Evaluating the potential of(epi)genotype‑by‑low pass nanopore sequencing in dairy cattle:a study on direct genomic value and methylation analysis

Background Genotype-by-sequencing has been proposed as an alternative to SNP genotyping arrays in genomic selection to obtain a high density of markers along the genome.It requires a low sequencing depth to be cost effective,which may increase the error at the genotype assigment.Third generation nanopore sequencing technology offers low cost sequencing and the possibility to detect genome methylation,which provides added value to genotype-by-sequencing.The aim of this study was to evaluate the performance of genotype-by-low pass nanopore sequencing for estimating the direct genomic value in dairy cattle,and the possibility to obtain methylation marks simultaneously.Results Latest nanopore chemistry(LSK14 and Q20)achieved a modal base calling accuracy of 99.55%,whereas previous kit(LSK109)achieved slightly lower accuracy(99.1%).The direct genomic value accuracy from genotype-by-low pass sequencing ranged between 0.79 and 0.99,depending on the trait(milk,fat or protein yield),with a sequencing depth as low as 2×and using the latest chemistry(LSK114).Lower sequencing depth led to biased estimates,yet with high rank correlations.The LSK109 and Q20 achieved lower accuracies(0.57-0.93).More than one million high reliable methylated sites were obtained,even at low sequencing depth,located mainly in distal intergenic(87%)and promoter(5%)regions.Conclusions This study showed that the latest nanopore technology in useful in a LowPass sequencing framework to estimate direct genomic values with high reliability.It may provide advantages in populations with no available SNP chip,or when a large density of markers with a wide range of allele frequencies is needed.In addition,low pass sequencing provided nucleotide methylation status of>1 million nucleotides at≥10×,which is an added value for epigenetic studies.

Chromosomal level assembly and population sequencing of the Chinese tree shrew genome

Chinese tree shrews (Tupaia belangeri chinensis) have become an increasingly important experimental animal in biomedical research due to their close relationship to primates. An accurately sequenced and assembled genome is essential for understanding the genetic features and biology of this animal. In this study, we used long-read single-molecule sequencing and high-throughput chromosome conformation capture (Hi-C) technology to obtain a high-qualitychromosome-scale scaffolding of the Chinese tree shrew genome. The new reference genome (KIZ version 2: TS_2.0) resolved problems in presently available tree shrew genomes and enabled accurate identification of large and complex repeat regions, gene structures, and species-specific genomic structural variants. In addition, by sequencing the genomes of six Chinese tree shrew individuals, we produced a comprehensive map of 12.8 M single nucleotide polymorphisms and confirmed that the major histocompatibility complex (MHC) loci and immunoglobulin gene family exhibited high nucleotide diversity in the tree shrew genome. We updated the tree shrew genome database (TreeshrewDB v2.0: http://www.treeshrewdb.org) to include the genome annotation information and genetic variations. The new high-quality reference genome of the Chinese tree shrew and the updated TreeshrewDB will facilitate the use of this animal in many different fields of research.

Current advances in genome sequencing of common wheat and its ancestral species

Common wheat is an important and widely cultivated food crop throughout the world.Much progress has been made in regard to wheat genome sequencing in the last decade.Starting from the sequencing of single chromosomes/chromosome arms whole genome sequences of common wheat and its diploid and tetraploid ancestors have been decoded along with the development of sequencing and assembling technologies. In this review, we give a brief summary on international progress in wheat genome sequencing, and mainly focus on reviewing the effort and contributions made by Chinese scientists.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer

Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 （EV71） and Coxsackievirus A16 （CA16） strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute （min） of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min （on average）, 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

First detection and complete genome of Soybean chlorotic mottle virus naturally infecting soybean in China by deep sequencing

Soybean chlorotic mottle virus(SbCMV)was first detected from soybean plants in Jiangxi Province of China by high throughput sequencing and was confirmed by PCR.The complete nucleotide sequence of NC113 was determined to be 8210 nucleotides,and shared the highest similarity(91.7%)with sequences of SbCMV that was only reported in Japan.It encodes nine putative open reading frames(ORFs Ia,Ib and Ⅱ-Ⅷ),and contains a large intergenic region located at nucleotide 5976-6512 between ORFs VI and VII.Sequence analysis and phylogenetic tree indicated that NC113 is an isolate of SbCMV,and is more related to the soymoviruses Blueberry red ringspot virus(BRRSV),Peanut chlorotic streak virus(PCSV)and Cestrum yellow leaf curling virus(CmYLCV)than to other representative members in the Caulimoviridae family.Field survey of 472 legume plants from Jiangxi and Zhejiang provinces showed SbCMV was only detected from soybean in Nanchang City with a low incidence rate.This is the first report of Soybean chlorotic mottle virus identified in China.

Current status and future perspectives for sequencing livestock genomes

Only in recent years, the draft sequences for several agricultural animals have been assembled. Assembling an individual animal＇s entire genome sequence or specific region（s） of interest is increasingly important for agricultura researchers to perform genetic comparisons between animals with different performance. We review the current status for several sequenced agricultural species and suggest that next generation sequencing （NGS） technology with decreased sequencing cost and increased speed of sequencing can benefit agricultural researchers. By taking advantage of advanced NGS technologies, genes and chromosomal regions that are more labile to the influence of environmental factors could be pinpointed. A more long term goal would be addressing the question of how animals respond at the molecular and cellular levels to different environmental models （e.g. nutrition）. Upon revealing important genes and gene-environment interactions, the rate of genetic improvement can also be accelerated. It is clear that NGS technologies will be able to assist animal scientists to efficiently raise animals and to better prevent infectious diseases so that overall costs of animal production can be decreased.

Identification and characterization of short tandem repeats in the Tibetan macaque genome based on resequencing data

The Tibetan macaque, which is endemic to China, is currently listed as a Near Endangered primate species by the International Union for Conservation of Nature （IUCN）（2017）. Short tandem repeats （STRs） refer to repetitive elements of genome sequence that range in length from 1-6 bp. They are found in many organisms and are widely applied in population genetic studies. To clarify the distribution characteristics of genome-wide STRs and understand their variation among Tibetan macaques, we conducted a genome-wide survey of STRs with next-generation sequencing of five macaque samples. A total of 1 077 790 perfect STRs were mined from our assembly, with an N50 of 4 966 bp. Mono-nucleotide repeats were the most abundant, followed by tetra- and di-nucleotide repeats. Analysis of GC content and repeats showed consistent results with other macaques. Furthermore, using STR analysis software （IobSTR）, we found that the proportion of base pair deletions in the STRs was greater than that of insertions in the five Tibetan macaque individuals （P〈0.05, t-test）. We also found a greater number of homozygous STRs than heterozygous STRs （P〈0.05, t-test）, with the Emei and Jianyang Tibetan macaques showing more heterozygous loci than Huangshan Tibetan macaques. The proportion of insertions and mean variation of alleles in the Emei and Jianyang individuals were slightly higher than those in the Huangshan individuals, thus revealing differences in STR allele size between the two populations The polymorphic STR loci identified based on the reference genome showed good amplification efficiency and could be used to study population genetics in Tibetan macaques. The neighbor-joining tree classified the five macaques into two different branches according to their geographical origin, indicating high genetic differentiation between the Huangshan and Sichuan populations. We elucidated the distribution characteristics of STRs in the Tibetan macaque genome and provided an effective method for screening polymorphic STRs. Our results also lay a foundation for future genetic variation studies of macaques.

BAC end sequencing of Pacific white shrimp Litopenaeus vannamei: a glimpse into the genome of Penaeid shrimp

Little is known about the genome of Pacific white shrimp (Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 paired- ends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for this species.

Genome-wide mining, characterization, and development of microsatellite markers in Marsupenaeus japonicus by genome survey sequencing

The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats(SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identified, among which dinucleotide repeats were the most frequent class(44.08%), followed by mononucleotides(29.67%), trinucleotides(18.96%), tetranucleotides(5.66%), hexanucleotides(1.07%), and pentanucleotides(0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci(90.0%) were successfully amplified with specific products and 24(80.0%) were polymorphic. For the amplified polymorphic loci, the alleles ranged from 5 to 17(with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had significant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identified SSRs significantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.

Isolation and the Full-length Genome Sequencing of Subgroup J Avian Leukosis Virus ZH-08 Isolate Associated with Hemangioma

A subgroup J avian leukosis virus (AVL-J), designated as ZH-08, was isolated from a breeder flock in Guangdong province with a novel hemangioma case. The identification results of ELISA test, PCR and immunofluoresecence assay (IFA) specific for ALV-J were all positive. Based on the public full-length proviral genome sequence of ALV-J prototype strain HPRS-103, three pairs of primers were synthesized. The full-length proviral genome sequence of ZH-08 isolate is 7597 bp, which has a little difference with that of published full-length genome sequences, but its organization corresponds with typical retroviral genome structure; and known oncogenes were not included in its genome. According to the gp85 sequence comparison of ZH-08 isolate with those of the other reference strains in China and abroad, the highest similarity (93.7%) was with the YZ9901 isolate. Phylogenetic analysis, based on the gp85 gene, showed that the ZH-08 isolated here had the closest linkage to the SD07LK1 isolate. This study provides the basis for the biological characterization and pathogenesis research of the ZH-08 isolate.

Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

Objective To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR （M-RTPCR）, for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing （NGS） platform. Methods Virus genome copy was quantified and seria（iy diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. Results The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing Iow-titer virus. Conclusion The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.