Application of Next Generation Sequencing for Rapid Identification of Lactic Acid Bacteria

The rapid identification of lactic acid bacteria,which are essential microorganisms in the food industry,is of great significance for industrial applications.The identification of lactic acid bacteria traditionally relies on the isolation and identification of pure colonies.While this method is well-established and widely used,it is not without limitations.The subjective judgment inherent in the isolation and purification process introduces potential for error,and the incomplete nature of the isolation process can result in the loss of valuable information.The advent of next generation sequencing has provided a novel approach to the rapid identification of lactic acid bacteria.This technology offers several advantages,including rapidity,accuracy,high throughput,and low cost.Next generation sequencing represents a significant advancement in the field of DNA sequencing.Its ability to rapidly and accurately identify lactic acid bacteria strains in samples with insufficient information or in the presence of multiple lactic acid bacteria sets it apart as a valuable tool.The application of this technology not only circumvents the potential errors inherent in the traditional method but also provides a robust foundation for the expeditious identification of lactic acid bacteria strains and the authentication of bacterial powder in industrial applications.This paper commences with an overview of traditional and molecular biology methods for the identification of lactic acid bacteria.While each method has its own advantages,they are not without limitations in practical application.Subsequently,the paper provides an introduction of the principle,process,advantages,and disadvantages of next generation sequencing,and also details its application in strain identification and rapid identification of lactic acid bacteria.The objective of this study is to provide a comprehensive and reliable basis for the rapid identification of industrial lactic acid bacteria strains and the authenticity identification of bacterial powder.

Next Generation Sequencing in Oncological Diagnostics: Hype or Hope?

The understanding of how genetic and epigenetic factors influence tumorigenesis, progression and invasion, is vastly growing since new technologies allow the analysis of the functional genome namely the exome, the transcriptome and the epigenome, besides enabling genome-wide assessment of genetic variations. With the advent of new drugs that are indicated tissue agnostic, depending on certain mutations, there is a growing demand for fast and cost-effective genetic diagnosis. The method in focus that already became an indispensable tool in viral diagnosis is next-generation sequencing (NGS). This approach allows sequencing of literally every DNA molecule in the sample and can either be used to assess numerous genetic markers of one patient at a time, or to assess fewer markers of many patients in parallel, which reduces costs. We submitted 23 samples of different tumor entities to four diagnostic companies with different analysis profiles. The results as disclosed and discussed in this report indicate that so far, the main application of NGS is rather in cancer research than in diagnosis, as none of the reports had a real impact on the therapeutic scheme. We are perfectly aware that such a small cohort cannot be generalized, but considering the costs vs. benefits, NGS should be engaged upon a very stringent evaluation only. However, in cases where obtaining a tissue biopsy is impossible or unfavorable, analysis of liquid biopsy by NGS provides a vital alternative.

Endoscopic ultrasound-guided fine-needle aspiration pancreatic adenocarcinoma samples yield adequate DNA for next-generation sequencing:A cohort analysis

BACKGROUND Genetic tests are increasingly performed for the management of unresectable pancreatic cancer.For genotyping aimed samples current guidelines recommend using core specimens,although based on moderate quality evidence.However,in clinical practice among the endoscopic ultrasound(EUS) guided tissue acquisition methods,fine needle aspiration(FNA) is the most widely performed.AIM To assess the adequacy for next generation sequencing(NGS) of the DNA yielded from EUS-FNA pancreatic adenocarcinoma(PDAC) samples.METHODS Between November 2018 and December 2021,105 patients with PDAC confirmed by EUS-FNA were included in the study at our tertiary gastroenterology center.Either 22 gauge(G) or 19G FNA needles were used.One pass was dedicated to DNA extraction.DNA concentration and purity(A260/280,A260/230) were assessed by spectrophotometry.We assessed the differences in DNA parameters according to needle size and tumor characteristics(size,location) and the adequacy of the extracted DNA for NGS(defined as A260/280 ≥ 1.7,and DNA yield:≥ 10 ng for amplicon based NGS,≥ 50 ng for whole exome sequencing [WES],≥ 100 ng for whole genome sequencing [WGS]) by analysis of variance and ttest respectively.Moreover,we compared DNA purity parameters across the different DNA yield categories.RESULTS Our cohort included 49% male patients,aged 67.02 ± 8.38 years.The 22G needle was used in 71%of the cases.The DNA parameters across our samples varied as follows:DNA yield:1289 ng(inter quartile range:534.75-3101),A260/280 = 1.85(1.79-1.86),A260/230 = 2.2(1.72-2.36).DNA yield was > 10 ng in all samples and > 100 ng in 93% of them(one sample < 50 ng).There were no significant differences in the concentration and A260/280 between samples by needle size.Needle size was the only independent predictor of A260/230 which was higher in the 22G samples(P =0.038).NGS adequacy rate was 90% for 19G samples regardless of NGS type,and for 22G samples it reached 89% for WGS adequacy and 91% for WES and amplicon based NGS.Samples with DNA yield > 100 ng had significantly higher A260/280(1.89 ± 0.32 vs 1.34 ± 0.42,P = 0.013).Tumor characteristics were not corelated with the DNA parameters.CONCLUSION EUS-FNA PDAC samples yield DNA adequate for subsequent NGS.DNA amount was similar between 22G and 19G FNA needles.DNA purity parameters may vary indirectly with needle size.

Next-generation Sequencing Study of Pathogens in Serum from Patients with Febrile Jaundice in Sierra Leone

Objective People in Western Africa suffer greatly from febrile jaundice, which is caused by a variety of pathogens. However, yellow fever virus(YFV) is the only pathogen under surveillance in Sierra Leone owing to the undeveloped medical and public health system there. Most of the results of YFV identification are negative. Elucidation of the pathogen spectrum is required to reduce the prevalence of febrile jaundice. Methods In the present study, we used Ion Torrent semiconductor sequencing to profile the pathogen spectrum in archived YFV‐negative sera from 96 patients in Sierra Leone who presented with unexplained febrile jaundice. Results The most frequently identified sequencing reads belonged to the following pathogens: cytomegalovirus(89.58%), Epstein‐Barr virus(55.21%), hepatitis C virus(34.38%), rhinovirus(28.13%), hepatitis A virus(20.83%), coxsackievirus(10.42%), Ebola virus(8.33%), hepatitis E virus(8.33%), lyssavirus(4.17%), leptospirosis(4.17%), chikungunya virus(2.08%), Crimean‐Congo hemorrhagic fever virus(1.04%), and hepatitis B virus(1.04%). Conclusion The distribution of sequencing reads suggests a broader spectrum of pathogens for consideration in clinical diagnostics and epidemiological surveillance in Sierra Leone.

A Multiplex PCR-Based Next-Generation Sequencing Approach Has Detected a Common Large Deletion in STS Gene in a Patient with X-Linked Ichthyosis

Several nuclear genes have been found to be linked to ichthyosis, and Next Generation Sequencing approach on panels of targeted genes has turned out to be particularly useful in analyzing diseases characterized by significant genetic and phenotypic heterogeneity. We developed a panel of 26 genes to be screened with the Ion Personal Genome Machine (PGM) for causative mutations relating to ichthyosis. Sequencing runs were obtained from a patient with ichthyosis using the Ion Torrent PGM and then processed with Ion Torrent Suite, Variant Caller, Coverage Analysis and wANNOVER tools. No causative mutations were found using Variant Caller and wANNOVER softwares, whereas the “Coverage Analysis” tool revealed a common large deletion in STS gene in a patient with X-linked ichthyosis. Identification of indels in Next Generation Sequencing (NGS) data is a veritable challenge. This study demonstrates the efficacy and effectiveness of using NGS approach to detect large deletions without resorting to specific algorithms for “indel” detection. Our results indicate that the NGS panel is a useful, rapid and cost-effective screening test for patients whose features are suggestive of a genetic etiology involving one of the genes embedded in the panel. It is an excellent alternative to Sanger sequencing as for costs, ease of analysis, and turnaround time.

Highly Aligned Ternary Nanofiber Matrices Loaded with MXene Expedite Regeneration of Volumetric Muscle Loss

Current therapeutic approaches for volumetric muscle loss(VML)face challenges due to limited graft availability and insufficient bioactivities.To overcome these limitations,tissue-engineered scaffolds have emerged as a promising alternative.In this study,we developed aligned ternary nanofibrous matrices comprised of poly(lactide-co-ε-caprolactone)integrated with collagen and Ti_(3)C_(2)T_(x)MXene nanoparticles(NPs)(PCM matrices),and explored their myogenic potential for skeletal muscle tissue regeneration.The PCM matrices demonstrated favorable physicochemical properties,including structural uniformity,alignment,microporosity,and hydrophilicity.In vitro assays revealed that the PCM matrices promoted cellular behaviors and myogenic differentiation of C2C12 myoblasts.Moreover,in vivo experiments demonstrated enhanced muscle remodeling and recovery in mice treated with PCM matrices following VML injury.Mechanistic insights from next-generation sequencing revealed that MXene NPs facilitated protein and ion availability within PCM matrices,leading to elevated intracellular Ca^(2+)levels in myoblasts through the activation of inducible nitric oxide synthase(i NOS)and serum/glucocorticoid regulated kinase 1(SGK1),ultimately promoting myogenic differentiation via the m TOR-AKT pathway.Additionally,upregulated i NOS and increased NO–contributed to myoblast proliferation and fiber fusion,thereby facilitating overall myoblast maturation.These findings underscore the potential of MXene NPs loaded within highly aligned matrices as therapeutic agents to promote skeletal muscle tissue recovery.

Review of current diagnostic methods and advances in Helicobacter pylori diagnostics in the era of next generation sequencing

Helicobacter pylori(H.pylori)infection is highly prevalent in the human population and may lead to severe gastrointestinal pathology including gastric and duodenal ulcers,mucosa associated tissue lymphoma and gastric adenocarcinoma.In recent years,an alarming increase in antimicrobial resistance and subsequently failing empiric H.pylori eradication therapies have been noted worldwide,also in many European countries.Therefore,rapid and accurate determination of H.pylori’s antibiotic susceptibility prior to the administration of eradication regimens becomes ever more important.Traditionally,detection of H.pylori and its antimicrobial resistance is done by culture and phenotypic drug susceptibility testing that are cumbersome with a long turn-around-time.Recent advances in diagnostics provide new tools,like real-time polymerase chain reaction(PCR)and line probe assays,to diagnose H.pylori infection and antimicrobial resistance to certain antibiotics,directly from clinical specimens.Moreover,high-throughput whole genome sequencing technologies allow the rapid analysis of the pathogen’s genome,thereby allowing identification of resistance mutations and associated antibiotic resistance.In the first part of this review,we will give an overview on currently available diagnostic methods for detection of H.pylori and its drug resistance and their implementation in H.pylori management.The second part of the review focusses on the use of next generation sequencing technology in H.pylori research.To this end,we conducted a literature search for original research articles in English using the terms“Helicobacter”,“transcriptomic”,“transcriptome”,“next generation sequencing”and“whole genome sequencing”.This review is aimed to bridge the gap between current diagnostic practice(histology,rapid urease test,H.pylori culture,PCR and line probe assays)and new sequencing technologies and their potential implementation in diagnostic laboratory settings in order to complement the currently recommended H.pylori management guidelines and subsequently improve public health.

A novel PIK3CD C896T mutation detected in bilateral sudden sensorineural hearing loss using next generation sequencing:An indication of primary immunodeficiency

Objective:To investigate immune-related genetic background in bilateral sudden sensorineural hearing loss (SSNHL). Case report and methods: The case is a 45-year-old man presenting with a 7-year history of bilateral profound SSNHL. Blood biochemical testing demonstrated increased levels of total cholesterol (5.88 mmol/L). Tests for hepatitis B showed a positive antibody against the hepatitis B core antigen. Complement C3 was below the normal value, and complement C4 and IgG were in the lower range of normal values. CT images showed a normal inner ear and vestibular aqueduct but round window membranous ossification on both sides. A total number of 232 immune-associated genes were sequenced using the next generation sequencing technique. Results: Mutations were detected in 5 genes, including the phosphoinositide 3-kinase catalytic subunit delta (PIK3CD), caspase recruitment domain-containing protein 9 (CARD9), complement factor H-related (CFHR2), immunoglobulin lambda-like polypeptide 1 Protein (IGLL1), and transmembrane channel-like gene family 8 (TMC8). In the PIK3CD gene, a C896T substitute in exon 7 was detected. This mutation causes primary immunodeficiency and is an autosomal dominant disease. Conclusion: The PIK3CD C896T mutation responsible for primary immunodeficiency may contribute to the onset of bilateral SSNHL with subsequent rapid progression.

Current scenario of the genetic testing for rare neurological disorders exploiting next generation sequencing

Next generation sequencing is currently a cornerstone of genetic testing in routine diagnostics,allowing for the detection of sequence variants with so far unprecedented large scale,mainly in genetically heterogenous diseases,such as neurological disorders.It is a fast-moving field,where new wet enrichment protocols and bioinformatics tools are constantly being developed to overcome initial limitations.Despite the as yet undiscussed advantages,however,there are still some challenges in data analysis and the interpretation of variants.In this review,we address the current state of next generation sequencing diagnostic testing for inherited human disorders,particularly giving an overview of the available high-throughput sequencing approaches;including targeted,whole-exome and whole-genome sequencing;and discussing the main critical aspects of the bioinformatic process,from raw data analysis to molecular diagnosis.

Next generation sequencing reveals co-existence of hereditary spherocytosis and Dubin–Johnson syndrome in a Chinese gril: A case report

BACKGROUND Hereditary spherocytosis(HS)is a hereditary disease of hemolytic anemia that occurs due to the erythrocyte membrane defects.Dubin–Johnson syndrome(DJS),which commonly results in jaundice,is a benign hereditary disorder of bilirubin clearance that occurs only rarely.The co-occurrence of HS and DJS is extremely rare.We recently diagnosed and treated a case of co-occurring HS and DJS.CASE SUMMARY A 21-year-old female patient presented to our department because of severe jaundice,severe splenomegaly,and mild anemia since birth.We eventually confirmed the diagnosis of co-occurring DJS and HS by next generation sequencing(NGS).The treatment of ursodeoxycholic acid in combination with phenobarbital successfully increased hemoglobin and reduced total bilirubin and direct bilirubin.CONCLUSION The routine application of NGS can efficiently render a definite diagnosis when inherited disorders are suspected.

Next generation sequencing in cardiovascular diseases

In the last few years,the advent of next generation sequencing(NGS) has revolutionized the approach to genetic studies,making whole-genome sequencing a possible way of obtaining global genomic information.NGS has very recently been shown to be successful in identifying novel causative mutations of rare or common Mendelian disorders.At the present time,it is expected that NGS will be increasingly important in the study of inherited and complex cardiovascular diseases(CVDs).However,the NGS approach to the genetics of CVDs represents a territory which has not been widely investigated.The identification of rare and frequent genetic variants can be very important in clinical practice to detect pathogenic mutations or to establish a profile of risk for the development of pathology.The purpose of this paper is to discuss the recent application of NGS in the study of several CVDs such as inherited cardiomyopathies,channelopathies,coronary artery disease and aortic aneurysm.We also discuss the future utility and challenges related to NGS in studying the genetic basis of CVDs in order to improve diagnosis,prevention,and treatment.

Next Generation Transcriptome Sequencing and Quantitative Real-Time PCR Technologies for Characterisation of the Bemisia tabaci Asia 1 mtCOI Phylogenetic Clade

A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemiptera：Aleyrodidae）,the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing（NGS）has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences（301 094 reads）using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes（e.g.,HSP90）but misassemblies in other datasets（e.g.,the vitellogenin gene family）,highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.

Large scale identification of SSR marker in perilla by next generation sequencing

Perilla frutescens (L.) is an edible, medicinal crop, and most popular in East Asia. Its molecular breeding and research are hampered by the paucity of molecular markers. Simple sequence repeat (SSR) markers are ubiquitous and widely used in eukaryotic genomes. EST-SSRs identification of perilla was performed in 116,387 reads generated by Illumina paired-end sequencing technology. In total 25,449 unigenes containing SSR and 33,867 SSR loci were identified, and 19,400 primer pairs were designed. Polymorphism of SSR primers was conducted by searching for insertions and deletions (INDELs), and 1,567 unique SSRs were predicted. Totally, 200 SSR primer pairs were selected for polymorphic validation among 23 perilla accessions. Results showed that 175 primer pairs produced amplicons, and 30 pairs exhibited polymorphism. Polymorphic ratio was higher by using INDEL method than using conventional primers. Phylogenetic analysis showed the 2 distinct groups: P. frutescens var. frutescens and P. frutescens var. crispa. Wrinkled leaf trait and seed trait were distinct between these 2 groups. However, no clear leaf color or geographic relationship was detected. The large scale development and identification of SSR marker in this research laid a foundation for genetic analysis and marker assisted breeding of cultivated perilla.

Fecal gene detection based on next generation sequencing for colorectal cancer diagnosis

BACKGROUND Colorectal cancer(CRC)is one of the most common malignancies worldwide.Given its insidious onset,the condition often already progresses to advanced stage when symptoms occur.Thus,early diagnosis is of great significance for timely clinical intervention,efficacy enhancement,and prognostic improvement.Featuring high throughput,fastness,and rich information,next generation sequencing(NGS)can greatly shorten the detection time,which is a widely used detection technique at present.AIM To screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC,and to establish a technological platform for CRC screening,diagnosis,and efficacy monitoring through fecal DNA detection.METHODS NGS was used to sequence the stool DNA of patients with CRC,which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease,as well as the tumor tissues of CRC patients.Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened,and their significances in diagnosing CRC and predicting patients'prognosis were comprehensively evaluated.RESULTS High mutation frequencies of TP53,APC,and KRAS were detected in the stools and tumor tissues of CRC patients prior to surgery.Contrastively,no pathogenic mutations of the above three genes were noted in the postoperative stools,the normal controls,or the benign intestinal disease group.This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients.The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent.Compared to the postoperative stools and the stools in the two control groups,the pathogenic mutation frequencies of TP53 and KRAS were significantly higher for the preoperative stools(χ^(2)=7.328,P<0.05;χ^(2)=4.219,P<0.05),suggesting that fecal TP53 and KRAS genes can be used for CRC screening,diagnosis,and prognostic prediction.No significant difference in the pathogenic mutation frequency of the APC gene was found from the postoperative stools or the two control groups(χ^(2)=0.878,P>0.05),so further analysis with larger sample size is required.Among CRC patients,the pathogenic mutation sites of TP53 occurred in 16 of 27 preoperative stools,with a true positive rate of 59.26%,while the pathogenic mutation sites of KRAS occurred in 10 stools,with a true positive rate of 37.04%.The sensitivity and negative predictive values of the combined genetic testing of TP53 and KRAS were 66.67%(18/27)and 68.97%,respectively,both of which were higher than those of TP53 or KRAS mutation detection alone,suggesting that the combined genetic testing can improve the CRC detection rate.The mutation sites TP53 exon 4 A84G and EGFR exon 20 I821T(mutation start and stop positions were both 7579436 for the former,while 55249164 for the latter)were found in the preoperative stools and tumor tissues.These"undetected"mutation sites may be new types of mutations occurring during the CRC carcinogenesis and progression,which needs to be confirmed through further research.Some mutations of"unknown clinical significance"were found in such genes as TP53,PTEN,KRAS,BRAF,AKT1,and PIK3CA,whose clinical values is worthy of further exploration.CONCLUSION NGS-based fecal genetic testing can be used as a complementary technique for the CRC diagnosis.Fecal TP53 and KRAS can be used as specific genes for the screening,diagnosis,prognostic prediction,and recurrence monitoring of CRC.Moreover,the combined testing of TP53 and KRAS genes can improve the CRC detection rate.

Development of Starfruit Simple Sequence Repeat (SSR) Using Next Generation Sequencing

Starfruit (Averrhoa carambola L.) is an important fruit for Malaysian export and great attention has been made to improve starfruit fruit quality at Malaysian Agricultural Research and Development Institute (MARDI). The current study used next generation sequencing (NGS) technologies to develop starfruit simple sequence repeat (SSR) from 2 varieties namely B11 and B17 using Illumina HiSeq. The pre-processed reads were de novo assembled to generate approximately 75,000 and 74,000 scaffolds respectively. Total genome size for B11 and B17 were around 345 Mbp and 342 Mbp based on K-mer distribution analysis. In-silico microsatellite mining of each variety has identified more than 17,000 SSR in B11 and B17 respectively. Dinucleotides were the most abundant, accounting for more than 70% of all SSR and repeat motif GA (49%) was most common. A total of 239 SSR primer pairs were designed from contigs larger than 350 nucleotides and tested for amplification. The 30 polymorphic SSRs were used to DNA fingerprint of 12 starfruit hybrids. Polymorphism information content (PIC) ranged from 0.1411 to 0.6838, with an average of 0.3919. The Unweighted Pair-Group Method for Arithmetic Averages (UPGMA) dendrogram clustered 12 starfruit accessions into 2 groups.

Comparison of next generation sequencing-based and methylated DNA immunoprecipitation-based approaches for fetal aneuploidy non-invasive prenatal testing

Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorionic villus and amniotic fluid sampling, which result in a significant risk for pregnancy loss. The discovery of cell free fetal DNA circulating in the maternal blood has great potential for the development of non-invasive prenatal testing(NIPT) methodologies. Such strategies have been successfully applied for the determination of the fetal rhesus status and inherited monogenic disease but the field of fetal aneuploidy investigation seems to be more challenging. The main reason for this is that the maternal cell free DNA in the mother's plasma is far more abundant, and because it is identical to half of the corresponding fetal DNA. Approaches developed are mainly based on next generation sequencing(NGS) technologies and epigenetic genetic modifications, such as fetal-maternal DNA differential methylation. At present, genetic services for non-invasive fetal aneuploidy detection are offered using NGS-based approaches but, for reasons that are presented herein, they still serve as screening tests which are not readily accessed by the majority of couples. Here we discuss the limitations of both strategies for NIPT and the future potential of the methods developed.

Next generation sequencing in oral disease diagnostics

DNA sequencing is the method of identifying the precise order of DNA nucleotides within a molecule. The information of DNA sequencing is of prime requisite for basic biological research as well as in various clinical specialties.They can be used to determine the individual genetic sequence, larger genetic regions, chromosomes as well as to sequence RNA and proteins. Since the first DNA sequencing in 1970s, there has been tremendous advancements in the technologies aimed to determine the entire human genome. The need for rapid and accurate sequencing of human genome has resulted in the introduction of next generation sequencing(NGS) technology. NGS refers to the secondgeneration DNA sequencing technologies where millions of DNA can be sequenced simultaneously. Some of the next gen sequencing methods employed are Roche/454 life science, Illumina/Solexa, SOLiD system and HeliScope.Application of NGS in decoding the genomic database of various oral diseases may possess therapeutic and prognostic value. This presentation provides an overview of the basics of NGS and their potential applications in oral disease diagnostics.

Identification of transgene insertions in two genetically modified soybeans using high throughput next generation sequencing

Genetically modified(GM) organisms are widely adopted. However, their safety assessments and control are still of special concern to the public. Identifying and localizing transgene insertion is an essentially prerequisite step. In this study, 2 independent transgene soybean lines were selected(LB4-AtDCGS-1-20-5-2 and CGS-ZG11) as typical cases. Both lines contained expression cassette of At-DCGS that encoding a feedback-insensitive cystathionine gamma-synthase to produce higher level methionine(Met). LB4-AtDCGS-1-20-5-2 was whole genome sequenced with one paired-end 500 bp library and two mate-paired 1 kb and 2 kb libraries using Illumina HiSeq sequencing platform. CGS-ZG11 was sequenced with only one paired-end 500 bp library. Both genomes were assembled,and 2 scaffold sequences(1 for each line) were screened out by aligning with transgene.Then the transgene insertion and its flanking regions in soybean genome were further identified and confirmed by PCR cloning and Sanger sequencing. Results showed that these 2 transgene lines had single copy of inserted transgene. Their transgene insertion contents were identified, which facilitates further safety assessment. These results indicated that genome assembly using high throughput sequencing is a powerful tool for identifying transgene insertions, even with limited knowledge.

DISCUSSION ON AVERAGE GENERATION OF TWODIMENSIONAL DATA SEQUENCE IN GREY SYSTEM THEORY

An unequal time interval sequence or a sequence with blanks is usually completed with average generation in grey system theory. This paper discovers that there exists obvious errors when using average generation to generate internal points of non-consecutive neighbours. The average generation and the preference generation of the sequence are discussed, the concave and convex properties show the status of local sequence and propose a new idea for using the status to build up the criteria of choosing generation coefficient. Compared with the general average method of the one-dimensional data sequence, the two-dimensional data sequence is defined and its average generation is discussed, and the coefficient decision method for the preference generation is presented.

Minimal-Length Interoperability Test Sequences Generation via Genetic Algorithm

A novel interoperability test sequences optimization scheme is proposed in which the genetic algorithm （GA） is used to obtain the minimal-length interoperability test sequences. During our work, the basic interoperability test sequences are generated based on the minimal-complete-coverage criterion, which removes the redundancy from conformance test sequences. Then interoperability sequences minimization problem can be considered as an instance of the set covering problem, and the GA is applied to remove redundancy in interoperability transitions. The results show that compared to conventional algorithm, the proposed algorithm is more practical to avoid the state space explosion problem, for it can reduce the length of the test sequences and maintain the same transition coverage.