Diagnostic Value of Targeted Next-generation Sequencing in Pulmonary Mycobacterial Infections

Objective This study aimed to explore the diagnostic value of novel technique-targeted next-generation sequencing(tNGS)of bronchoalveolar lavage fluid(BALF)in pulmonary mycobacterial infections.Methods This retrospective study was conducted on patients who underwent bronchoscopy and tNGS,smear microscopy,and mycobacterial culture of BALF.Patients with positive Mycobacterium tuberculosis(MTB)culture or GeneXpert results were classified into the tuberculosis case group.Those diagnosed with nontuberculous mycobacteria(NTM)-pulmonary disease(NTM-PD)composed the case group of NTM-PD patients.The control group comprised patients without tuberculosis or NTM-PD.Sensitivity,specificity,and receiver operating characteristic(ROC)curves were used to evaluate the diagnostic performance.Results For tuberculosis patients with positive mycobacterial culture results,the areas under the ROC curves(AUCs)for tNGS,GeneXpert,and smear microscopy were 0.975(95%CI:0.935,1.000),0.925(95%CI:0.859,0.991),and 0.675(95%CI:0.563,0.787),respectively.For tuberculosis patients with positive GeneXpert results,the AUCs of tNGS,culture,and smear microscopy were 0.970(95%CI:0.931,1.000),0.850(95%CI:0.770,0.930),and 0.680(95%CI:0.579,0.781),respectively.For NTM-PD,the AUCs of tNGS,culture,and smear-positive but GeneXpert-negative results were 0.987(95%CI:0.967,1.000),0.750(95%CI:0.622,0.878),and 0.615(95%CI:0.479,0.752),respectively.The sensitivity and specificity of tNGS in NTM-PD patients were 100%and 97.5%,respectively.Conclusion tNGS demonstrated superior diagnostic efficacy in mycobacterial infection,indicating its potential for clinical application.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

Objective:Plant-derived cytotoxic transgene expression,such as trichosanthin(tcs),regulated by recombinant adeno-associated virus(r AAV)vector is a promising cancer gene therapy.However,the cytotoxic transgene can hamper the vector production in the r AAV producer cell line,human embryonic kidney(HEK293)cells.Here,we explored micro RNA-122(miR122)and its target sequence to limit the expression of the cytotoxic gene in the r AAV producer cells.Methods:A miR122 target(122 T)sequence was incorporated into the 30 untranslated region of the tcs c DNA sequence.The firefly luciferase(fluc)transgene was used as an appropriate control.Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells.The effects of miR122 overexpression on cell growth,transgene expression,and r AAV production were determined.Results:The presence of 122 T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line(in vitro),fresh human hepatocytes(ex vivo),and mouse liver(in vivo).Also,the normal liver physiology was unaffected by delivery of 122 T sequence by r AAV vectors.Compared with the parental cells,the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122 T,as well as the ability to produce liver-targeting r AAV vectors.Fascinatingly,the yield of r AAV vectors carrying the tcs-122 T gene was increased by 77.7-fold in HEK293-mir122 cells.Moreover,the tcs-122 T-containing r AAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.Conclusion:HEK293-mir122 cells along with the 122 T sequence provide a potential tool to attenuate the cytotoxic transgene expression,such as tcs,during r AAV vector production.

Research on Financial Targets Sequence of Impacting on Business Value—— Based on Data Analysis from Chinese High-tech Listed Firms of Unmodified Auditing Opinions

The high-tech listed firms are not only representative for mainstream of nongovernmental businesses, but also afflux new livingness into the capital market development in China. By considering our country＇s high-tech listed firms as study object, we have disclosed different degree between diverse financial targets and business value, built a financial targets sequence model of impact on business value. The study supplies a certain extent reference to valid supervising and exalting of firms＇ financial core.

Construction of SNP genetic maps based on targeted next-generation sequencing and QTL mapping of vital agronomic traits in faba bean(Vicia faba L.)

Owing to the limitation of a large genome size(~13 Gb),the genetic and gene mapping studies on faba bean(Vicia faba L.)are lagging far behind those for other legumes.In this study,we selected three purified faba bean lines(Yundou 8137,H0003712,and H000572)as parents and constructed two F2 populations.These two F2 populations,namely 167 F2 plants in Pop1(Yundou 8137×H0003712)and 204 F2 plants in Pop2(H000572×Yundou 8137),were genotyped using a targeted next-generation sequencing(TNGS)genotyping platform,and two high-density single nucleotide polymorphisms(SNP)genetic linkage maps of faba bean were constructed.The map constructed from Pop1 contained 5103 SNPs with a length of 1333.31 cM and an average marker density of 0.26 cM.The map constructed from Pop2 contained 1904 SNPs with a greater length of 1610.61 cM.In these two F2 populations,QTL mapping identified 98 QTLs for 14 agronomic traits related to the flowers,pods,plant types and grains.The two maps were then merged into an integrated genetic linkage map containing 6895 SNPs,with a length of 3324.48 cM.These results not only lay the foundation for fine mapping and map-based cloning of related genes,but can also accelerate the molecular marker-assisted breeding of faba bean.

Omadacycline in the treatment of scrub typhus:Three case reports

BACKGROUND Scrub typhus is a naturally occurring acute infectious disease that is primarily transmitted through the bites of chiggers or larval mites infected by Orientia tsutsugamushi(O.tsutsugamushi).Omadacycline,a novel tetracycline,exhibits potent antibacterial efficacy against both typical bacteria and atypical pathogens.However,omadacycline application in the treatment of scrub typhus remains limited.CASE SUMMARY In the present work,we report several cases of scrub typhus,with the main clinical symptoms being fever,the formation of eschars or ulcers,local or systemic lymphadenopathy,headache,myalgia and rash.Blood samples were collected before omadacycline was administered,and O.tsutsugamushi infection was confirmed through targeted next-generation sequencing(tNGS).After two days of treatment,the patients’symptoms,including fever,were alleviated,with no adverse drug reactions.CONCLUSION tNGS is an effective method for diagnosing scrub typhus.Omadacycline can be considered an alternative option for antiinfective therapy in patients with O.tsutsugamushi infections.

VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens

Objective Unbiased next generation sequencing（NGS） is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. Methods A viral Sequence Independent Targeted Amplification（VSITA） approach using a set of non-ribosomal and virus-enriched octamers（V8） was developed and compared with traditionally used random hexamers（N6）. Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR（qP CR）. Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. Results A minimum 30 hexamers matching to viral reference sequences（sense and antisense） were selected from a dataset of random 4,096（4~6） hexamers（N6）. Two random nucleotides were added to the 5＇ end of the selected hexamers, and 480（30 × 4~2） octamers（V8） were obtained. In general, VSITA approach showed higher enrichment of virus-targeted c DNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. Conclusion The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.

Identification of 8 Rare Deleterious Variants in ADAMTS13 by Next-generation Sequencing in a Chinese Population with Thrombotic Thrombocytopenic Purpura

Objective Thrombotic thrombocytopenic purpura(TTP)is a rare and fatal disease caused by a severe deficiency in the metalloprotease ADAMTS13 and is characterized by thrombotic microangiopathy.The present study aimed to investigate the genes and variants associated with TTP in a Chinese population.Methods Target sequencing was performed on 220 genes related to complements,coagulation factors,platelets,fibrinolytic,endothelial,inflammatory,and anticoagulation systems in 207 TTP patients and 574 controls.Subsequently,logistic regression analysis was carried out to identify the TTP-associated genes based on the counts of rare deleterious variants in the region of a certain gene.Moreover,the associations between common variants and TTP were also investigated.Results ADAMTS13 was the only TTP-associated gene(OR=3.77;95%CI:1.82–7.81;P=3.6×10^(-4))containing rare deleterious variants in TTP patients.Among these 8 variants,5 novel rare variants that might contribute to TTP were identified,including rs200594025,rs782492477,c.T1928G(p.I643S),c.3336_3361del(p.Q1114Afs*20),and c.3469_3470del(p.A1158Sfs*17).No common variants associated with TTP were identified under the stringent criteria of correction for multiple testing.Conclusion ADAMTS13 is the primary gene related to TTP.The genetic variants associated with the occurrence of TTP were slightly different between the Chinese and European populations.

Co-infection of Chlamydia psittaci and Tropheryma whipplei: A case report

BACKGROUND The co-infection of Chlamydia psittaci(C.psittaci)and Tropheryma whipplei(T.whipplei)is unusual,and the detection of pathogenic microorganisms is particularly important for patients with severe diseases or poor experience in treatment.Early identification of pathogens can significantly improve the prognosis of the patients.Targeted next-generation sequencing(tNGS)is currently widely used in clinical practice for various infectious diseases,including respiratory infections,to achieve early,accurate,and rapid microbial diagnosis.CASE SUMMARY We report a case of a 40-year-old female patient with a history of contact with parrots who was diagnosed with C.psittaci and T.whipplei infection through bronchial lavage fluid targeted next generation sequencing.After moxifloxacin treatment,the patient's symptoms improved significantly,and the imaging changes were obviously resolved.CONCLUSION Coinfection with C.psittaci and T.whipplei is not common.In this case,timely and accurate identification of both pathogens was achieved using tNGS.Moreover,the efficacy of monotherapy with moxifloxacin was confirmed.

Formation Conditions and Exploration-favoring Areas of Subtle Reservoirs in West Taibei Sag

At the initial stage of subtle reservoir exploration in the shallow to medium formations of the Taibei Sag, Tuha Basin, so far, three kinds of subtle reservoirs (stratigraphic, lithologic and structural-lithologic reservoirs) have been found. Since both the amount and reserve of the discovered subtle reservoirs are small, a great exploration potential remains in the Taibei Sag. The shallow to medium formations in the west of the Taibei Sag were divided into three second-order sequences and ten third-order sequences, of which the rising cyclothems of SQ(J2q) and SQ8 (J2s2) datum level are major exploration targets for the subtle reservoirs (lithologic). The depositional systems of Qiquanhu and Subashi braided river delta were developed there, where the sedimentary sand body is dominated by the regional slopes and slope break belts. There are four main modes the formation of lithologic traps: deltaic front onlap on the slope belt, deltaic front toplap on the slope break belt, lately tilted frontal sand body and sublacustrine fan—slope fan on the slope break belt, of which the first three modes are the major styles of the formation of subtle reservoirs. Major targets for subtle reservoir exploration in the near future include Putaogou Member on the north slope of Huoyanshan, the east slope of Pubei, the nose-like palaeohigh regions of Huobei, Subashi and Lianmuqin, around the Shengbei secondary sag.

Isolation and Identification of Porcine Circovirus Type 2 in Taizhou

In September 2011, an infectious disease suspected to be postweaning multisystemic wasting syndrome （PMWS） broke out in some pig farm in Taizhou. The inguinal lymph node, liver and lung tissues were collected and grinded into tissue suspension. The suspension was subjected to PCR detection, and the positive product was sequenced. The suspension of positive samples was filtered with 0.22 μm filter membrane, and the filtrate was inoculated onto PK15 cells. After five generations of blind passages, the cell viral liquid was collected and extracted for DNA, which was subjected to PCR detection and indirect immunofluorescence. The results showed that the isolate was porcine circovirus type 2 （PCV2） and designated as TAIZ110926. The target sequence was committed to NCBI with a serial number： KF039888.

Comprehensive analysis of genetic variations in strictly-defined Leber congenital amaurosis with whole-exome sequencing in Chinese

AIM：To make a comprehensive analysis of the potential pathogenic genes related with Leber congenital amaurosis（LCA） in Chinese.METHODS：LCA subjects and their families were retrospectively collected from 2013 to 2015.Firstly,whole-exome sequencing was performed in patients who had underwent gene mutation screening with nothing found,and then homozygous sites was selected,candidate sites were annotated,and pathogenic analysis was conducted using softwares including Sorting Tolerant from Intolerant（SIFT）,Polyphen-2,Mutation assessor,Condel,and Functional Analysis through Hidden Markov Models（FATHMM）.Furthermore,Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of pathogenic genes were performed followed by co-segregation analysis using Fisher exact Test.Sanger sequencing was used to validate single-nucleotide variations（SNVs）.Expanded verification was performed in the rest patients.RESULTS：Totally 51 LCA families with 53 patients and24 family members were recruited.A total of 104 SNVs（66 LCA-related genes and 15 co-segregated genes）were submitted for expand verification.The frequencies of homozygous mutation of KRT12 and CYP1A1 were simultaneously observed in 3 families.Enrichment analysis showed that the potential pathogenic genes were mainly enriched in functions related to cell adhesion,biological adhesion,retinoid metabolic process,and eye development biological adhesion.Additionally,WFS7 and STAU2 had the highest homozygous frequencies.CONCLUSION：LCA is a highly heterogeneous disease.Mutations in KRT12,CVP1A1,WFS1,and STAU2 may be involved in the development of LCA.

Nanopore techniques as a potent tool in the diagnosis and treatment of endophthalmitis:a literature review

Endophthalmitis is a serious ophthalmic disease characterized by changes in the eye's posterior segment,such as hypopyon and intraocular inflammation,vitritis being a hallmark.Infection-caused endophthalmitis can lead to irreversible vision loss,accompanied by eye pain or eye distention,and in the most severe cases the removal of the eyeball.Microorganisms such as bacteria,fungi,viruses,and parasites typically account for the disease and the entry pathways of the microbial can be divided into either endogenous or exogenous approaches,according to the origin of the etiological agents.Exogenous endophthalmitis can be derived from various occasions(such as postoperative complications or trauma)while endogenous endophthalmitis results from the bloodstream which carries pathogens to the eye.This review aims to summarize the application of new technology in pathogen identification of endophthalmitis so as to prevent the disease and better guide clinical diagnosis and treatment.

A systematic approach for synthesizing a low-temperature distillation system

In this paper, by combining a stochastic optimization method with a refrigeration shaft work targeting method,an approach for the synthesis of a heat integrated complex distillation system in a low-temperature process is presented. The synthesis problem is formulated as a mixed-integer nonlinear programming(MINLP) problem,which is solved by simulated annealing algorithm under a random procedure to explore the optimal operating parameters and the distillation sequence structure. The shaft work targeting method is used to evaluate the minimum energy cost of the corresponding separation system during the optimization without any need for a detailed design for the heat exchanger network(HEN) and the refrigeration system(RS). The method presented in the paper can dramatically reduce the scale and complexity of the problem. A case study of ethylene cold-end separation is used to illustrate the application of the approach. Compared with the original industrial scheme, the result is encouraging.

A comparative genomics analysis of lung adenocarcinoma for Chinese population by using panel of recurrent mutations

Previous studies have demonstrated that Chinese lung adenocarcinoma(LUAD)patients have unique genetic characteristics,however,the specific genomic features relating to the development and treatment of LUAD in the Chinese population are not fully understood.Here,we applied the ultra-deep targeted sequencing to 66 Chinese LUAD samples,accompanied by comparative analysis with 162 Caucasian LUAD in The Cancer Genome Atlas.We focused on the 68 recurrently mutated genes and results revealed that the panel-based tumor mutational burden(pTMB)is significantly higher in the Chinese LUAD(P=0.0017).Additionally,the percentage of smoking-associated C>A transversion is significantly lower in Chinese LUAD(15.5%vs.39.7%,P=5.69×10^(-27)),while C>T transition is more frequent in Chinese LUAD(35.8%vs.25.7%,P=2.67×10^(-5)),which indicated the ethnic difference in mutation types.Notably,novel driver genes(GNAS and JAK1)that are peculiar to Chinese LUAD were identified,and a more convergent distribution of mutations was observed in the Chinese cohort(P=0.012)compared with scattered mutations in Caucasian LUAD.Our results present a distinct genomic profile of Chinese LUAD compared to Caucasians LUAD and elucidate the ethnic difference in mutation distribution besides the type and rate.

A Few Questions that Should be Clarified in the Polymerase Chain Reaction

The polymerase chain reaction is one of the most useful technical ad- vance and inventions in modern molecular biology. Developed in 1983 by Kary Mullis, PCR is now a common and indispensable technique used in medical and bi- ology research labs for a variety of applications. A large number of articles relat- ed to PCR are available on the internet and other places. People know well about the basic principle and are very familiar with the procedures of the PCR. But, some details were neglected on the numbers of the target sequence and other DNA strands number after 30 to 35 cycles of the PCR. In most papers, the number of newly synthesized DNA strands including target DNA and non target DNA is am- biguous and even wrong. In this paper, highlights were given to the theoretical number of target DNA number in details and the exact number of the target DNA number can be concluded by analysis.

The next generation of cancer management

As readers of Cancer Biology and Medicine well know,there has been a seismic shift in human molecular biology over the past few years,as momentous in its own way as the discovery of the double-helical structure of DNA by Watson and Crick 60 years ago,the elucidation of the genetic code shortly thereafter,the advent of recombinant DNA and gene cloningin the 1970s, and the introduction of the polymerase chain reaction in the mid-1980s.

Genetic Susceptibility Study of Chinese Sudden Sensorineural Hearing Loss Patients with Vertigo

Objective To investigate the genetic causes of sudden sensorineural hearing loss(SSNHL)patients in China.This study focused on analyzing variations of coding sequence of common genes related to deafness,revealing the molecular pathogenesis of sudden deafness from a genomics perspective,discovering molecular markers associated with the onset of deafness,and then supplying prevention to high-risk populations,classifying disease according to accurate etiology,and choosing a much more precision therapy.Methods We retrospectively analyzed the clinical characteristics of 51 patients diagnosed as SSNHL with vertigo treated in the Chinese PLA General Hospital.In this study,mutation screening of 307 nuclear genes and mitochondrial genome responsible for human or mouse deafness was performed on the 51 cases of unilateral sudden deafness patients with vertigo.Results We identified 51 cases of unilateral sudden deafness,including 2 cases of low-mid frequency hearing impairment,18 cases of mid-high frequency hearing loss,11 cases of flat-type hearing loss,and 20 cases of all frequency hearing loss.Among the 51 cases,8(15.69%)cases of GJB2 heterozygous variations,1(1.96%)case of GJB3 heterozygous variations,5(9.8%)cases of SLC26A4 heterozygous variations,2(3.92%)cases of COCH heterozygous variations,14(27.45%)cases of CDH23 heterozygous variations,14(27.45%)cases of OTOF heterozygous variations,1(1.96%)case of SLC17A8 heterozygous variations and 2(3.92%)cases of KCNE1 heterozygous variations.No mtDNA gene variations were identified.Conclusion SSNHL has some relationship with hereditary in Chinese population,but its complex genetic pathogenic mechanisms need further study.

Targeted deep sequencing reveals the genetic heterogeneity in well-differentiated pancreatic neuroendocrine tumors with liver metastasis

Background:Pancreatic neuroendocrine tumor is a rare and heterogeneous entity,and approximately half of the patients harbored liver metastasis when initially diagnosed,whose prognosis is dismal.High-throughput sequencing has largely uncovered the genomic features of pancreatic neuroendocrine tumor,but the genetic alterations in the metastatic cases remain relatively unclear,which we aimed to study.Methods:Pathologically confirmed well-differentiated pancreatic neuroendocrine tumor samples resected in our hospital from 2000 to 2019 were collected.We performed deep sequencing on the exome of 341 tumor-related genes,and compared the differences of genetic alterations between the metastatic and the non-metastatic cases,as well as between the primary and the paired liver metastatic tumors.Results:Sequencing data of 79 samples from 29 pancreatic neuroendocrine tumor patients were included into analysis.A total of 2,471 somatic variants were identified,75.5%of which were considered as low-abundance.NOTCH1 was the most frequently mutated gene,altered in 26(53.1%)pancreatic neuroendocrine tumor samples from 18(62.1%)patients.Compared with the non-metastatic pancreatic neuroendocrine tumors,the metastatic cases were discovered with more single nucleotide variants and copy number variations,indicating the increased genomic instability.In addition,among the paired metastatic cases,the primary and the metastatic lesions shared limited mutated genes.Conclusions:Through the targeted deep sequencing,we identified the intratumor,intraindividual,and interindividual heterogeneity in the pancreatic neuroendocrine tumor patients,particularly in the metastatic cases,bringing potential challenges for the current biopsy strategies in guiding clinical treatments.

Newborn screening with targeted sequencing:a multicenter investigation and a pilot clinical study in China

Different newborn screening(NBS) programs have been practiced in many countries since the 1960 s. It is of considerable interest whether next-generation sequencing is applicable in NBS. We have developed a panel of 465 causative genes for 596 early-onset, relatively high incidence, and potentially actionable severe inherited diseases in our Newborn Screening with Targeted Sequencing(NESTS) program to screen 11,484 babies in 8 Women and Children’s hospitals nationwide in China retrospectively. The positive rate from preliminary screening of NESTS was 7.85%(902/11,484). With 45.89%(414/902) follow-up of preliminary positive cases, the overall clinically confirmative diagnosis rate of monogenic disorders was 12.07%(50/414), estimating an average of 0.95%(7.85% × 12.07%) clinical diagnosis rate, suggesting that monogenic disorders account for a considerable proportion of birth defects. The disease/gene spectrum varied in different regions of China. NESTS was implemented in a hospital by screening 3923 newborns to evaluate its clinical application. The turn-around time of a primary report, including the sequencing period of < 7 days, was within 11 days by our automatic interpretation pipeline. Our results suggest that NESTS is feasible and cost-effective as a first-tier NBS program, which will change the status of current clinical practice of NBS in China.

Development of high-resolution multiple-SNP arrays for genetic analyses and molecular breeding through genotyping by target sequencing and liquid chip

Genotyping platforms,as critical supports for genomics,genetics,and molecular breeding,have been well implemented at national institutions/universities in developed countries and multinational seed companies that possess high-throughput,automatic,large-scale,and shared facilities.In this study,we integrated an improved genotyping by target sequencing(GBTS)system with capture-in-solution(liquid chip)technology to develop a multiple single-nucleotide polymorphism(mSNP)approach in which mSNPs can be captured from a single amplicon.From one 40K maize mSNP panel,we developed three types of markers(40K mSNPs,251K SNPs,and 690K haplotypes),and generated multiple panels with various marker densities(1K–40K mSNPs)by sequencing at different depths.Comparative genetic diversity analysis was performed with genic versus intergenic markers and di-allelic SNPs versus non-typical SNPs.Compared with the one-amplicon-one-SNP system,mSNPs and within-mSNP haplotypes are more powerful for genetic diversity detection,linkage disequilibrium decay analysis,and genome-wide association studies.The technologies,protocols,and application scenarios developed for maize in this study will serve as a model for the development of mSNP arrays and highly efficient GBTS systems in animals,plants,and microorganisms.