From the Cover: PNAS Plus: Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.DNA sequencing is a fundamental technology in the biological and medical sciences (1). Advances in sequencing technology have enabled the growth of interest in individualized medicine with the hope of better treating human disease. The cost of genome sequencing has dropped by five orders of magnitude over the last decade but still remains out of reach as a conventional clinical tool (2, 3). Thus, the development of new, high-throughput, accurate, low-cost DNA-sequencing technologies is a high priority. Ensemble sequencing-by-synthesis (SBS) platforms dominate the current landscape. During SBS, a DNA polymerase binds and incorporates a nucleotide analog complementary to the template strand. Depending on the instrumentation, this nucleotide is identified either by its associated label or the appearance of a chemical by-product upon incorporation (4). These platforms take advantage of a high-fidelity polymerase reaction but require amplification and have limited read lengths (5). Recently, single-molecule strategies have been shown to have great potential to achieve long read lengths, which is critical for highly scalable and reliable genomic analysis (6–9). Pacific Biosciences’ SMRT SBS approach has been used for this purpose but has lower throughput and higher cost compared with current second-generation technology (10).Since the first demonstration of single-molecule characterization by a biological nanopore two decades ago (11), interest has grown in using nanopores as sensors for DNA base discrimination. One approach is strand sequencing, in which each base is identified as it moves through an ion-conducting channel, ideally producing a characteristic current blockade event for each base. Progress in nanopore sequencing has been hampered by two physical limitations. First, single-base translocation can be too rapid for detection (1–3 μs per base), and second, structural similarities between bases make them difficult to identify unambiguously (12). Some attempts to address these issues have used enzymes as molecular motors to control single-stranded DNA (ssDNA) translocation speeds but still rely on identifying multiple bases simultaneously (13–15). Other approaches used exonuclease to cleave a single nucleoside-5′-monophosphate that then passes through the pore (16), or modified the pore opening with a cyclodextrin molecule to slow translocation and increase resolution for individual base detection (17, 18). All of these techniques rely on detecting similarly sized natural bases, which produce relatively similar current blockade signatures. Additionally, no strategies for covalently linking a single enzyme to a multimeric nanopore have been published.Recently, we reported a method for SBS with nanopore detection (19, 20). This approach has two distinct features: the use of nucleotides with specific tags to enhance base discrimination and a ternary DNA polymerase complex to hold the tagged nucleotides long enough for tag recognition by the nanopore. As shown in Fig. 1, a single DNA polymerase is coupled to a membrane-embedded nanopore by a short linker. Next, template and four uniquely tagged nucleotides are added to initiate DNA synthesis. During formation of the ternary complex, a polymerase binds to a complementary tagged nucleotide; the tag specific for that nucleotide is then captured in the pore. Each tag is designed to have a different size, mass, or charge, so that they generate characteristic current blockade signatures, uniquely identifying the added base. This system requires a single polymerase coupled to each nanopore to ensure any signal represents sequencing information from only one DNA template at a time. Kumar et al. (19) demonstrated that nucleotides tagged with four different polyethylene glycol (PEG) molecules at the terminal phosphate were good substrates for polymerase and that the tags could generate distinct signals as they translocate through the nanopore. These modifications enlarge the discrimination of the bases by the nanopore relative to the use of the natural nucleotides. We recently expanded upon this work by replacing the four PEG polymers with oligonucleotide-based tags and showed that a DNA polymerase coupled to the nanopore could sequentially add these tagged nucleotides to a growing DNA strand to perform Nanopore-SBS (20). Although this work showcased the promise of this technology, it did not describe in detail how to build a protein construct capable of Nanopore-SBS and did not obtain enough data to develop a statistical framework to uniquely distinguish the tagged nucleotides from each other.Open in a separate windowFig. 1.Principle of single-molecule DNA sequencing by a nanopore using tagged nucleotides. Each of the four nucleotides carries a different polymer tag (green square, A; red oval, T; blue triangle, C; black square, G). During SBS, the complementary nucleotide (T shown here) forms a tight complex with primer/template DNA and the nanopore-coupled polymerase. As the tagged nucleotides are incorporated into the growing DNA template, their tags, attached via the 5′-phosphate, are captured in the pore lumen, which results in a unique current blockade signature (Bottom). At the end of the polymerase catalytic reaction, the tag is released, ending the current blockade, which returns to open-channel reading at this time. For the purpose of illustration, four distinct tag signatures are shown in the order of their sequential capture. A large array of such nanopores could lead to highly parallel, high-throughput DNA sequencing.Here, we describe the design and characterization of a protein construct capable of carrying out Nanopore-SBS (Fig. 1). A porin attached to a single DNA polymerase molecule is inserted into a lipid bilayer formed on an electrode array. The polymerase synthesizes a new DNA strand using four uniquely tagged nucleotides. The DNA polymerase is positioned in such a way that when the ternary complex is formed with the tagged nucleotide, the tag is captured by the nanopore and identified by the resulting current blockade signature. We first describe the construction and purification of an α-hemolysin (αHL) heptamer covalently attached to a single ϕ29 DNA polymerase using the SpyTag/SpyCatcher conjugation approach (21), followed by binding of this conjugate with template DNA and its insertion into a lipid bilayer array. We confirm that this complex is stable and retains adequate pore and polymerase activities. We verify that the tagged nucleotides developed by Fuller et al. (20) can be bound by the polymerase and accurately discriminated by the nanopore. We develop an experimental approach and computational methods to uniquely and specifically distinguish true tagged-nucleotide captures from background and from other tagged nucleotides. We address ways that tagged-nucleotide captures may be misidentified and demonstrate approaches to correct for these. We further show this protein construct can capture tagged nucleotides during template-directed DNA synthesis in the presence of Mn²⁺, demonstrating its utility for Nanopore-SBS.     

A case of genotype‐3b hepatitis C virus in which the whole genome was successfully analyzed using third‐generation nanopore sequencing

A 42‐year‐old Chinese man with chronic hepatitis C virus (HCV) infection visited our hospital for antiviral therapy. The subgenotype could not be determined using the HCV GENOTYPE Primer Kit (Institute of Immunology, Tokyo, Japan), which can identify genotype 3a HCV exclusively among genotype 3 HCV. Thus, the whole‐genome sequence of HCV was analyzed using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK), a third‐generation single‐molecule sequencing platform. Consequently, a total of 9442 bases with a 73.6 mean depth, corresponding to the sequences between nt25 and PolyU/UC were determined (LC414155.2). The similarity analysis revealed that the obtained sequence was classified into genotype 3b HCV and showed nucleotide identities from 87.6% to 93.9% with those of 12 previously reported strains. Furthermore, possible resistance‐associated substitutions in non‐structural protein (NS)3, NS5A, and NS5B based on consensus sequences of 12 genotype 3b HCV strains, including NS5A‐Y93H and NS5B‐S282 T substitutions, were absent. In conclusion, the MinION nanopore sequencer is useful for analyzing the HCV genome, especially the genomes of genotype 3 HCV strains for which standardized real‐ time PCR methods for all subgenotypes have not been established.     

Structural characterization and functional effects of a circulating heparan sulfate in a patient with hepatocellular carcinoma

A circulating anticoagulant was isolated from the plasma of a 42-year-old man with cirrhosis and hepatocellular carcinoma who had an unusual coagulation test profile. The patient developed a fatal coagulopathy, unresponsive to protamine therapy or plasma exchange following liver biopsy. However, at presentation, routine hemostasis assays were normal. The patient had mucocutaneous bleeding but the sole laboratory abnormality was a prolonged thrombin time (TT = 99 s, normal 25–35 s). Protamine titration indicated activity equivalent to a heparin concentration of 6–7 U/ml. Antithrombin III (AT III) antigen and activity were markedly elevated. The anticoagulant activity, purified from plasma by DEAE chromatography, was identified as a glycosaminoglycan (GAG). GAG anti-thrombin activity was completely abolished by heparin lyase III. Based on the degree of sulfation and HPLC pattern, the GAG was classified as heparan sulfate. Low levels (4 μM) of purified GAG markedly prolonged the TT (>120 s) but not the activated partial thromboplastin time (PTT) (31.4 s). In a Factor Xa assay, the GAG exhibited a potency equivalent to 0.06 U of low molecular weight heparin per nmol of uronic acid. Patients with endogenous circulating glycosaminoglycans can present with unusual laboratory coagulation test profiles. These reflect complex dysfunction of hemostasis, leading to difficulty in providing diagnosis and effective care. Am. J. Hematol. 58:285–292, 1998. © 1998 Wiley-Liss, Inc.     

人博卡病毒全基因组序列测定与种系分析

目的了解福州地区人博卡病毒(HBoV)在儿童呼吸道感染中的检出情况,并对其进行全基因组序列测定和种系分析。方法收集2007年11月至2008年10月在福建省妇幼保健院因下呼吸道感染住院的重症监护病房的57例小儿鼻咽抽取物标本,用一对特异引物通过PCR扩增法对HBoV基因片段进行检测,对检测出的2例HBoV(FZ1和FZ40)用7对全序列引物进行扩增和拼接,获得这两株病毒全基因组序列,上传GenBank并与基因库中国内外其它10株HBoV的全基因组序列和各氨基酸序列进行比对,并做种系分析。结果FZ1株基因组序列全长为5299bp,与HBoV参考株st2株序列长度相同;而FZ40株的基因组序列全长少2bp。病毒全基因组编码4种蛋白,分别是非结构蛋白NS1、核蛋白NP-1和衣壳蛋白VP1、VP2。结论种系分析显示福州的FZ40株与浙江温岭的WLL-1株关系较近,而FZ1株与北京的两株及泰国的CU6株关系较近。     

STROBE-sequencing analysis of the vaginal microecology of 4- to 6-year-old girls in Southwest China

We investigated the vaginal flora diversity of preschool-aged (ie, 4–6-year-old) girls in southwest China.Fourteen preschool-aged girls were enrolled in this study. The statuses and differences in their vaginal flora were evaluated by Gram staining, bacterial culturing, and sequencing analysis.Gram staining and microbial culturing showed that the main vaginal flora of the preschool-aged girls were Gram-negative bacilli, whereas the main vaginal flora of healthy adult controls were large Gram-positive bacilli such as Lactobacillus crispatus. Shannon and Simpson indexes indicated that the bacterial diversity tended to decrease with age. The species abundance heat map showed that the vaginal microecology of the girls differed slightly at different ages but mainly comprised Pseudomonas, Methylobacterium, Sphingomona,s and Escherichia. The functional abundance heat map indicated that the bacterial functions increased with age.The vaginal microecology of preschool-aged girls differs from that of adults. A comprehensive understanding of the vaginal flora diversity of preschool-aged girls will aid in clinically diagnosing vulvovaginitis in preschool-aged girls.     

Added value of systemic inflammation markers in predicting pulmonary infection in stroke patients: A retrospective study by machine learning analysis

Exploring candidate markers to predict the clinical outcomes of pulmonary infection in stroke patients have a high unmet need. This study aimed to develop machine learning (ML)-based predictive models for pulmonary infection.Between January 2008 and April 2021, a retrospective analysis of 1397 stroke patients who had CT angiography from skull to diaphragm (including CT of the chest) within 24 hours of symptom onset. A total of 21 variables were included, and the prediction model of pulmonary infection was established by multiple ML-based algorithms. Risk factors for pulmonary infection were determined by the feature selection method. Area under the curve (AUC) and decision curve analysis were used to determine the model with the best resolution and to assess the net clinical benefits associated with the use of predictive models, respectively.A total of 889 cases were included in this study as a training group, while 508 cases were as a validation group. The feature selection indicated the top 6 predictors were procalcitonin, C-reactive protein, soluble interleukin-2 receptor, consciousness disorder, dysphagia, and invasive procedure. The AUCs of the 5 models ranged from 0.78 to 0.87 in the training cohort. When the ML-based models were applied to the validation set, the results also remained reconcilable, and the AUC was between 0.891 and 0.804. The decision curve analysis also showed performed better than positive line and negative line, indicating the favorable predictive performance and clinical values of the models.By incorporating clinical characteristics and systemic inflammation markers, it is feasible to develop ML-based models for the presence and consequences of signs of pulmonary infection in stroke patients, and the use of the model may be greatly beneficial to clinicians in risk stratification and management decisions.     

Predictive ability of current machine learning algorithms for type 2 diabetes mellitus: A meta‐analysis

Aims/IntroductionRecently, an increasing number of cohort studies have suggested using machine learning (ML) to predict type 2 diabetes mellitus. However, its predictive ability remains inconclusive. This meta‐analysis evaluated the current ability of ML algorithms for predicting incident type 2 diabetes mellitus.Materials and MethodsWe systematically searched longitudinal studies published from 1 January 1950 to 17 May 2020 using MEDLINE and EMBASE. Included studies had to compare ML’s classification with the actual incidence of type 2 diabetes mellitus, and present data on the number of true positives, false positives, true negatives and false negatives. The dataset for these four values was pooled with a hierarchical summary receiver operating characteristic and a bivariate random effects model.ResultsThere were 12 eligible studies. The pooled sensitivity, specificity, positive likelihood ratio and negative likelihood ratio were 0.81 (95% confidence interval [CI] 0.67–0.90), 0.82 [95% CI 0.74–0.88], 4.55 [95% CI 3.07–6.75] and 0.23 [95% CI 0.13–0.42], respectively. The area under the summarized receiver operating characteristic curve was 0.88 (95% CI 0.85–0.91).ConclusionsCurrent ML algorithms have sufficient ability to help clinicians determine whether individuals will develop type 2 diabetes mellitus in the future. However, persons should be cautious before changing their attitude toward future diabetes risk after learning the result of the diabetes prediction test using ML algorithms.     

Use of machine learning techniques in the development and refinement of a predictive model for early diagnosis of ankylosing spondylitis

Clinical Rheumatology - To develop a predictive mathematical model for the early identification of ankylosing spondylitis (AS) based on the medical and pharmacy claims history of patients with and...     

两株犬源狂犬病病毒街毒株全基因组序列测定与分析

目的对2株犬源狂犬病病毒街毒株全基因组测序,了解国内狂犬病病毒的流行和变异情况。方法采用乳鼠脑内接种方法分离2株天津地区狂犬病病毒街毒株,RT-PCR扩增病毒DNA覆盖全基因组12个相互重叠的基因片段,PCR产物平端克隆后进行全基因组核苷酸序列测定,然后对其分子变异和进化特点进行分析。进行两分离株的序列相似性、氨基酸同源性、主要功能位点的变异比较和种系发生分析。结果 TJD04和TJD05两株狂犬病病毒全基因组长均为11 924 nts,全基因组序列的组成和结构均符合弹状病毒科狂犬病病毒属的特征。病毒基因组由5个编码区组成,基因起始位点和终止位点高度保守,氨基酸编码长度未发生变异,5个编码蛋白序列较少发生变化,仅有个别主要功能位点发生变异,且大部分变异均属于无意义沉默突变。多序列相似性比较和种系发生分析显示,两株病毒均属于基因1型,N基因与全基因进化树保持一致,与中国犬源狂犬病病毒BD06同源性最高(99.6%),进化关系近,并具有较为独特的中国地域特点。结论两天津株狂犬病毒可能是自然界中固有的狂犬病病毒街毒株。     

C组轮状病毒主要基因全片段扩增及武汉地方株Wu82的VP6和VP7编码基因序列分析

目的建立C组轮状病毒VP6、VP7、VP4和NSP4基因全片段扩增方法;明确武汉地方株Wu82与其他毒株的系统发生关系。腹泻患者大便。方法PAGE筛查病毒;设计9对引物RT-PCR扩增VP6、VP7、VP4和NSP4基因;VP6、VP7序列及亲水位点分析。结果Wu82VP6和VP7基因核酸序列与其它人C组轮状病毒相比同源性分别为98.45%～97.12%和99.34%～94.83%。结论C组轮状病毒基因非常保守,进化缓慢。2001年武汉地方株Wu82与1995年武汉地方株208株相比,VP7和VP6抗原表位存在差异。     

九江市HIV-1流行毒株基因序列测定和亚型分析

目的了解九江市艾滋病病毒1型(HIV-1)流行毒株的亚型分布情况。方法对采集的6份九江市HIV-1感染者的血浆样品进行RT-PCR扩增,获得gag基因的核酸片断进行核苷酸序列分析。结果在6份样品中发现B’亚型HIV-1毒株4株,B亚型和A/E重组亚型HIV-1毒株各1株。结论HIV-1流行毒株亚型在九江市分布情况复杂,防控形势严峻。     

福建省登革1型病毒基因组序列测定及进化分析

目的对分离自福建省的1株登革1型病毒（FJ231／04）进行全基因组序列测定，并同其它病毒株全基因序列进行比较，了解其序列特征和可能的传播来源。方法采取分段扩增、克隆、测序的方案，设计10对引物分别扩增不同的片段，基因组5’末端序列采用RACE技术进行扩增，扩增产物随后克隆到质粒载体并测序。通过末端重叠序列进行拼按后组成全长病毒基因组序列。结果拼接后的病毒全基因组全长10735nt，编码一个长的开放读码框。参考已公布的登革1型病毒的全基因序列，对该病毒株进行进化分析。序列的进化分析表明，该福建省分离株属登革1型病毒中的第1群（基因1型）病毒。在遗传距离上，该毒株和2002年的泰国分离株最为接近（差异为0．53％）。根据病毒发现时间及其核酸差异，推测该病毒可能是通过在东南亚一带感染者或病人带入福建。结论通过对登革病毒金基因组序列的测定可以获得毒株的特征。此外病毒序列的进化分析，还可为了解病毒可能的来源以及传播途径，正确了解登革病毒在我省的流行病学概况提供重要的线索。     

Genome Profiling of SARS-CoV-2 in Indonesia,ASEAN and the Neighbouring East Asian Countries: Features,Challenges and Achievements

Whole-genome sequencing (WGS) has played a significant role in understanding the epidemiology and biology of SARS-CoV-2 virus. Here, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asian countries as a genomic surveillance during the COVID-19 pandemic. Nottingham–Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative has facilitated collaboration between the University of Nottingham and a team in the Research Center for Biotechnology, National Research and Innovation Agency (BRIN), to carry out a small number of SARS-CoV-2 WGS in Indonesia using Oxford Nanopore Technology (ONT). Analyses of SARS- CoV-2 genomes deposited on GISAID reveal the importance of clinical and demographic metadata collection and the importance of open access and data sharing. Lineage and phylogenetic analyses of two periods defined by the Delta variant outbreak reveal that: (1) B.1.466.2 variants were the most predominant in Indonesia before the Delta variant outbreak, having a unique spike gene mutation N439K at more than 98% frequency, (2) Delta variants AY.23 sub-lineage took over after June 2021, and (3) the highest rate of virus transmissions between Indonesia and other countries was through interactions with Singapore and Japan, two neighbouring countries with a high degree of access and travels to and from Indonesia.