Detection Of Single Nucleotide Polymorphisms Research Articles

Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems.

Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how “self-avoiding molecular recognition systems” (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases. This should allow primers holding SAMRS components to avoid primer–primer interactions, preventing primer dimers, allowing more sensitive SNP detection, and supporting higher levels of multiplex PCR. The experiments here examine the PCR performances of primers containing different numbers of SAMRS components placed strategically at different positions, and put these performances in the context of estimates of SAMRS:standard pairing strengths. The impact of these variables on primer dimer formation, the overall efficiency and sensitivity of SAMRS-based PCR, and the value of SAMRS primers when detecting single nucleotide polymorphisms (SNPs) are also evaluated. With appropriately chosen polymerases, SNP discrimination can be greater than the conventional allele-specific PCR, with the further benefit of avoiding primer dimer artifacts. General rules guiding the design of SAMRS-modified primers are offered to support medical research and clinical diagnostics products.

Thiopurine S-methyltransferase genetic polymorphisms in adult patients with inflammatory bowel diseases in the Latvian population.

Background:Thiopurine methyltransferase (TPMT) plays a significant role in the metabolism of thiopurines, and, for patients with inflammatory bowel disease (IBD), it is useful to perform TPMT genotyping prior to azathioprine (AZA) treatment. In this study, we determined TPMT gene polymorphisms in a cohort of IBD patients in Latvia.Methods:DNA samples were obtained from 244 IBD patients, and qPCR was performed for detection of rs1800462, rs1800460, and rs1142345 single-nucleotide polymorphisms (SNPs). Three common, non-functional TPMT alleles (TPMT*2, *3B, and *3C) were identified (women, 51%; men, 49%). TPMT*2, *3A, *3B, and *3C allelic variants detected using qPCR were consistent with restriction fragment length polymorphism (RFLP) data.Results:Among patients, 78% had ulcerative colitis and 22% had Crohn’s disease, with 93.9% of the former carrying a wild-type homozygous TPMT*1/*1 genotype and 6.1% carrying heterozygous genotypes. The most frequent polymorphisms were TPMT*1/*3A (5.3%: two variants: TPMT*3B and TPMT*3C), TPMT*1/*3C (0.4%), and TPMT*1/*2 (0.4%). None of the patients carried a TPMT*3B polymorphism and no patients were homozygous for any mutation.Conclusion:This is the first study to identify TPMT gene polymorphisms in adult IBD patients in Latvia. The results indicate that the frequency of common TPMT alleles is similar to that of other European populations.

Nucleotide Identification in DNA Using Dielectrophoresis Spectroscopy.

We show that negative dielectrophoresis (DEP) spectroscopy is an effective transduction mechanism of a biosensor for the detection of single nucleotide polymorphism (SNP) in a short DNA strand. We observed a frequency dependence of the negative DEP force applied by interdigitated electrodes to polystyrene microspheres (PM) with respect to changes in both the last and the second-to-last nucleotides of a single-strand DNA bound to the PM. The drift velocity of PM functionalized to single-strand DNA, which is proportional to the DEP force, was measured at the frequency range from 0.5 MHz to 2 MHz. The drift velocity was calculated using a custom-made automated software using real time image processing technique. This technology for SNP genotyping has the potential to be used in the diagnosis and the identification of genetic variants associated with diseases.

One-Step Synthesis of Tunable Zinc-Based Nanohybrids as an Ultrasensitive DNA Signal Amplification Platform

Herein, we demonstrated a one-step route for the manufacturing of polypyrrole (PPy)/zinc nanohybrids with tunable elemental composition and nanoscale component mixing resolution by using ultrafast (within tens of seconds) microwave approach for ultrasensitive DNA biosensors. The zinc based nanoparticles (i.e. MWPPy/ZnO and MWPPy/ZnS) were produced by loading zinc acetate (ZnAc2) on PPy under the electromagnetic environment of microwave with or without sulfur powder in one pot. Then, the signal amplification platforms were fabricated by modifying the glassy carbon electrode (GCE) with the obtained nanohybrids. It was found that both of the resultant MWPPy/ZnO and MWPPy/ZnS were suitable for ultrasensitive DNA molecules detection of gastric carcinoma related PIK3CA gene ascribing to their unique hybrid nanostructures and surface characteristics. Experimental results revealed that the proposed GCE/MWPPy/ZnO sensor showed a linear range of 1.0 × 10-10-1.0 × 10-13 M. Notably, GCE/MWPPy/ZnS sensor was endowed with promising DNA hybrids selection with a minimum concentration response of 1.0 × 10-18 M. The corresponding detection limit was respectively found to be 2.90 × 10-11 M and 7.73 × 10-21 M for MWPPy/ZnO and MWPPy/ZnS based biosensors. Furthermore, reliable determination of single-base and two-base mismatched DNA are more attractive, which greatly supported the application of the constructed zinc based nanohybrids for the detection of single nucleotide polymorphism in genetic diseases, biological infectious pathogens or warning against bio-warfare agents.

Obstrüktif Uyku Apne Sendromuna Sahip Türk Hastalarda Vitamin D Reseptör Polimorfizmlerinin İncelenmesi

Objective: Numerous studies investigating Vitamin D receptor (VDR) polymorphisms in different populations have been present in current literature. For this reason, we designed a study to investigate the role of three known common VDR genetic polymorphisms (Apa-I/rs7975232, Bsm-I/ rs1544410 and Taq-I/rs731236) in Turkish individuals affected by Obstructive sleep apnea syndrome (OSAS). Methods: The study was carried out on a total of 175 consecutive subjects, including 80 OSAS patients and 95 healthy participants. Single Nucleotide Polymorphism (SNP) Detection was performed with the iPLEX® Assay and the MassARRAY® System for detection of Apa-I (rs7975232), Bsm-I (rs1544410), and Taq-I (rs731236) polymorphisms. Results: Fifty-seven C alleles (71.3%) and 59 T alleles (73.8%) were detected in the group of 80 OSAS patients in terms of rs1544410 polymorphism. When the patient and control groups were evaluated statistically at allelic level, it was observed that the T allele increased the risk of disease and this increase was statistically significant (Odds Ratio (OR) = 1.549 [Confidence Interval (CI): 1.012-2.371], (p=0.043). However, there were no significant differences between other VDR polymorphisms (rs7975232 and rs731236) and OSAS clinical data (p=0.6 and p=0.9, respectively). Discussion: As a conclusion, no statistically significant relationship was found between all three VDR polymorphisms and OSAS patients' clinical features. Further studies should be performed by creating a large sampling group. Finally, population studies should be given importance considering the variability of polymorphism according to ethnic origin.

Exonuclease III-Regulated Target Cyclic Amplification-Based Single Nucleotide Polymorphism Detection Using Ultrathin Ternary Chalcogenide Nanosheets

Herein, we report that the ternary chalcogenide nanosheet exhibits different affinity toward oligonucleotides with different lengths and efficiently quenches the fluorescence of dye-labeled DNA probes. Based on these findings, as a proof-of-concept application, the ternary chalcogenide nanosheet is used as a target cyclic amplification biosensor, showing high specificity in discriminating single-base mismatch. This simple strategy is fast and sensitive for the single nucleotide polymorphism detection. Ultralow detection limit of unlabeled target (250 fM) and high discrimination ratio (5%) in the mixture of perfect match (mutant-type) and single-base mismatch (wild-type) target are achieved. This sensing method is extensively compatible for the single nucleotide polymorphism detection in clinical samples, making it a promising tool for the mutation-based clinical diagnostic and genomic research.

Exploring the Potential and Limitations of PCR-RFLP and PCR-SSCP for SNP Detection: A Review

Polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) and PCR–restriction fragment length polymorphism (PCR-RFLP) are two independent methods used in the post-amplification genotyping of DNA variations. Both techniques are used in a wide range of screening applications to characterize single nucleotide polymorphisms (SNPs). The PCR-SSCP enables the identification of a potentially causative unknown SNP that could not be identified by PCR-RFLP. However, because complicated steps are not required to perform PCR-RFLP, it is used in many applications. On the other hand, PCR-RFLP is easier to process in terms of time and handover experience, the detection of a particular unknown SNP by PCR-SSCP has further chances. The simplicity of PCR-RFLP does not mean that it is better than PCR-SSCP. The reason is the limited ability of PCR-RFLP to detect nucleotide variations, which often go undetected because each restriction enzyme (RE) scans only a few recognition sequences, and other sequences are ignored. Furthermore, the efficacy of PCR-SSCP is sometimes hindered by many optimizations and also lack of experience. As PCR-SSCP allows other sequences within an amplicon to be separated and characterized, the choice between PCR-RFLP and PCR-SSCP is largely dependent on the reason for each genotyping experiment. This review provides a useful guide for comparing PCR-RFLP and PCR-SSCP in terms of their concepts, efficiency, ease of use, interpretation, and sensitivity as well as several other parameters. The comparison is extended to the practical applications of both techniques in terms of their utilization in molecular diagnostics and related applications.

Genome-Wide Association Studies for Methane Production in Dairy Cattle.

Genomic selection has been proposed for the mitigation of methane (CH4) emissions by cattle because there is considerable variability in CH4 emissions between individuals fed on the same diet. The genome-wide association study (GWAS) represents an important tool for the detection of candidate genes, haplotypes or single nucleotide polymorphisms (SNP) markers related to characteristics of economic interest. The present study included information for 280 cows in three dairy production systems in Mexico: 1) Dual Purpose (n = 100), 2) Specialized Tropical Dairy (n = 76), 3) Familiar Production System (n = 104). Concentrations of CH4 in a breath of individual cows at the time of milking (MEIm) were estimated through a system of infrared sensors. After quality control analyses, 21,958 SNPs were included. Associations of markers were made using a linear regression model, corrected with principal component analyses. In total, 46 SNPs were identified as significant for CH4 production. Several SNPs associated with CH4 production were found at regions previously described for quantitative trait loci of composition characteristics of meat, milk fatty acids and characteristics related to feed intake. It was concluded that the SNPs identified could be used in genomic selection programs in developing countries and combined with other datasets for global selection.

The analysis of pharmacokinetic and pharmacogenomic impact on gefitinib efficacy in advanced non-small cell lung cancer patients: results from a prospective cohort study.

The current study is aimed to examine the impact of pharmacokinetics and gene polymorphisms of enzymes involving in absorption, distribution, metabolism and excretion (ADME) on the efficacy of gefitinib in non-small cell lung cancer (NSCLC) patients. Eligible patients with indication of gefitinib treatment were prospectively enrolled in this study. Two peripheral blood samples at baseline and before cycle 2 day 1 were collected for the detection of single nucleotide polymorphisms (SNPs) of drug ADME enzymes and trough drug concentration (Ctrough) at steady state. Thirteen SNPs were genotyped using the Sequenom Massarray system. Ctrough was determined by validated high-performance liquid chromatographic method with tandem mass spectrometric (LC-MS/MS). Fifty-eight NSCLC patients were enrolled in this study. The median of Ctrough was 175ng/mL (range from 47.8 to 470 ng/mL). The trough concentration was not associated with either objective response or progression free survival (PFS). Ctrough was significantly lower in CYP3A4 rs2242480 CC + CT genotype than in TT genotype (P=0.019) and in ABCG2 rs2231142 AA genotype than in AC + CC genotype (P=0.031). ABCB1 rs2032582 dominant model was significantly correlated with overall response rate (ORR) and patients with GG phenotype respond better than patients with GT + TT phenotypes (84.6% vs. 51.2%, P=0.032). ABCB1 rs10256836 recessive model was significantly correlated with PFS and patients with GG phenotype achieved longer PFS than patients with GC + CC phenotypes (17.40 vs. 10.33 months, P=0.040). The Ctrough of gefitinib was significantly different between CYP3A4 and ABCG2 genotypes, but not with the efficacy of gefitinib treatment. ABCB1 rs2032582 and rs10256836 polymorphisms were correlated treatment outcome. Polymorphisms analysis of ABCB1 could be a predictive biomarker for gefitinib treatment.

Detection of Single-Nucleotide Polymorphism in AGPAT6 Gene, Associated with Milk Fat Content, using Tetra-Primer ARMS PCR-Based Assay, in Karan Fries Breeding Bulls.

Background: The bovine AGPAT6 gene is one of the potential candidate genes governing milk fat synthesis.Objectives: Identification of single nucleotide polymorphisms (SNP) in the targeted region of AGPAT6 gene and their effect on expected breeding values (EBV) of first lactation milk production traits viz. fat %, fat yield and 305 days milk yield in Karan Fries (KF) breeding bulls were sought.Materials and Methods: A tetra-primer ARMS PCR technique was adapted to genotype an SNP, g.36,175,805C>T located on 5’ flanking region of AGPAT6 gene. The relationship between EBV of milk production traits and polymorphic locus of AGPAT6 gene was assessed. Results: Three kinds of genotype (CC, CT, and TT) with respect to g.36,175,805C>T SNP locus were observed. The identified SNP had significant (P<0.05) influence on EBV of fat % (EBV-FP). The KF bulls with CC and CT genotype had comparatively higher EBV-FP than the bulls with the TT genotype. The substitution of “C” allele by “T” allele led to a decrease of 0.0045 % in the EBV-FP.Conclusion: The identified SNP was significantly associated with EBV-FP, thus it may be utilized as a molecular marker for developing marker-assisted selection strategy to enhance the milk fat content in KF cattle population.

TSLRF: Two-Stage Algorithm Based on Least Angle Regression and Random Forest in genome-wide association studies

One of the most important tasks in genome-wide association analysis (GWAS) is the detection of single-nucleotide polymorphisms (SNPs) which are related to target traits. With the development of sequencing technology, traditional statistical methods are difficult to analyze the corresponding high-dimensional massive data or SNPs. Recently, machine learning methods have become more popular in high-dimensional genetic data analysis for their fast computation speed. However, most of machine learning methods have several drawbacks, such as poor generalization ability, over-fitting, unsatisfactory classification and low detection accuracy. This study proposed a two-stage algorithm based on least angle regression and random forest (TSLRF), which firstly considered the control of population structure and polygenic effects, then selected the SNPs that were potentially related to target traits by using least angle regression (LARS), furtherly analyzed this variable subset using random forest (RF) to detect quantitative trait nucleotides (QTNs) associated with target traits. The new method has more powerful detection in simulation experiments and real data analyses. The results of simulation experiments showed that, compared with the existing approaches, the new method effectively improved the detection ability of QTNs and model fitting degree, and required less calculation time. In addition, the new method significantly distinguished QTNs and other SNPs. Subsequently, the new method was applied to analyze five flowering-related traits in Arabidopsis. The results showed that, the distinction between QTNs and unrelated SNPs was more significant than the other methods. The new method detected 60 genes confirmed to be related to the target trait, which was significantly higher than the other methods, and simultaneously detected multiple gene clusters associated with the target trait.

Adverse Drug Reactions Associated with CYP 2B6 Polymorphisms in HIV/AIDS-Treated Patients in Yaoundé, Cameroon.

PurposeThe metabolism of antiretroviral drugs is subject to individual variations of the CYP 2B6 gene. The objective of this study was to evaluate the prevalence of CYP 2B6 516 G>T and 983 T>C polymorphisms and investigate their association with the development of adverse drug reactions (ADRs) in people living with HIV/AIDS in Cameroon.Patients and methodsA total number of 122 patients, attending the Yaoundé Central Hospital HIV Day Clinic, consented to take part in this study. Blood specimens were collected and DNA was extracted using the Chelex method. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was performed for the detection of CYP 2B6 Single-Nucleotide Polymorphisms (SNPs). Genotype frequencies were compared between groups with or without ADRs. Logistic regression analysis was performed to assess association between genotype and adverse effects of antiretroviral therapy (ART).ResultsThree types of metabolizers were identified: extensive, intermediate and slow. For the 516G>T polymorphism, prevalences of 8.2% GG, 65.6% GT and 26.2% TT were obtained. For the 983T>C polymorphism, 89.3% TT, 4.1% CT and 6.6% CC prevalences were obtained. Those homozygous for the wild-type allele (516GG) were less likely to develop ADR with a statistically significant difference (OR=0.885, P=0.029). For the CYP2B6 T983C SNP, homozygous mutants (CC) may present a higher risk (threefold) of developing adverse reactions (OR=2.677, P=0.164).ConclusionThese findings demonstrate that ADRs among HIV/AIDS patients under ART may be associated with the genetic variability of the metabolizing enzyme CYP 2B6. Genotyping for this gene may guide the better administration of Efavirenz and Nevirapine to Cameroonian patients.

Detection and discrimination of single nucleotide polymorphisms by exploiting metal ion-mediated DNA duplex

Detection of mutant DNA is necessary for early diagnosis of disease. In particular, detection of single nucleotide polymorphisms (SNPs) has become an important method for monitoring cancer and obtaining theragnostic information about it. SNPs are very difficult to detect selectively because of their similarity with wildtype DNA. In this paper, the specific binding between metal ion and DNA mismatch was used to achieve highly sensitive and selective SNP detection. Cytosine-cytosine (C-C) mismatched nucleobases capture silver (Ag) ions, and thymine-thymine (T-T) mismatched nucleobases capture mercury (Hg) ions. Metal ions bound between DNA could be reduced using a reducing agent (hydroquinone). In addition, gold (Au) amalgam could be formed around the DNA mismatch by the reduction of Au ion and mercury ion bound between T-T mismatches. Differential pulse voltammetry (DPV) was used to measure signals which are mediated by silver and Au amalgam. The detection limit of point mutation DNA obtained by the proposed detection method is 20 pM. In addition, by comparing the values of the DPV peaks by silver and Au amalgam, the types of DNA point mutations including cytosine or thymine (CT, CC, TT, AC, and TG) could be distinguished. The results obtained from our proposed method suggest its potential for diagnosing cancer patients.

Non-hybridization single nucleotide polymorphism detection and genotyping assay through direct discrimination of single base mutation by capillary electrophoretic separation of single-stranded DNA.

The significant demands for single nucleotide polymorphism detection and genotyping assays have grown. Most common assays are based on the recognition of the target sequence by the hybridization with its specific probe having the complementary sequence of the target. Herein, a simple, label-free, and economical non-hybridization assay was developed for single nucleotide polymorphism detection and genotyping, based on the direct discrimination of single base mutation by simple capillary electrophoresis separation for single-stranded DNA in an acidic electrophoretic buffer solution containing urea. Capillary electrophoresis separation of single-base sequential isomers of DNA was achieved due to charge differences resulting from the different protonation properties of the DNA bases. Single nucleotide polymorphism detection and genotyping were achieved by discriminating the electropherogram pattern change, that is, peak number in the electropherogram, obtained by the proposed method. The successful practical application of the proposed method was demonstrated through single nucleotide polymorphism detection and genotyping on a known gene region of 84-mer, in which guanine to adenine single-base mutation is commonly observed, using a human hair sample in combination with genomic DNA extraction, polymerase chain reaction amplification, DNA purification from polymerase chain reaction products, and capillary electrophoresis separation.

Prevalence of mutations in Plasmodium falciparum genes associated with resistance to different antimalarial drugs in Nyando, Kisumu County in Kenya

Resistance to the mainstay antimalarial drugs is a major concern in the control of malaria. Delayed Plasmodium falciparum parasite clearance has been associated with Single Nucleotide Polymorphisms (SNPs) in the kelch propeller region (K13). However, SNPs in the Pf-adaptor protein complex 2 mu subunit (Pfap2-mu), Pfcrt and Pfmdr1 are possible markers associated with multi-drug resistance. Here, we explored the prevalence of SNPs in the K13, Pfap2-mu, Pfcrt, and Pfmdr1 in 94 dried blood spot field isolates collected from children aged below 12 years infected with P. falciparum during a cross-sectional study. The samples were collected in 2015 during the peak malaria transmission season in the Nyando region of Western Kenya before treatment with Artemether-Lumefantrine, the first-line artemisinin-based combination therapy (ACT) in Kenya. However, 47 of the 94 samples had recurrent parasitemia and were interrogated for the presence of the SNPs in K13 and Pfap2-mu. We used PCR amplification and sequencing to evaluate specific regions of K13 (codons 432–702), Pfap2-mu (codons 1–350), Pfmdr1 (codons 86, 1034–1246), and Pfcrt (codons 72–76) gene(s). The majority of parasites harbored the wild type K13 sequence. However, we found a unique non-synonymous W611S change. In silico studies on the impact of the W611S predicted structural changes in the overall topology of the K13 protein. Of the 47 samples analyzed for SNPs in the Pfap2-mu gene, 14 (29%) had S160 N/T mutation. The CVIET haplotype associated with CQ resistance in the Pfcrt yielded a 7.44% (7/94), while CVMNK haplotype was at 92.56%. Mutations in the Pfmdr1 region were detected only in three samples (3/94; 3.19%) at codon D1246Y. Our data suggest that parasites in the western part of Kenya harbor the wildtype strains. However, the detection of the unique SNP in K13 and Pfap2-mu linked with ACT delayed parasite clearance may suggest slow filtering of ACT-resistant parasites.

Dual-Color Emissive AIEgen for Specific and Label-Free Double-Stranded DNA Recognition and Single-Nucleotide Polymorphisms Detection.

Simple, rapid, and sensitive assays of DNA sequence hold great importance in genetic analysis, clinical diagnosis, and molecular biology research. Most current methods for DNA detection, based on the complementary base pairing, require hybridization with intricately modified single-stranded DNA (ssDNA) probes or analytes. Herein, we have developed a powerful molecule with aggregation-induced emission (AIE) characteristic, namely, TPBT, which can specifically recognize double-stranded DNA (dsDNA) by emitting out a unique dual-color fluorescent signal of red (∼640 nm) and green (∼537 nm). The red-color emission at around 640 nm is observed when TPBT binds with dsDNA, ssDNA, proteins, and other polyanionic analytes. However, the green emission at around 537 nm is demonstrated to be the exclusive response of TPBT to dsDNA, which is closely related to the conformational change of TPBT upon groove binding. More strikingly, TPBT can distinguish single-nucleotide polymorphisms (SNPs) in a dsDNA sequence and detect the DNA damage suffered from UV light with ultrahigh sensitivity and specificity. This label-free, AIEgen-based dsDNA assay method is facile, robust, and universal, which will lead to major advances in genomic and disease diagnosis.

A DNA Switch for Detecting Single Nucleotide Polymorphism within a Long DNA Sequence Under Denaturing Conditions

DNA detection is usually conducted under nondenaturing conditions to favor the formation of Watson-Crick base-paring interactions. However, although such a setting is excellent for distinguishing a single-nucleotide polymorphism (SNP) within short DNA sequences (15-25 nucleotides), it does not offer a good solution to SNP detection within much longer sequences. Here we report on a new detection method capable of detecting SNP in a DNA sequence containing 35-90 nucleotides. This is achieved through incorporating into the recognition DNA sequence a previously discovered DNA molecule that forms a stable G-quadruplex in the presence of 7 molar urea, a known condition for denaturing DNA structures. The systems are configured to produce both colorimetric and fluorescent signals upon target binding.

Target-Triggered Polymerization of Branched DNA Enables Enzyme-free and Fast Discrimination of Single-Base Changes.

SummarySingle-base changes lead to important biological and biomedical implications; however, the discrimination of single-base changes from normal DNA always remains a grand challenge. Herein we developed a DNA recognition and amplification system based on artificial branched DNA, namely, target-triggered polymerization (TTP), to realize enzyme-free and fast discrimination of single-base changes. Branched DNA as monomers rapidly polymerized into DNA nanospheres only with the trigger of specific DNA. Our TTP system worked reliably over a wide range of conditions. Remarkably, our TTP system was capable of discriminating base-changing DNA from normal DNA, including distinguishing 1–4 nucleotide changes and positions of single base, which was attributed to the significant amplification of small differences in hybridization thermodynamics and kinetics. We further proposed a theoretical method for calculating the hybridization probability of nucleic acids, which performed highly consistent with experimental results.

Relationship Between Single Nucleotide Polymorphisms and the Toxicy and Side Effects of Paclitaxel and Platinum-Based Chemotherapy in Patients with Malignant Tumors

Objective: Investigating the relationship between single nucleotide polymorphisms (SNPs) and the toxic and adverse effects of paclitaxel and platinum-based chemotherapy in patients with malignant tumors, to provide recommendations for individualized treatment. Methods: Determinate 17 patients with malignant tumor DNA site and analysis. Results: 1. All 17 selected specimens’ fluorouracil related genes 18DPYD*2A(476002G&gt;A)GG type、153DPYD*13(1679T&gt;G)TT type 154DPYD(2846A&gt;T)TT type, and the synthesis of DPYD enzyme activity. 21GSTP1(313A&gt;G) polymorphism site mutation rate was 25.0%(4 cases), 29XRCC1(16323944T&gt;C) polymorphism site mutation rate was 90.9%(10 cases), 62ABCB1(3435C&gt;T) polymorphism site mutation rate was 52.9%(9 cases), and 68MTHFR(677C&gt;T) polymorphism site mutation rate was 50.0%(8 cases). 2. Fluorouracil related genes 18DPYD*2A(476002G&gt;A)GG type, 153DPYD*13(1679T&gt;G)TT type, 154DPYD(2846A&gt;T)TT type ,and the synthesis DPYD enzyme activity is normal. 3. Paclitaxel related genes 62ABCB1(3435T&gt;C) CC type has a lower incidence of hematotoxicity and neurotoxicity, than CT type and TT type. 13ABCB1(2677T&gt;G)GG type has a higher rate of drug resistance than TT type. 14CYP1B1*3(C&gt;G)CC type has a higher progression-free survival. Platinum-related genes 21GSTP1(313A&gt;G)AA is homozygous wild type and has a higher incidence of hematotoxicity than GA type. 29XRCC1(1196T&gt;C)CC is homozygous mutant and has a higher risk of serious neutropenia than CT type. 62ABCB1 (3435T&gt;C)CC is homozygous mutant and has a higher risk of lymphatic metastasis than TC type and TT type. 68MTHFR(677C&gt;T)TT type is homozygous mutant and has a higher mucosal toxicity and toxic and side effects than CT type and CC type. Conclusion: Single nucleotide polymorphism is related to the toxic and side effects of chemotherapy，the detection of SNP to predict the toxicity risk of drug users can be an important reference index to guide clinical individualized treatment.

Novel algorithms for efficient subsequence searching and mapping in nanopore raw signals towards targeted sequencing.

Genome diagnostics have gradually become a prevailing routine for human healthcare. With the advances in understanding the causal genes for many human diseases, targeted sequencing provides a rapid, cost-efficient and focused option for clinical applications, such as single nucleotide polymorphism (SNP) detection and haplotype classification, in a specific genomic region. Although nanopore sequencing offers a perfect tool for targeted sequencing because of its mobility, PCR-freeness and long read properties, it poses a challenging computational problem of how to efficiently and accurately search and map genomic subsequences of interest in a pool of nanopore reads (or raw signals). Due to its relatively low sequencing accuracy, there is no reliable solution to this problem, especially at low sequencing coverage. Here, we propose a brand new signal-based subsequence inquiry pipeline as well as two novel algorithms to tackle this problem. The proposed algorithms follow the principle of subsequence dynamic time warping and directly operate on the electrical current signals, without loss of information in base-calling. Therefore, the proposed algorithms can serve as a tool for sequence inquiry in targeted sequencing. Two novel criteria are offered for the consequent signal quality analysis and data classification. Comprehensive experiments on real-world nanopore datasets show the efficiency and effectiveness of the proposed algorithms. We further demonstrate the potential applications of the proposed algorithms in two typical tasks in nanopore-based targeted sequencing: SNP detection under low sequencing coverage, and haplotype classification under low sequencing accuracy. The project is accessible at https://github.com/icthrm/cwSDTWnano.git, and the presented bench data is available upon request. Supplementary data are available at Bioinformatics online.