PCR-based Sequencing Method Research Articles

Recovery of microbial community profile information hidden in chimeric sequence reads

The next frontier in the field of microbiome studies is identification of all microbes present in the microbiome and accurate determination of their abundance such that microbiome profiles can serve as reliable assessments of health or disease status. PCR-based 16S rRNA gene sequencing and metagenome shotgun sequencing technologies are the prevailing approaches used in microbiome analyses. Each poses a number of technical challenges associated with PCR amplification, sample availability, and cost of processing and analysis. In general, results from these two approaches rarely agree completely with each other. Here, we compare these methods utilizing a set of vaginal swab and lavage specimens from a cohort of 42 pregnant women collected for a pilot study exploring the effect of the vaginal microbiome on preterm birth. We generated the microbial community profiles from the sequencing reads of the V3V4 and V4V5 regions of the 16S rRNA gene in the vaginal swab and lavage samples. For a subset of the vaginal samples from 12 subjects, we also performed metagenomic shotgun sequencing analysis and compared the results obtained from the PCR-based sequencing methods. Our findings suggest that sample composition and complexity, particularly at the species level, are major factors that must be considered when analyzing and interpreting microbiome data. Our approach to sequence analysis includes consideration of chimeric reads, by using our chimera-counting BlastBin program, and enables recovery of microbial content information generated during PCR-based sequencing methods, such that the microbial profiles more closely resemble those obtained from metagenomic read-based approaches.

Complete mitochondrial genome of Anthus richardi (Passeriformes: Motacillidae)

Richard’s Pipit Anthus richardi is a medium-sized passerine bird which breeds in open grasslands in northern Asia with a long-distance migrant moving to open lowlands in the Indian subcontinent and Southeast Asia. In this study, the mitochondrial genome of this bird species was obtained by the PCR-based sequencing method. The mitogenome is 16,871 bp in size, including 22 tRNAs, 2 rRNAs, 13 protein-coding genes and a D-loop. The A, T, G, and C base composition of the mitochondrial DNA are 29.73%, 23.12%, 14.59% and 32.56%, respectively. The starting codon of protein-coding genes is typical ATG, except for the ND2 which starts with ATA. The D-loop locates between genes of tRNAGlu and tRNAPhe. The result of the phylogenetic analysis indicates that A. richardi has close phylogenetic relationship with A. novaeseelandiae.

Use of a bioinformatic-assisted primer design strategy to establish a new nested PCR-based method for Cryptosporidium

BackgroundThe accurate tracking of Cryptosporidium in faecal, water and/or soil samples in water catchment areas is central to developing strategies to manage the potential risk of cryptosporidiosis transmission to humans. Various PCR assays are used for this purpose. Although some assays achieve specific amplification from Cryptosporidium DNA in animal faecal samples, some do not. Indeed, we have observed non-specificity of some oligonucleotide primers in the small subunit of nuclear ribosomal RNA gene (SSU), which has presented an obstacle to the identification and classification of Cryptosporidium species and genotypes (taxa) from faecal samples.ResultsUsing a novel bioinformatic approach, we explored all available Cryptosporidium genome sequences for new and diagnostically-informative, multi-copy regions to specifically design oligonucleotide primers in the large subunit of nuclear ribosomal RNA gene (LSU) as a basis for an effective nested PCR-based sequencing method for the identification and/or classification of Cryptosporidium taxa.ConclusionThis newly established PCR, which has high analytical specificity and sensitivity, is now in routine use in our laboratory, together with other assays developed by various colleagues. Although the present bioinformatic workflow used here was for the specific design of primers in nuclear DNA of Cryptosporidium, this approach should be broadly applicable to many other microorganisms.

Methylation status of DJ-1 in leukocyte DNA of Parkinson's disease patients.

BackgroundDJ-1 has been thought as a candidate biomarker for Parkinson’s disease (PD). It was found reduced in PD brains, CSF and saliva, although there were conflicting results. How DJ-1 expression may be regulated is not clear. Recently, blood-based DNA methylation represents a highly promising biomarker for PD by regulating the causative gene expression. Thus, in this study, we try to explore whether blood-based DNA methylation of DJ-1 could be used as a biomarker to differentiate PD patients from normal control (NC), and whether DNA methylation could regulate DJ-1 expression in a SH-SY5Y cell model.MethodsForty PD patients and 40 NC were recruited in this study. DNA was extracted from peripheral blood leukocytes (PBLs). Methylation status of two CpG islands (CpG1 and CpG2) in promoter region of DJ-1 was explored by bisulfite specific PCR-based sequencing method. Methylation inhibitor 5-Aza-dC was used to treat SH-SY5Y cell line, DJ-1 level was detected in both mRNA and protein level.ResultsCpG sites in these two CpG islands (CpG1 and CpG2) of DJ-1 were unmethylated in both PD and NC group. In SH-SY5Y cell model treated by methylation inhibitor, there was no significant change of DJ-1 expression in either mRNA level or protein level.ConclusionsOur results indicated that DNA methylation inhibitor didn’t alter DJ-1 gene expression in SH-SY5Y cell model, and DNA methylation of DJ-1 promoter region in PBLs level might not be an efficient biomarker for PD patients.

The complete mtDNA genome of Erythroculter ilishaeformis: genome characterization and phylogenetic analysis

The complete mitochondrial genome sequence of the hybrid of Erythroculter ilishaeformis was first determined by a PCR-based sequencing method in this study. The mitochondrial was 16 624 bp in length, including 13 protein genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and one control region. All genes were encoded on the heavy strain except for ND6 and eight tRNA genes. Base composition of the heavy strain was A (30.09%), T (24.96%), C (27.67%), G (16.41%) and with A + T bias of 55.05%. All the genes in E. ilishaeformis were distributed on the H-strand, except for the ND6 subunit gene and nine tRNA genes which were encoded on the L-strand. The phylogenetic relationships of 14 Cyprinidae species were reconstructed based on the complete mtDNA sequences using the MEGA inference method.

Complete mitochondrial genome of the Ptychobarbus chungtienensis (Teleostei: Cyprinidae)

The complete mitochondrial genome of Ptychobarbus chungtienensis was first determined by using a PCR-based sequencing method. Its mitochondrial genome is 16,970bp in length, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a non-coding control region, and its gene order was consistent with other fishes. In the control region, there was a 54 bp tandem repeat array identified at 3′ end. Phylogenetic analysis showed that P. chungtiensensis was clustered with other three species of Ptychobarbus.

Complete mitochondrial genomes of the critically endangered Ship sturgeon Acipenser nudiventris from two seas

Two complete mitochondrial genome sequences of ship sturgeon Acipenser nudiventris from the Caspian Sea and from Balkhash Lake (introduced from the Aral Sea during the last century) were determined by PCR-based sequencing method. The whole mitogenome sequences have been deposited in GenBank under accession numbers KU321568 and KU321569.

The complete mtDNA genome of suspected natural Bagridae hybridization (Pelteobagrus olimcorporis sp.nov): genome characterization and phylogenetic analysis

The complete mitochondrial genome sequence of the suspected new species (Pelteobagrus olimcorporis sp.nov) was first determined by a PCR-based sequencing method in this study. The complete mtDNA genome of this suspected new species is 16,533 bp in length and consists of 13 protein-coding genes, 22 transfer RNA genes (tRNA), two ribosomal RNA genes (rRNA) and a non-coding control region (D-loop). Compared with the 13 proteins genome of the Bagridae and Cyprinidae, results showed that this suspected new species was clustered together and from a sister group with fishes of Bagridae. Blasting the special region on NCBI, we found 99% of the sequence was the same with Pseudolaubuca sinensis. It indicated that P. olimcorporis sp.nov is a hybrid and the mitochondrial genomes of the father may permeate to the hybrid.

Complete mitochondrial genome of the hybrid of Acipenser schrenckii (♀) × Huso dauricus (♂)

The complete mitochondrial genome sequence of the hybrid of Acipenser schrenckii (♀) × Huso dauricus (♂) (A × H) was first determined by a PCR-based sequencing method in this study. The mitochondrial was 16 687 bp in length, including 13 protein genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and one control region. All genes were encoded on the heavy strain except for ND6 and eight tRNA genes. Base composition of the heavy strain was A (29.80%), T (24.42%), C (28.94%), G (16.82%), and with A + T bias of 54.26%. Compared with the complete mitochondrial genome of the parents, results showed the hybrid sturgeon was consistent with a maternal inheritance; however, we also found ND6 and tRNA-Glu which were species-specific for the male parent H. dauricus. The complete mitochondrial genome sequence of the A × H provided an important data set for further study in mitochondrial inheritance mechanism.

The complete mitochondrial genome of the Megalobrama pellegrini (Teleostei: Cyprinidae)

The complete mitochondrial genome of Megalobrama pellegrini was determined by using a PCR-based sequencing method. The mitochondrial was 16,620 bp in length, including 13 protein genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and one control region. All genes were encoded on the heavy strain except for ND6 and eight tRNA genes. The gene arrangement of M. pellegrini was similar to those found in the other Megalobrama species. Base composition of the heavy strain was A (31.19%), T (24.69%), C (46.37%), G (16.22%) and with A + T bias of 55.88%.

Mutation screening of mitochondrial DNA as well as OPA1 and OPA3 in a Chinese cohort with suspected hereditary optic atrophy.

Leber's hereditary optic atrophy (LHON) and autosomal dominant optic atrophy (DOA) are the two most common forms. The objective of this study was to define the fractional prevalence of LHON and DOA in a cohort of Chinese patients with suspected hereditary optic neuropathy. We recruited 520 unrelated patients with bilateral optic atrophy for genetic analysis: 174 patients had a positive family history of visual failure and 346 were sporadic cases. A total of 14 primary LHON-causing mtDNA mutations was screened by PCR-based sequencing methods for all patients except the individuals with a paternal family history. All coding exons and exon-intron boundaries of the OPA1 and OPA3 gene were screened for mutations by PCR-based DNA sequencing for all patients with paternal family history and for the LHON-negative patients. A large genomic DNA arrangement of the OPA1 gene was detected further by multiplex ligation probe amplification (MLPA) assay for the patients with paternal family history, but results were negative for the OPA1 and OPA3 mutation screenings. We found molecular defects in 323 (62%) of the 520 probands screened. Among these, 271 patients (83.9%) had an mtDNA mutation, 50 patients (15.5%) carried an OPA1 mutation, and 2 patients (0.6%) had an OPA3 mutation. Coexistence m.3460 G>A and m.11778G>A was found in one patient. We identified 40 intragenic mutations and six large genomic DNA arrangements of the OPA1 gene, 23 of which were novel. The LHON-mtDNA mutations are the most common genetic defects, followed by the OPA1 mutations, in this Chinese cohort.

Clonal Analysis of Staphylococcus hominis Strains Isolated from Hospitalized Patients

Staphylococcus hominis is a part of normal skin flora, but it is also a cause of nosocomial infections. The aim of this study was to investigate the genetic relatedness of 62 strains of S. hominis obtained from hospitalised patients during an 11-year period. For the discrimination of these clinical strains we used repetitive sequence-based PCR method (BOX-PCR) and multiple-locus variable-number tandem repeat analysis (MLVA). BOX-PCR analysis revealed a large genetic diversity among clinical strains and we did not find a predominant clone with the ability to persist in a hospital environment. MLVA is not as discriminatory as BOX fingerprinting and would not be a useful method for epidemiological studies.

Methylation of α-synuclein and leucine-rich repeat kinase 2 in leukocyte DNA of Parkinson's disease patients

BackgroundRecent studies highlight the role of DNA methylation in the pathogenesis of Parkinson's disease (PD). However, there is a paucity of studies exploring the role of blood-based DNA methylation in PD. We aimed to explore identifiable epigenetic biomarkers for PD by analyzing the methylation status of α-synuclein (SNCA) and leucine-rich repeat kinase 2 (LRRK2) in leukocytes. MethodsBisulfite Specific PCR-based Sequencing method was used for semi-quantitative detection of methylation status of CpG islands in SNCA and LRRK2 promoter regions. Bisulfite Specific Cloning-based Sequencing method was used for further quantitative examination of CpG-2 methylation of SNCA. mRNA level was also detected in leukocytes. ResultsSemi-quantitative detection showed that the methylation status of SNCA CpG-2 differed between PD patients and normal controls, while there was no difference in CpG-1 of SNCA or in LRRK2 promoter. Further quantitative analysis by clonal assay showed that the CpG-2 of SNCA was hypomethylated in PD patients compared with the normal control (5.90% versus 7.69%, P=0.034). Moreover, among the 14 CpG sites of CpG-2, the 2nd, 4th and 9th CpG sites were significantly hypomethylated in PD patients. In subgroups of PD, the methylation level decreased in the early-onset PD patients (P=0.001). RT-PCR examination showed that SNCA mRNA was increased in PD patients compared with normal control (P=0.003). ConclusionsOur results indicated that the methylation level of SNCA CpG-2, especially that of the 2nd, 4th and 9th CpG sites in leukocytes might have great potential to be a useful and informative biomarker in PD diagnosis and treatment.

Development and application of specific polymerase chain reaction assay targeting the gyrB gene for rapid detection of Riemerella anatipestifer

A pair of PCR primers was designed and synthesized to amplify a gyrB gene sequence from Riemerella anatipestifer (RA). A fragment of 194 bp was detected in RA-positive isolates, whereas other isolates were negative, which confirmed the high specificity of the primers and PCR conditions. As little as 1.6 × 10(4) cfu/mL of cultural liquid was required by this method. We compared a 16S rRNA sequence-based PCR method and a Biolog bacterial identification system used in the detection and identification of suspicious isolates of RA in clinical tests. The results showed that the gyrB-based PCR was consistent with the results of the Biolog identification system and was more specific. By applying the gyrB-PCR to detect RA strains in 56 duck livers, a positive rate of 46% (26/56) was observed, whereas the positive rate of 85 throat swabs from clinically healthy ducks was 11%. Thus, this method could be used for the epidemiological investigation and preliminary isolate identification of RA.

A novel multiplex real time PCR for rapid and accurate identification of the genus Mycobacterium and Mycobacterium tuberculosis complex species

Mycobacteria are a heterogeneous group of bacteria in terms of their genotypic features and disease association. The accurate and rapid detection of genus Mycobacterium andMycobacterium tuberculosis complex (MTC) isolates from primary culture is important due to timely and appropriate antibiotic therapy. In this study, we developed a novel multiplex real time PCR for rapid identification of Mycobacteria from other genus of bacteria and Mycobacterium tuberculosis complex from non-tuberculosis Mycobacteria(NTMs). Genetic targets for primer designing and identification of genus Mycobacteriumand Mycobacterium tuberculosis complex (MTC) included the 16SrDNA and 16S-23S internal transcribed spacer (ITS). Multiplex real time PCR was setup with reference toMycobacterium strains. Results of real time PCR were analyzed with melting curves and determined melting temperature of genus Mycobacterium and Mycobacterium tuberculosis complex. For the assessment of the sensitivity and specificity of technique, fifty three clinical isolates of Mycobacteria were collected from different Tuberculousis Centers in Iran. Multiplex real time PCR results were compared by PCR-based sequencing method with amplification and sequencing of 16S–23S ribosomal RNA internal transcribed spacer (ITS) for identification of Mycobacterium species. Of 53 isolates, 52 (98%) isolates identified as genus Mycobacterium and 34 isolates as MTC (65%). Sensitivity and specificity of multiplex real time PCR were 96%, 100% for genusMycobacterium and 100%, 100% for Mycobacterium tuberculosis complex (MTC), respectively. The multiplex real time PCR with melting curve analysis in the present study is a reliable and suitable method for rapid and accurate identification of genusMycobacterium and Mycobacterium tuberculosis complex. Key words: Mycobacterium tuberculosis complex, multiplex real time PCR, rapid identification

Identification of the first germline HRPT2 whole‐gene deletion in a patient with primary hyperparathyroidism

Germline mutations in the HRPT2 gene are associated with the hereditary hyperparathyroidism-jaw tumour syndrome (HPT-JT) and a subset of familial isolated hyperparathyroidism (FIHP). Somatic HRPT2 mutations are detected in sporadic parathyroid carcinomas and less frequently in cystic adenomas. The purpose of this study was to investigate the underlying HRPT2 defect in a young patient with symptomatic hyperparathyroidism due to an apparently sporadic parathyroid adenoma with cystic features. HRPT2 mutations in the patient's genomic and parathyroid tumour DNA were screened by PCR-based sequencing. Tumour loss of heterozygosity (LOH) at the HRPT2 locus was assessed with microsatellite markers. A large germline HRPT2 deletion was investigated by real-time quantitative PCR analysis (qPCR). Genomic DNA losses were also appraised by chromosomal comparative genomic hybridization (cCGH). No germline HRPT2 point mutation was detected by direct sequencing. A novel hemizygous HRPT2 somatic mutation (c.32delA) was identified in the tumour. Apparent constitutional homozygosity for HRPT2 flanking microsatellite markers, and absence of LOH at a distal marker, suggested a large germline deletion. Gene dose mapping by qPCR unveiled a de novo deletion of the whole HRPT2 gene and adjacent loci (<9·3 Mb in size). cCGH confirmed germline DNA loss involving the HRPT2 locus. We report the first large germline deletion of the HRPT2 gene, which was not detectable by conventional PCR-based sequencing methods. This finding emphasizes that qPCR should be implemented in HRPT2 molecular analysis, which may improve genetic assessment and clinical management of patients with FIHP and HPT-JT.

Higher incidence of perineal community acquired MRSA infections among toddlers

BackgroundA six-fold increase in pediatric MRSA infections, prompted us to examine the clinical profile of children with MRSA infections seen at Mercy Children's Hospital, Toledo, Ohio and to characterize the responsible strains.MethodsRecords were reviewed of pediatric patients who cultured positive for MRSA from June 1 to December 31, 2007. Strain typing by pulsed field gel electrophoresis (PFT) and DiversiLab, SCCmec typing, and PCR-based lukSF-PV gene (encodes Panton-Valentine leukocidin), arginine catabolic mobile element (ACME) and cap5 gene detection was performed.ResultsChart review of 63 patients with MRSA infections revealed that 58(92%) were community acquired MRSA (CAMRSA). All CAMRSA were skin and soft tissue infections (SSTI). Twenty five (43%) patients were aged < 3 yrs, 19(33%) aged 4-12 and 14(24%) aged 13-18. Nineteen (76%) of those aged < 3 yrs had higher incidence of perineal infections compared to only 2(11%) of the 4-12 yrs and none of the 13-18 yrs of age. Infections in the extremities were more common in the older youth compared to the youngest children. Overall, there was a significant association between site of the infection and age group (Fisher's Exact p-value < 0.001). All CAMRSA were USA300 PFT, clindamycin susceptible, SCCmec type IVa and lukSF-PV gene positive. Nearly all contained ACME and about 80% were cap5 positive. Of the 58 USA300 strains by PFT, 55(95%) were also identified as USA300 via the automated repetitive sequence-based PCR method from DiversiLab.ConclusionsCAMRSA SSTI of the perineum was significantly more common among toddlers and that of the extremities in older children. The infecting strains were all USA300 PFT. Further studies are needed to identify the unique virulence and colonization characteristics of USA300 strains in these infections.

Screening and purification for novel cytochrome b5from uncultured environmental micro-organisms

We describe a sequence-based PCR method suitable for the isolation of a novel soluble heme-binding domain of cytochrome b(5) (cyt b(5)) gene directly from metagenomic DNA is described. Using the degenerate primer set, a cyt b(5) gene was isolated directly from metagenomic DNA. Based on the sequence-based PCR method, the similar conserved motif of cyt b(5) from Rhodopseudomonas palustris strain makes the novel target gene. The gene encoding cyt b(5) was cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized. Sequence-based strategy is an effective method for application of the novel gene from metagenomic DNA. Investigation of novel genes from metagenome, most of the micro-organism species are largely untapped, could represent an interesting and useful reservoir for biological processes.

Real-time Polymerase Chain Reaction To Diagnose Lymphogranuloma Venereum

Real-time Polymerase Chain Reaction To Diagnose Lymphogranuloma Venereum

Frequency of H‐ras mutations in human bladder cancer detected by direct sequencing

To determine the frequency of mutations of the H-ras gene in transitional cell carcinomas of the human urinary bladder using direct DNA sequencing based on the polymerase chain reaction (PCR) method and to compare the results with those of other methods. In addition, the relationship of the mutation frequency to tumour stage and grade was examined. Bladder tumour samples, taken by cystoscopic resection from 50 patients with newly diagnosed transitional cell carcinoma of the urinary bladder, were analysed by PCR-based direct DNA sequencing for point mutations in the H-ras gene at codon 12. Point mutations were found in 9 of 50 tumours examined (18%). The most frequent mutation (8/9) was a G to T transversion converting GGC to GTC, which would result in a glycine to valine substitution. The remaining mutations was a G to A transition altering GGC to GAC, producing a glycine to aspartic acid substitution, which has not previously been reported in bladder cancer. In all tumour samples examined the wild-type allele (GGC) was also evident. Variation in the proportion of wild-type to mutated sequence was found within tumour samples. No relationship between mutations and tumor grade and stage was apparent. The frequency of H-ras mutations detected in this first large scale study using the highly sensitive and rapid PCR-based sequencing method was comparable to that reported by earlier studies with the nude mouse tumorigenesis variation of the 3T3 transfection assay. H-ras mutations can be early events in the development and progression of a significant proportion of human bladder cancer cases.