Multiplex Sequencing Technology Research Articles

HLA genotypes associated with gastrointestinal symptoms in patients with spondyloarthritis without inflammatory bowel disease

This study aimed to establish the association between HLA-A, B, DR genotypes and gastrointestinal variables in patients with SpA without inflammatory bowel disease (IBD). Retrospective study of 91 patients with SpA and 401 healthy controls, with typing by Illumina Sequencing/PacBio and LIFECODES HLA-PCR/SSO multiplex sequencing technology. The presence of gastrointestinal symptoms was evaluated by administering a survey, and those who presented 2 or more symptoms were taken for clinical evaluation by rheumatology and gastroenterology, colonoscopy and histopathological study. (Ethics committee approval). The 59,3% of the patients were men, with a mean age of 43,9±11.4 years; 80,2% were classified as ankylosing spondylitis. 14, 28 and 19 genotypes for the HLA-A*, HLA-B* and HLA-DR* loci were identified in both groups, of which a relationship with gastrointestinal symptoms was identified: A*26, A*29 and B*27 were associated to abdominal pain, DRB1*11 and DRB1*16 with abdominal distention, A*30, B*38, DRB1*13 and DRB1*14 with weight loss, B*40 with diarrhea >4 weeks, and presence of mucus in the stools with A*02 and DRB1*11 (p<0.05). Furthermore, the presence of B*15 had a statistical relationship with intolerance to some food, highlighting the B*27 genotype in relation to grains and dairy products, A*23 with grains, vegetables and meats, and B*49 with vegetables and dairy (p<0.05). Regarding the endoscopic variables, macroscopic changes were found in the ileum mucosa related to A*02, B*48, DRB1*14 and the relationship between B*27 and ulcers at this level should be highlighted. Macroscopic changes in the sigmoid colon with B*48 and the rectum with A*30. In microscopic changes, inflammatory alterations of the ileum are mentioned with genotypes DRB1*07, DRB1*13 and DRB1*14, a genotype that is related to changes in the ileum both endoscopically and histologically (p<0.05). These findings indicate a potential genetic predisposition related to HLA genotypes that may increase the likelihood of food intolerance, gastrointestinal symptoms, and even visible and microscopic changes, specifically in the ileal tissue. The study highlights the presence of B*27 and other noteworthy HLA class I and class II genes (such as DRB1*14) in the diverse Colombian population.

Inhibition of the fibroblast growth factor receptor (FGFR) pathway: the current landscape and barriers to clinical application.

The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) is a tyrosine kinase signaling pathway that has a fundamental role in many biologic processes including embryonic development, tissue regeneration, and angiogenesis. Increasing evidence indicates that this pathway plays a critical role in oncogenesis via gene amplification, activating mutations, or translocation in tumors of various histologies. With multiplex sequencing technology, the detection of FGFR aberrations has become more common and is tied to cancer cell proliferation, resistance to anticancer therapies, and neoangiogenesis. Inhibition of FGFR signaling appears promising in preclinical studies, suggesting a pathway of clinical interest in the development of targeted therapy. Phase I trials have demonstrated a manageable toxicity profile. Currently, there are multiple FGFR inhibitors under study with many non-selective (multi-kinase) inhibitors demonstrating limited clinical responses. As we progress from the first generation of non-selective drugs to the second generation of selective FGFR inhibitors, it is clear that FGFR aberrations do not behave uniformly across cancer types; thus, a deeper understanding of biomarker strategies is undoubtedly warranted. This review aims to consolidate data from recent clinical trials with a focus on selective FGFR inhibitors. As Phase II clinical trials emerge, concentration on patient selection as it pertains to predicting response to therapy, feasible methods for overcoming toxicity, and the likelihood of combination therapies should be utilized. We will also discuss qualities that may be desirable in future generations of FGFR inhibitors, with the hope that overcoming these current barriers will expedite the availability of this novel class of medications.

Rapid screening of SNPs in metastatic colorectal cancer (mCRC) utilizing multiplex sequencing technology (Sequenom).

418 Background: Accurate and fast screening of mutations is essential for designing individualized therapy necessary and critical for efficient disease management and better patient outcome in mCRC. Detection of hotspots by gold standard direct sequencing (DS) is time consuming and cost ineffective. Pyrosequencing (PS) technique is rapid and precisely committed towards SNP detection. Recent introduction of high throughput multiplex PCR based extension on microarray (Sequenom, SEQ) offers a robust platform capable of detecting multiple SNPs simultaneously in a rapid and cost effective manner. The current study analyzes the concordance and efficacy of the cutting edge SEQ technique to the well established DS and PS methods. Methods: DNA isolated from 122 specimens from 76 mCRC patients were sequenced by all three methods. DS and PS were performed on 4 genes at 10 hotspots. SEQ multiplexing was performed on 31 hotspots in 19 genes by 4 multiplex reactions. Results: We were able to make "calls" for all samples by DS and PS. With the multiplex system, the “calls” rate was 97.8% of successful reactions. Using PS data as our standard in the assay we calculated the percent concordance of DS and SEQ. Futhermore SEQ offered a more accurate identification of the substituted nucleotide in Kras codon 12 as compared to PS. Conclusions: The multiplexing of PCR reactions offers an excellent advantage of high throughput with strong feasibility of analyzing several samples for multiple SNPs simultaneously. The concordance rate of &gt; 90% when compared to PS along with the ability to analyze multiple samples/ hotspots plexed together in a time effective rapid mode provided a trifold advantage of the sequenom technology. It is therefore the next generation technology for rapid genetic evaluation of cancer patients. [Table: see text]

Modeling Transcriptome Based on Transcript-Sampling Data

BackgroundNewly-evolved multiplex sequencing technology has been bringing transcriptome sequencing into an unprecedented depth. Millions of transcript tags now can be acquired in a single experiment through parallelization. The significant increase in throughput and reduction in cost required us to address some fundamental questions, such as how many transcript tags do we have to sequence for a given transcriptome? How could we estimate the total number of unique transcripts for different cell types (transcriptome diversity) and the distribution of their copy numbers (transcriptome dynamics)? What is the probability that a transcript with a given expression level to be detected at a certain sampling depth?Methodology/Principal FindingsWe developed a statistical model to evaluate these parameters based on transcriptome-sampling data. Three mixture models were exploited for their potentials to model the sampling frequencies. We demonstrated that relative abundances of all transcripts in a transcriptome follow the generalized inverse Gaussian distribution. The widely known beta and gamma distributions failed to fulfill the singular characteristics of relative abundance distribution, i.e., highly skewed toward zero and with a long tail. An estimator of transcriptome diversity and an analytical form of sampling growth curve were proposed in a coherent framework. Experimental data fitted this model very well and Monte Carlo simulations based on this model replicated sampling experiments in a remarkable precision.ConclusionsTaking human embryonic stem cell as a prototype, we demonstrated that sequencing tens of thousands of transcript tags in an ordinary EST/SAGE experiment was far from sufficient. In order to fully characterize a human transcriptome, millions of transcript tags had to be sequenced. This model lays a statistical basis for transcriptome-sampling experiments and in essence can be used in all sampling-based data.

Molecular Typing of West Nile Virus, Dengue, and St. Louis Encephalitis Using Multiplex Sequencing

We report the development of an assay to simultaneously identify three of the clinically important flaviviruses (West Nile Virus, Dengue, and St. Louis encephalitis). This assay is based on the nucleotide sequence variations within a 266-bp region of the non-structural protein 5. Further, based on the nucleotide variations in the same region of the non-structural protein 5, four of the present Dengue serotypes were identified. To identify some of the subtypes of WNV we have developed a second assay using multiplex sequencing technology. The format of the result of this assay is an electropherogram of two genomic segments of the WNV genome: a 48-nucleotide sequence from the anchored core protein C and a 45-nucleotide sequence coding for the non-structural proteins (proteinase and putative helicase genes).

Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome.

The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.

Nebularine (9-2′-deoxy-β- d-ribofuranosylpurine) has the template characteristics of adenine in vivo and in vitro

Nebularine (9-β- d-ribofuranosylpurine; Nb) is a naturally occurring nucleoside with structural features suggestive of a universal base. However, previous observations based on thermal melting characteristics of oligonucleotides suggested that Nb formed stable pairs only with thymine. To determine the template characteristics of Nb, we constructed M13 viral single-stranded DNA (ssDNA) molecules bearing a single site-specific deoxynebularine (9-2′-deoxy-β- d-ribofuranosylpurine) residue. The ssDNA constructs were transfected into Escherichia coli cells to determine the specificity of base insertion opposite Nb, as well as to determine the effect of Nb on the replicability of the transfected DNA. Base insertion opposite Nb, analyzed by a multiplex sequencing technology, suggests that Nb has the template characteristics of adenine. Analysis of DNA replicability, measured as transfection efficiency, indicates that Nb does not block DNA replication. UV irradiation of host cells before transfection did not significantly affect survival or base insertion specificity within the limits of multiplex sequencing technology employed, suggesting that inducible mutagenic phenomena appear to have only minor effects on translesion synthesis across Nb. In addition, in vitro DNA elongation experiments on oligonucleotide templates using E. coli DNA polymerase I (Klenow fragment) as the model polymerase showed that Nb templates for T, but not for other bases under the tested conditions. The data reported in this communication underscore the importance of base-pair geometry as a specificity-determinant during base insertion by replicative polymerases.

Role of mismatch repair in the Escherichia coli UVM response.

Mutagenesis at 3,N4-ethenocytosine (epsilonC), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2. This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents. Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis. To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E. coli cells defective for methyl-directed mismatch repair, for very-short-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific epsilonC lesion. Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology. The results showed normal UVM induction patterns in all the repair-defective strains tested. In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS. All strains displayed UVM reactivation, the term used to describe the increased survival of epsilonC-containing DNA in UVM-induced cells. Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E. coli and may thus represent a previously unrecognized misrepair or misreplication pathway.