Analysis Kit Research Articles

Multi-Institutional FASTQ File Exchange as a Means of Proficiency Testing for Next-Generation Sequencing Bioinformatics and Variant Interpretation

Next-generation sequencing is becoming increasingly common in clinical laboratories worldwide and is revolutionizing clinical molecular testing. However, the large amounts of raw data produced by next-generation sequencing assays and the need for complex bioinformatics analyses present unique challenges. Proficiency testing in clinical laboratories has traditionally been designed to evaluate assays in their entirety; however, it can be alternatively applied to separate assay components. We developed and implemented a multi-institutional proficiency testing approach to directly assess custom bioinformatics and variant interpretation processes. Six clinical laboratories, all of which use the same commercial library preparation kit for next-generation sequencing analysis of tumor specimens, each submitted raw data (FASTQ files) from four samples. These 24 file sets were then deidentified and redistributed to five of the institutions for analysis and interpretation according to their clinically validated approach. Among the laboratories, there was a high rate of concordance in the calling of single-nucleotide variants, in particular those we considered clinically significant (100% concordance). However, there was significant discordance in the calling of clinically significant insertions/deletions, with only two of seven being called by all participating laboratories. Missed calls were addressed by each laboratory to improve their bioinformatics processes. Thus, through our alternative proficiency testing approach, we identified the bioinformatic detection of insertions/deletions as an area of particular concern for clinical laboratories performing next-generation sequencing testing.

Global Optimization Under Uncertainty and Uncertainty Quantification Applied to Tractor-Trailer Base Flaps

Using a global optimization evolutionary algorithm (EA), propagating aleatory and epistemic uncertainty within the optimization loop, and using computational fluid dynamics (CFD), this study determines a design for a 3D tractor-trailer base (back-end) drag reduction device that reduces the wind-averaged drag coefficient by 41% at 57 mph (92 km/h). Because it is optimized under uncertainty, this design is relatively insensitive to uncertain wind speed and direction and uncertain deflection angles due to mounting accuracy and static aeroelastic loading. The model includes five design variables with generous constraints, and this study additionally includes the uncertain effects on drag prediction due to truck speed and elevation, steady Reynolds-averaged Navier–Stokes (RANS) approximation, and numerical approximation. This study uses the Design Analysis Kit for Optimization and Terascale Applications (DAKOTA) optimization and uncertainty quantification (UQ) framework to interface the RANS flow solver, grid generator, and optimization algorithm. The computational model is a simplified full-scale tractor-trailer with flow at highway speed. For the optimized design, the estimate of total predictive uncertainty is +15/−42%; 8–10% of this uncertainty comes from model form (computation versus experiment); 3–7% from model input (wind speed and direction, flap angle, and truck speed); and +0.0/−28.5% from numerical approximation (due to the relatively coarse, 6 × 106 cell grid). Relative comparison of designs to the no-flaps baseline should have considerably less uncertainty because numerical error and input variation are nearly eliminated and model form differences are reduced. The total predictive uncertainty is also presented in the form of a probability box, which may be used to decide how to improve the model and reduce uncertainty.

Chemical composition of urinary stones in Rahim Yar Khan and adjacent districts of Sindh and Baluchistan

Aim: To find out the constituents of the urinary stones, so that preventive measures would be taken against recurrence. Patients and methods: A total No. of 200 consecutive patients with upper urinary tract calculi, that were operated in the urology and general surgical wards of Sh. Zayed Hospital Rahim Yar Khan were included in the study. In addition to Rahim Yar Khan the patients also came from adjacent districts of Sindh and Baluchistan. Qualitative Chemical analysis was carried out using Merchognost (Germany) urinary calculi analysis kit. All the tests were performed according to the instruction of manufacturers of the kit. Results: Stone samples were collected from 200 patients, out of these 138 (69%) were male and 62 (31%) of patients were female with male to female ratio 2.2:1 age range was between 9-72 years. Results of stone analysis indicate that calcium oxalate is the most common ingredient found in 100% of the stone samples. In 83% of patients it was found mixed with other varieties of stones while in 17% samples it was seen in pure form. Uric acid was the second most common ingredient seen in 73% of patients mixe d with calcium oxalate and magnesium ammonium phosphate but it was not found as pure form. Magnesium Ammonium phosphate found in 13% of stones samples in mixed form only, not as isolated stone.

Novel versatile smart phone based Microplate readers for on-site diagnoses

Microplate readers are important diagnostic instruments, used intensively for various readout test kits (biochemical analysis kits and ELISA kits). However, due to their expensive and non-portability, commercial microplate readers are unavailable for home testing, community and rural hospitals, especially in developing countries. In this study, to provide a field-portable, cost-effective and versatile diagnostic tool, we reported a novel smart phone based microplate reader. The basic principle of this devise relies on a smart phone's optical sensor that measures transmitted light intensities of liquid samples. To prove the validity of these devises, developed smart phone based microplate readers were applied to readout results of various analytical targets. These targets included analanine aminotransferase (ALT; limit of detection (LOD) was 17.54 U/L), alkaline phosphatase (AKP; LOD was 15.56 U/L), creatinine (LOD was 1.35μM), bovine serum albumin (BSA; LOD was 0.0041mg/mL), prostate specific antigen (PSA; LOD was 0.76pg/mL), and ractopamine (Rac; LOD was 0.31ng/mL). The developed smart phone based microplate readers are versatile, portable, and inexpensive; they are unique because of their ability to perform under circumstances where resources and expertize are limited.

The Use of Kits in the Analysis of Tissue Lipids Requires Validation.

The ready availability and ease of use of kits for the measurement of serum lipids has greatly facilitated these measurements. In many cases it would be convenient to use these kits in the determination of lipid concentrations in tissues. The successful application of serum kits in tissue analysis requires that two important issues be considered. First, the solvent system for the extraction of the lipids and the solvent used for analysis by the kit must be compatible with the reactions in the kit. Second, the concentration range in the analyzed solution must be within the range for which the kit is used. We report here that lipids in liver and adipose tissues may be significantly underestimated by the use of some kits. We recommend that the use of kits for tissue analysis of lipids be validated for the specific analysis.

법과학 현장시료에서 Yfiler<sup>®</sup> PLUS PCR Amplification Kit의 민감도 연구

A variety of Y-STR analysis kits have been developed and used in the forensic field. Prior to the forensic application of a new kit, laboratory validation and sensitivity tests are essential processes in selecting suitable alternatives and for assuring that standard operating procedures are followed. In this paper, we have performed a sensitivity study of a new commercial kit, the Yfiler ® PLUS PCR Amplification Kit (Yfiler plus kit, released in 2014) by comparing it with the AmpF/STR ® Yfiler TM PCR Amplification Kit (Yfiler kit, released in 2004). The Yfiler plus kit includes the 17 Y-STR loci of the Yfiler kit and has been supplemented with 10 new Y-STR loci. First, we analyzed the sensitivity difference between the two kits using commercial control DNA 2800M and 007. In addition, we compared the detection rate between the two kits from the 16 selected forensic casework samples of less than 0.5 ng concentrations. The results show that the sensitivity and detection rate of the Yfiler plus kit are higher than the corresponding rates of the Yfiler kit. In addition, we were able to obtain more Y-STR profiles with the use of the new kit. Thus, we suggest that Yfiler plus kit is a more effective forensic tool to detect Y-STR profiles from forensic casework samples of low concentrations. 요약 : 법과학 분야에서 다양한 Y-STR 분석 키트가 개발되어 사용되고 있고, 새로운 키트의 법과학적 적용에 앞서 DNA 감정에 적절한 분석 키트들의 선정과 표준작업절차서의 작성을 위해 실험실 내의 내 부적 유효성 검증 및 민감도 시험은 필수적인 과정이다. 본 논문에서는 새로운 상업용 키트인 Yfiler ®

The Development and Use of Internal Amplification Controls (IACs) with DNA Profiling Kits for Forensic DNA Analysis.

Biological samples recovered for forensic investigations are often degraded and/or have low amounts of DNA; in addition, in some instances the samples may be contaminated with chemicals that can act as PCR inhibitors. As a consequence this can make interpretation of the results challenging with the possibility of having partial profiles and false negative results. Because of the impact of DNA analysis on forensic investigations, it is important to monitor the process of DNA profiling, in particular the amplification reaction. In this chapter we describe a method for the in-house generation and use of internal amplification controls (IACs) with DNA profiling kits to monitor the success of the PCR proces. In the example we show the use of the SGM Plus® kit. These controls can also be used to aid the interpretation of the DNA profile.

地球周回軌道に投入された超小型衛星の月への移動手法

When we develop a space probe, are needed to carry out its orbit analysis. Space industries and space agencies they have been using the System Tool Kit (STK) for these orbit analysis. Most of satellite projects were used this software. An allowable mass demand of a rocket to an ultra-small space probe is very severe, for this reason a throughput and a number of a microcomputer are restricted. Since this probe cruises deep space, advanced autonomous control capability is required. It is important that an orbital prediction method as highly precise as possibly simple is built. An analyzed result by this new method is agreement with an analysis result by this STK. This may use be utilized for an autonomous control type ultra-small probe. This paper shows an outline of this new orbital prediction method.

Comparison of FXII contact activation with or without proteolytic cleavage

Event Abstract Back to Event Comparison of FXII contact activation with or without proteolytic cleavage Yuan Yan1, Lichong Xu1 and Christopher A. Siedlecki1 1 The Pennsylvania State University Hershey Medical Center, Department of Surgery and Bioengineering, United States Introduction: Surface-induced plasma coagulation is potentiated by contact activation of the blood zymogen factor XII (FXII, Hageman factor). Recent studies revealed that FXII surface activation exhibited different characteristics in neat-buffer and in plasma[1]. aFXIIa and βFXIIa are well known products of FXII contact activation via enzymatic cleavage in the presence of plasma proteins such as prekallikrein, plasmin, and trypsin[2]. Activation products of FXII in buffer (FXII autoactivation) are yet to be fully understood. This study compared the products of FXII activation with or without adding prekallikrein (PK) with regard to activated FXII composition, procoagulant and amidolytic activities. Materials and Methods: Activators used were glass particles (425-600 µm, Sigma Aldrich) in either cleaned or silanized form. 500 µL of 30 µg/ml FXII in PBS was brought into contact with 100 mg of different activators with or without added PK for 1 hr on a rotating hematology mixer. The products present in the supernatant were then assessed by high-resolution electrophoresis (Agilent Protein 230 kit) for molecular weight analysis, a plasma coagulation time assay for procoagulant yield, and a chromogenic assay with Pefa-5963 for amidolytic yield. Results and Discussion: Electrophoretic analyses of FXII activation products in neat-buffer with or without PK. In the absence of PK, the products of FXII activation showed only one band in the size of 95-98 kDa regardless of surface activator, indicating that no proteolytic cleavage takes place in FXII autoactivation. Instead, these FXII-derived products appear to be of the same molecular weight as initial FXII. Note that they present as~ 20% higher molecular weight as observed by automated electrophoresis due to the glycosylation of FXII[3]. In the presence of PK, FXII-derived fragments with smaller molecular weight of about 33 kDa were observed. This matches the size of shared catalytic domain by both aFXIIa and βFXIIa. Yields of FXII-derived products by different activators are shown in Table 1 according to the concentrations calculated by electrophoresis software. Functional analyses of FXII activation products in buffer with or without PK (Figure 1). FXII autoactivation takes place at all surface types, not only at anionic, as demonstrated by measurable procoagulant and amidolytic activities. However, procoagulant yields from OTS and APTES were much lower than that of Glass, suggesting activators of varying surface energy induce different FXII conformers displaying remarkably different procoagulant activity. The presence of PK (or proteolytic cleavage) amplified both procoagulant and amidolytic yields from all surface types in a manner roughly proportional to the amount of βFXIIa and total FXIIa, respectively. Conclusion: FXII contact activation in buffer does not produce proteolytic fragments. It is likely that FXII undergoes non-enzymatic conformational changes in response to surface contact, thus showing different procoagulant and amidolytic activities depending on surface energy. The presence of prekallikrein amplifies both activities, likely as the result of subsequent enzymatic cleavage of FXII into aFXIIa and βFXIIa.

Effect of increasing maximal aerobic exercise on serum gonadal hormones and alpha-fetoprotein in the luteal phase of professional female soccer players.

[Purpose] The performance of female athletes during their menstrual period has attracted the attention of researchers for many years. It is known that the menstrual period changes with exercise. Alpha-fetoprotein (AFP) is an oncofetal protein. In this study, the effect of maximal aerobic exercise in the luteal phase on some hormones and AFP in female athletes was researched. [Subjects and Methods] Twelve volunteers and healthy female footballers with normal menstrual cycles volunteered for this study as subjects. All the participants performed a shuttle run test. Blood samples were taken before, after, and one hour after exercise. Serum AFP, estrogen, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) values were measured using an auto analyzer and original kits. Heart rate measurements were performed before and after the exercise. [Results] AFP activity had significantly decreased after 1 h of recovery from the exercise in the female soccer players, and estrogen and LH activity had significantly increased immediately after the exercise. Progesterone activity had significantly decreased immediately after the exercise. FSH values had significantly increased immediately after the exercise. [Conclusion] The results of the present study show there were significant decreases in the values of AFP, which is a cancer parameter, 1 hour after the exercise. This result may be valuable in future physiotherapy studies on the relationship between exercise and cancer.

Effect and mechanism of inhibition of lipopolysaccharide-induced pulmonary fibrosis by butyric acid

To evaluate the inhibitory effect of butyric acid (BA) as a histone deacetylase (HDAC) inhibitor on lipopolysaccharide (LPS)-induced pulmonary fibrosis and its mechanism. Thirty C57/BL6 mice were randomly divided into three groups according to the random number method, namely control group (physiological saline was given intraperitoneally and by gavage), LPS challenge group (LPS-induced murine model of pulmonary fibrosis was reproduced with intraperitoneal injection of 10 mg/kg LPS), and BA preconditioning + LPS challenge group (10 mg/kg BA was given followed by intraperitoneal injection of 10 mg/kg LPS), with 10 mice in each group. Mice were sacrificed painlessly, and lung tissue samples were harvested at 2 weeks and 4 weeks respectively (five samples every group each time). HDAC activity was evaluated with fluorescence analysis kit. Protein expression of acetylated-histone H3 (Ace-H3), acetylated-histone H4 (Ace-H4) and thymocyte differentiation antigen 1 (Thy-1) were determined by Western Blot. The mRNA expression of Thy-1 was assessed by real-time reverse transcription- polymerase chain reaction (real-time RT-PCR). The degree of lung inflammation and fibrosis were microscopic detected after hematoxylin-eosin (HE) staining and Masson collagen staining. The deposition of lung collagen was detected by hydroxyproline content measurement kit. Compared to control group, the degree of lung inflammation and fibrosis was aggravated after LPS challenge, as manifested by increased hydroxyproline content (μg/mg, 2 weeks: 8.384±0.632 vs. 4.388±0.334, 4 weeks: 8.308±0.244 vs. 4.370±0.342, both P < 0.01), increased HDAC activity (μmol/L, 2 weeks: 7.243±0.384 vs. 3.628±0.641, 4 weeks: 6.479±0.202 vs. 3.238±0.524, both P < 0.01), increased deacetylation degree of histone H3 and H4 [relative expression of Ace-H3 (gray value): 0.516±0.115 vs. 1.005±0.359 at 2 weeks, 0.633±0.143 vs. 1.092±0.193 at 4 weeks, both P < 0.05; relative expression of Ace-H4 (gray value): 0.402±0.164 vs. 0.759±0.187 at 2 weeks, P > 0.05; 0.426±0.098 vs. 0.858±0.177 at 4 weeks, P < 0.01], and lowered Thy-1 mRNA and protein expression [Thy-1 mRNA (2(-ΔΔCt)): 0.606±0.066 vs. 1.005±0.109 at 2 weeks, P < 0.01; 0.824±0.101 vs. 1.210±0.400 at 4 weeks, P > 0.05; relative expression of Thy-1 protein (gray value): 0.725±0.284 vs. 1.249±0.297 at 2 weeks, 0.589±0.139 vs. 1.372±0.343 at 4 weeks, both P < 0.05]. Compared with LPS group, BA precondition could inhibit above processes, as manifested by decreased hydroxyproline content (μg/mg: 5.943±0.726 vs. 8.384±0.632 at 2 weeks, 4.938±0.209 vs. 8.308±0.244 at 4 weeks, both P < 0.01), decreased HDAC activity (μmol/L: 4.386±0.117 vs. 7.243±0.384 at 2 weeks, 4.863±0.096 vs. 6.479±0.202 at 4 weeks, both P < 0.01), increased Thy-1 mRNA expression at 2 weeks (2(-ΔΔCt): 0.884±0.216 vs. 0.606±0.066, P < 0.05), increased acetylation degree of histone H4 and Thy-1 protein expression at 4 weeks [relative expression of Ace-H4 (gray value): 0.715±0.145 vs. 0.426±0.098, P < 0.05; relative protein expression of Thy-1 (gray value): 0.939±0.098 vs. 0.589±0.139, P < 0.01]. LPS-induced pulmonary fibrosis was related with activation of HDAC, deacetylation of histone H3 and H4 and Thy-1 gene silencing. HDAC inhibitor BA could inhibit LPS-induced pulmonary fibrosis and Thy-1 gene silencing through inhibiting activation of HDAC and deacetylation of histone H4.

The Influence on Soil Classification of Processing Soil With a Coffee Grinder

Abstract The rapid soils analysis kit uses a coffee grinder to break apart aggregated soil particles in fine-grained soils. This leads to the question of potential particle cutting or breaking due to the grinder. Hence, we performed a laboratory investigation to determine if the use of a coffee grinder to break apart aggregated soil particles produces fines from cutting or breaking sand-size and smaller particles and whether the production of more fines sometimes changes the USCS classification by some combination of adding soil fines and altering the Atterberg limits. Processing Ottawa Sand for 90 s in a coffee grinder established that the coffee grinder breaks down sand particles into fines. A silty sand (SM) sand was tested in the same way and the fines content increased significantly. Several tests were performed on fine-grained soils, and no significant increase in fines due to processing in the coffee grinder was noted. The Atterberg limits of all soils tested changed little due to processing with the grinder. In particular, the plastic limit was not changed by more than 2 (% water content). Several recommendations were made for potential future investigations.

Overexpression of X-Box Binding Protein 1 (XBP1) Correlates to Poor Prognosis and Up-Regulation of PI3K/mTOR in Human Osteosarcoma

Increasing evidence demonstrates that dysregulation of XBP1 function contributes to tumorigenesis in some cancers. However, little is known about the role of XBP1 in the progression of osteosarcoma (OS). The expression of XBP1 in OS samples was measured by quantitative RT-PCR and Western blotting assays. Cell cycle analysis and cell counting kit 8 (CCK8) assays were performed to determine the effects of XBP1 expression on cells growth capacity. Cell apoptosis coassay was applied to determine cell survival. The expression of genes affected by XBP1 was examined by quantitative RT-RCR and validated by Western blotting assays. XBP1 was overexpressed in OS clinical samples compared with corresponding non-cancerous tissues. Overexpression of XBP1 was significantly associated with advanced clinical stages, high degree of malignancy and low tumor necrosis rate. Furthermore, hypoxia activated XBP1, and silencing XBP1 significantly enhanced OS cell apoptosis. Knock-down of XBP1 resulted in inhibition of OS growth. Most importantly, knockdown of XBP1 led to down-regulation of PIK3R3 and mTOR. Taken together, XBP1 is up-regulated and has a pro-tumor effect in OS with activation of PI3K/mTOR signaling. Thus, targeting XBP1 may provide a new potential therapeutic method for OS.

Cytology smears as diagnostic material for EGFR gene testing in non-small cell lung cancer.

Cytology smears can be effectively used for EGFR mutation testing in the qualification of NSCLC patients for EGFR tyrosine kinase inhibitor therapy. However, tissue specimens are preferred for EGFR mutation analysis. The aim of this study was to estimate the effectiveness of the real-time PCR method for EGFR testing in histology and cytology materials obtained simultaneously from NSCLC patients. Fourteen adenocarcinoma patients with EGFR-mutation-positive primary tumor tissues were included in the study. Corresponding cytological smears of metastatic lymph nodes obtained by EBUS-TBNA were examined. EGFR Mutation Analysis Kit (EntroGen, USA) and real-time PCR (m2000rt system, Abbott, USA) were used for EGFR mutation analysis in both types of material. In primary tumor tissues, 12 deletions in exon 19 and 2 substitutions in exon 21 (L858R mutation) of the EGFR gene were found. Except for 1 deletion in exon 19, the same EGFR gene mutations were detected in all corresponding cytology samples. The percentage of tumor cells, DNA concentration, percentage of mutated DNA as well as ΔCt values were similar in cytology slides and histology material. In both types of materials, no significant correlations were found between the percentage of tumor cells and the percentage of mutated DNA nor between the DNA concentration and the percentage of mutated DNA. We demonstrated the high effectiveness of a sensitive real-time PCR method in EGFR gene mutation detection in cytology smears.

A panel of PCR-inhibitory reference materials for quality evaluation of multiplex STR analysis kits

PCR inhibition is a critical parameter in forensic DNA analysis. Substances interfering with amplification of short tandem repeats (STRs) may generate partial and/or ambiguous DNA profiles. We present a strategy for developing a broad panel of PCR-inhibitory reference materials (RMs), representing common casework samples. Our panel, including solutions prepared from for example cigarettes, chewing gum and soil, is a tool for in-house validation and lot testing of STR systems. PowerPlex ESX 16 Fast System tolerated high levels of some RMs, but several substances caused amplification problems. Humic acid had a negative effect on amelogenin, moist snuff hindered amplification of longer fragments, and chewing gum caused generally lowered allele peak heights. Applying a broad panel of RMs ensures that a wide range of inhibitory substances are tested, giving an improved understanding of inhibitor tolerance and effects.

Initial experience in the application of the PowerQuant™ system and the Investigator® ESSplex SE QS kit for aDNA analysis

Degradation-related poor quality of ancient DNA from long-time stored biological material (e.g. historical skeletal remains) often leads to a loss of information in molecular genetic characterisation. A new generation of DNA quantification and STR technologies consider mentioned issue by an optimised primer design combined with active master mix components. In this respect, the efficiency of the new PowerQuant™ (Promega) quantification kit and the Investigator® ESSplex SE QS STR kit (QIAGEN) was tested and analysed. Both analyses tools were evaluated by their applicability to monitor the presence of possible PCR inhibitors and DNA degradation that influences downstream STR analyses and further result interpretation. Characteristic features that reflect mentioned phenomena of both systems where correlated with respect to a combined use in aDNA analyses.Here, especially the Investigator® ESSplex SE QS kit, which features an intelligent system design comprising both DNA typing and quality control, has shown to be valuable. Some given result interpretation thresholds were adapted with regard to aDNA applicability in forensic case work and anthropological interest.

AB029. Next generation sequencing analysis in hereditary muscle diseases

Still many cases with probable hereditary myopathy are genetically undiagnosed for two major reasons: (I) muscle genes are often big in size and thus it is difficult to at least routinely sequence such genes even though mutations in those genes have been known to cause muscle diseases; and (II) most cases are sporadic without parental consanguinity, which makes us difficult to do linkage study to identify new causative genes. However, the advent of next-generation sequencers is changing the diagnostic and research scenes in myology just as in many other fields. We have set up target resequencing panels that basically cover all known causative genes for hereditary muscle diseases (as of early 2014). We started providing genetic analysis in September 2014 using this system to the cases whose pathological analysis was done at NCNP. So far we have reached diagnosis in 27% of those cases. To identify new causative genes, we applied whole exome sequencing (WES) analysis. In the last 3 years, we have performed more than 750 exome analyses but we found causative mutations in only 11% of cases. This low rate appears to be at least partly due to the fact that Japanese variation database is not well established, in addition to the fact that currently available WES analysis kits do not fully, albeit mostly, cover all exonic regions. In my talk, I will demonstrate some of our analysis data, including the identification of ORAI1 as a causative gene for tubular aggregate myopathy, and discuss current status and yet-to-be-solved issues of next-generation sequencing in myology field.

Pushing the limits of preimplantation genetic diagnosis (PGD): a Karyomapping and Next Generation Sequencing (NGS) stress test

We sought to examine the limits of embryo genetic analysis by performing PGD and aneuploidy screening (PGS) in 1) a consanguineous mating for, 2) two disease-causing point mutations and 3) HLA-genotyping to match 3 affected children while 4) detecting genetic recombination and potential maternal cell contamination, and 5) 24-chromosome aneuploidy screening with NGS. Karyomapping utilizes ∼300,000 Single Nucleotide Polymorphisms (SNPs) across the human genome and allows high-resolution analysis for nearly every gene. We used karyomapping to rapidly design a PGD test targeting the HBB gene and the highly recombinant HLA locus to identify embryos with one or both healthy HBB alleles and an HLA genotype matching the children in need of transplantation. Cheek-swab kits were used to collect DNA samples from the parents and affected children. Mutation status of each individual was confirmed by direct sequencing. DNA samples were analyzed using the Infinium HumanKaryomap-12 DNA Analysis Kit from Illumina. BluFuse Multi software was used to compare the SNP profiles of each family member to identify informative SNPs, regions of consanguinity, and determine HLA phase for each affected child. NGS was used for 24-chromosome aneuploidy screening. We identified sufficient informative SNPs surrounding the HBB gene and HLA region to track the inheritance of familial haplotypes. As expected, the same HBB haplotypes were observed in each affected child. Two children were found to have the same set of HLA haplotypes, while the third inherited the opposite paternal HLA haplotype. This is consistent with the HLA-typing reported by an external laboratory. Recombination and consanguineous regions were observed but did not impact our ability to determine genetic phase. We successfully used karyomapping to build a complex PGD test, which would have been highly challenging using previous methods. Moreover, we determined the HLA phases of all three children simultaneously and confirmed that recombination and consanguinity would not interfere with diagnosis or HLA matching. Our success addresses concerns about using karyomapping for HLA matching and diagnosing embryos of consanguineous families. Karyomapping combined with PGS by Next-Generation Sequencing represents a great advance to the field, 25 years after its inception.

Gene expression profiling of archived dried blood spot samples from the Danish Neonatal Screening Biobank.

A large part of the human genome is transcribed into various forms of RNA, and the global gene expression profile (GEP) has been studied for several years using technology such as RNA-microarrays. In this study, we evaluate whether neonatal dried blood spot (DBS) samples stored in the Danish Neonatal Screening Biobank (DNSB) can be used for GEP. This paper is divided into sub-studies examining the effects of: 1) different whole transcriptome amplification kits (WTA); 2) years of storage and storage in room temperature (RT) versus freezers (-20°C) on DNSB DBS samples; 3) effects of RT storage vs freezer storage on DBS samples from the USA and DNSB, and 4) using smaller disc sizes, thereby decreasing DBS use. We present evidence that reliable and reproducible GEPs can be obtained using neonatal DBS samples. The main source of variation is the storage condition. When samples are stored at -20°C, the dynamic range is increased, and Pearson correlations are higher. Differential analysis reveals no statistically significant differences between samples collected a decade apart and stored at -20°C. However, samples stored at RT show differential expression for a third of the gene-specific probes. Our data also suggests that using alternate WTA kits significantly changes the GEP. Finally, the amount of input material, i.e., the size and number of DBS discs used, can be reduced to preserve this valuable and limited material. We conclude that DNSB DBS samples provide a reproducible resource for GEP. Results are improved if the cards are stored at -20°C. Furthermore, it is important to use a single type of kit for analysis because using alternate kits introduces differential expression.

Quantification of hypoglycin A in serum using aTRAQ(®) assay.

Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine's biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication. Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition. Outlined accuracy profiles demonstrated very reliable data. A good linearity was observed from 0.09 to 50μmol/L and precision was very good with coefficient of variation below 8%. Fifty-five samples collected from 25 confirmed AM horses revealed significant hypoglycin A concentrations, while toxin was not found in serum of 8 control animals. The described aTRAQ variant method has been analytically and clinically validated. The reliability of our approach is thus demonstrated into the workup of atypical myopathy.