Analysis of archived residual newborn screening blood spots after whole genome amplification

Background

Deidentified newborn screening bloodspot samples (NBS) represent a valuable potential resource for genomic research if impediments to whole exome sequencing of NBS deoxyribonucleic acid (DNA), including the small amount of genomic DNA in NBS material, can be overcome. For instance, genomic analysis of NBS could be used to define allele frequencies of disease-associated variants in local populations, or to conduct prospective or retrospective studies relating genomic variation to disease emergence in pediatric populations over time. In this study, we compared the recovery of variant calls from exome sequences of amplified NBS genomic DNA to variant calls from exome sequencing of non-amplified NBS DNA from the same individuals.

Results

Using a standard alignment-based Genome Analysis Toolkit (GATK), we find 62,000–76,000 additional variants in amplified samples. After application of a unique kmer enumeration and variant detection method (RUFUS), only 38,000–47,000 additional variants are observed in amplified gDNA. This result suggests that roughly half of the amplification-introduced variants identified using GATK may be the result of mapping errors and read misalignment.

Conclusions

Our results show that it is possible to obtain informative, high-quality data from exome analysis of whole genome amplified NBS with the important caveat that different data generation and analysis methods can affect variant detection accuracy, and the concordance of variant calls in whole-genome amplified and non-amplified exomes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1747-2) contains supplementary material, which is available to authorized users.     

Discovery stage pharmacokinetics using dried blood spots

Early in the discovery stage, the measurement of drug candidates in biological fluids as a function time provides important information used in decision making for lead optimization. The detection methodology primarily used is liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS). Sample preparation is an important aspect of these experiments and robotic-based automation is commonly used. The often overlooked aspect of these experiments is the sample collection itself. Typically, several hundred microliters of whole blood is collected and the plasma fraction separated for each time-point. The plasma is then transferred to an appropriate vessel for subsequent aliquoting and processing. We describe a method for performing discovery stage pharmacokinetic analysis using whole blood dried onto filter paper. The use of dried blood spots is a well established technique for neo-natal screening, and its application to early screening of drug candidates proves to be robust, reliable and reproducible.     

Novel approach for deriving genome wide SNP analysis data from archived blood spots

ABSTRACT: BACKGROUND: The ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman[trade mark sign] technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman[trade mark sign] cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored. FINDINGS: DNA was extracted from FTA Whatman[trade mark sign] cards (following adaptations of the manufacturer's instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way. CONCLUSIONS: DNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies.     

DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening

Summary Microextraction of DNA from dried blood specimens would ease specimen transport to centralized laboratory facilities for recombinant DNA diagnosis in the same manner as use of dried blood spots allowed the broad application of screening tests to newborn populations. A method is described which reproducibly yields 0.5g DNA from the dried equivalent of 50l whole blood. Though DNA yields decreased with storage of dried specimens at room temperature, good-quality DNA was still obtained. Sufficient DNA was routinely obtained for Southern blot analysis using repetitive and unique sequences. This microextraction procedure will allow immediate application of molecular genetic technology to direct newborn screening follow-up of disorders amenable to DNA diagnosis, such as sickle cell anemia, and may eventually permit primary DNA screening for specific mutations.     

Improved batch correction in untargeted MS-based metabolomics

Introduction

Batch effects in large untargeted metabolomics experiments are almost unavoidable, especially when sensitive detection techniques like mass spectrometry (MS) are employed. In order to obtain peak intensities that are comparable across all batches, corrections need to be performed. Since non-detects, i.e., signals with an intensity too low to be detected with certainty, are common in metabolomics studies, the batch correction methods need to take these into account.

Objectives

This paper aims to compare several batch correction methods, and investigates the effect of different strategies for handling non-detects.

Methods

Batch correction methods usually consist of regression models, possibly also accounting for trends within batches. To fit these models quality control samples (QCs), injected at regular intervals, can be used. Also study samples can be used, provided that the injection order is properly randomized. Normalization methods, not using information on batch labels or injection order, can correct for batch effects as well. Introducing two easy-to-use quality criteria, we assess the merits of these batch correction strategies using three large LC–MS and GC–MS data sets of samples from Arabidopsis thaliana.

Results

The three data sets have very different characteristics, leading to clearly distinct behaviour of the batch correction strategies studied. Explicit inclusion of information on batch and injection order in general leads to very good corrections; when enough QCs are available, also general normalization approaches perform well. Several approaches are shown to be able to handle non-detects—replacing them with very small numbers such as zero seems the worst of the approaches considered.

Conclusion

The use of quality control samples for batch correction leads to good results when enough QCs are available. If an experiment is properly set up, batch correction using the study samples usually leads to a similar high-quality correction, but has the advantage that more metabolites are corrected. The strategy for handling non-detects is important: choosing small values like zero can lead to suboptimal batch corrections.

     

Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies

A reversed phase HPLC-MS/MS method has been developed and validated for the quantitative bioanalysis of acetaminophen in dried blood spots (DBS) prepared from small volumes (15 microL) of dog blood. Samples were extracted for analysis with methanol. Detection was by positive ion TurboIonSpray ionisation combined with selected reaction monitoring MS. The analytical concentration range was 0.1-50 microg/mL. The intra-day precision and bias values were both less than 15%. Acetaminophen was stable in DBS stored at room temperature for at least 10 days. The methodology was applied in a toxicokinetic (TK) study where the data obtained from DBS samples was physiologically comparable with results from duplicate blood samples (diluted 1:1 (v/v) with water) analysed using identical HPLC-MS/MS conditions. This work demonstrates that quantitative analysis of a drug extracted from DBS can provide high quality TK data while minimising the volume of blood withdrawn from experimental animals, to an order of magnitude lower than is current practice in the pharmaceutical industry. This is the first reported application of DBS analysis to a TK study in support of a safety assessment study. The success of this and similar, related studies has led to the intent to apply DBS technology as the recommended analytical approach for the assessment of pharmacokinetics (PK)/TK for all new oral small molecule drug candidates, which have previously demonstrated a successful bioanalytical validation.     

A dried blood spots technique based LC-MS/MS method for the analysis of posaconazole in human whole blood samples

A rugged and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method utilizing dried blood spots (DBS) was developed and validated for the analysis of posaconazole in human whole blood. Posaconazole fortified blood samples were spotted (15 μL) onto Ahlstrom Alh-226 DBS cards and dried for at least 2h. Punched spots were then extracted by using a mixture of acetonitrile and water containing stable labeled internal standard (IS). Posaconazole and its IS were separated from endogenous matrix components on a Kinetex? C18 column under gradient conditions with a mobile phase A consisting of 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (70/30, v/v). The analyte and IS were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray? source operated in the positive ion mode. The assay was linear over the concentration range of 5-5000 ng/mL. The inter-run accuracy and precision of the assay were -1.8% to 0.8% and 4.0% to 10.4%, respectively. Additional assessments unique to DBS were investigated including sample spot homogeneity, spot volume, and hematocrit. Blood spot homogeneity was maintained and accurate and precise quantitation results were obtained when using a blood spot volume of between 15 and 35 μL. Human blood samples with hematocrit values ranging between 25% and 41% gave acceptable quantitation results. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible.     

Newborn screening by DNA analysis of dried blood spots

Summary Amplification of DNA recovered from a dried blood spot was used to genotype individuals with sickle cell disease, sickle cell carriers, and controls. A single 200-l blood spot applied to a filter paper provides sufficient material for more than 20 genetic analyses. In addition, the stability of the DNA is such that adequate material for amplification can be isolated from dried blood spots up to a year following collection. The DNA analysis methods described in this study could be applied to large-scale screening of newborns for genetic disorders.     

Sensitive high-performance liquid chromatographic method using coulometric electrode array detection for measurement of phytoestrogens in dried blood spots

As the epidemiological and physiological investigation of isoflavones and lignans expands, the need for sensitive methods for analyzing large numbers of samples intensifies. We have developed a method using high-performance liquid chromatography (HPLC) equipped with a coulometric electrode array detector for separation and sensitive detection of daidzein (Da), equol (Eq), genistein (Ge) and enterolactone (Enl) in dried blood spots (DBS). Detection limits ranged from 4.5 pg or 0.09 ng/mL (Eq) to 19 pg or 0.38 ng/mL (Ge) on column. Signal linearities ranged from detection limits to 200 ng/mL (Eq, Enl) and 600 ng/mL (Da, Ge) sample concentration. Correlations between DBS and serum concentrations were 0.66 (Enl), 0.88 (Eq), 0.98 (Ge) and 0.99 (Da). Intra-assay coefficients of variation (CVs) were less than 8% and inter-assay CVs ranged from 2.4 to 20.2% for Da, Eq and Ge for three levels of controls. Enl intra-assay CV was 13.6% for the low pooled control. Analytic recovery ranged from 87% (inter-assay Ge) to 98% (inter-assay Enl). DBS concentrations of Da, Ge and Eq were stable for at least 8 weeks at 4 and 25 degrees C, and at 37 degrees C for at least 5 weeks, with Enl showing greater variability at all temperatures but relative stability for 7 weeks. Measurement of samples from 135 perimenopausal Japanese women consuming habitual diets in Kyoto and Fukushima prefectures showed the former to have the expected lower concentrations of Da and Eq (416 and 87 nM) as well as Enl (49 nM) compared to the latter locale (566, 145 and 72 nM, respectively). This method could be useful in large epidemiological research or detailed physiological studies.     

Simultaneous diagnostic method for phenylketonuria and galactosemia from dried blood spots using high-performance liquid chromatography-pulsed amperometric detection

We developed a simultaneous diagnostic method for phenylketonuria (PKU) and galactosemia through simultaneous determination of phenylalanine (Phe) and galactose (Gal) by high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD). The intra- and inter-day precisions were <5.8%, with satisfactory mean recoveries (98.2–105%). For all PKU-positive samples, Phe levels were above the cut-off value (>30.0 mg/L), but Gal levels were nearly zero. For 77% of galactosemia-positive samples, Phe levels were above the cut-off value, but Gal levels were above the cut-off value (>80.0 mg/L) for all samples. Our HPLC-PAD method can reduce the false-positive rate of misdiagnosis for PKU and galactosemia.     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Use of dried blood spots and inductively coupled plasma mass spectrometry for multi-element determination in blood

The paper describes the development of an inductively coupled plasma mass spectrometry (ICP MS) method for multitrace element determination in dried blood spots (DBSs). The analytical conditions were optimized using Seronorm™ L-3 and L-1 Certified Reference Materials. The best results were obtained by sampling blood drops on a decontaminated PVDF filter membrane. After drying under metal-free conditions, the DBSs underwent acidic digestion and were analyzed with ICP MS. The method was then validated for As, Cd, Cu, Pb, Mo, Se and Zn. Using a matrix-matched calibration curve, the recovery levels ranged from 96% to 117%. The repeatability and reproducibility were generally below 15%. Limits of quantification ranging from 0.5 to 50 μg/L. In order to investigate the analytical procedure under real sampling conditions, the results obtained from DBSs and liquid blood aliquots (less subject to contamination) from two adult subjects were compared.     

A comparison of high-throughput plasma NMR protocols for comparative untargeted metabolomics

Metabolomics - Understanding the interaction between organisms and the environment is important for predicting and mitigating the effects of global phenomena such as climate change, and the fate,...     

Microdetermination of galactose and galactose 1-phosphate in dried blood spots

Newborn screening for galactosemia (galactose-1-phosphate uridyltransferase deficiency), as well as for other defects in galactose metabolism (galactokinase deficiency and uridine diphosphogalactose 4-epimerase deficiency), requires a method of determining both galactose and galactose 1-phosphate in dried blood. We have developed a sequential quantitative method for the microdetermination of galactose and galactose 1-phosphate that can be applied to 3-mm-diameter disks of dried blood and that can be used with a Technion Autoanalyser II equipped with a fluorometer.Galactose is determined by the fluorescence of NADH following treatment with β-galactose dehydrogenase and with the consequent reduction of NAD. The complete system includes alkaline phosphatase for the hydrolysis of galactose 1-phosphate, so that the total amounts of a galactose and galactose 1-phosphate are determined. For the measurement of galactose alone, alkaline phosphate is omitted from the system. The difference in fluorescence between that from the complete system and that from the alkaline phosphatase-omitted system yields the concentration of galactose 1-phosphate.     

DHPR activity decrease in dried blood spots stored at 4 degrees C

Dihydropteridine reductase (DHPR) activity has been measured in dried blood spots by the Arai method in 286 subjects divided into 4 groups: 45 newborns aged between 4 and 7 days; 40 subjects with hyperphenylalainaemia; 199 apparently normal subjects, and 2 patients with DHPR deficiency. Furthermore, a study of the persistence of enzymatic activity on the spots stored at 4 degrees C revealed that after 1 year it decreased to one third of the original value, corresponding to the mean value of controls minus 2 SD.     

Cytomegalovirus antibodies in dried blood spots: a minimally invasive method for assessing stress,immune function,and aging

Background  

Cytomegalovirus (CMV) is a prevalent herpesvirus with links to both stress and aging. This paper describes and validates a minimally invasive method for assessing antibodies against CMV in finger stick whole blood spot samples for use as an indirect marker of an aspect of cell-mediated immunity.     

Mass appeal: metabolite identification in mass spectrometry-focused untargeted metabolomics

Metabolomics has advanced significantly in the past 10 years with important developments related to hardware, software and methodologies and an increasing complexity of applications. In discovery-based investigations, applying untargeted analytical methods, thousands of metabolites can be detected with no or limited prior knowledge of the metabolite composition of samples. In these cases, metabolite identification is required following data acquisition and processing. Currently, the process of metabolite identification in untargeted metabolomic studies is a significant bottleneck in deriving biological knowledge from metabolomic studies. In this review we highlight the different traditional and emerging tools and strategies applied to identify subsets of metabolites detected in untargeted metabolomic studies applying various mass spectrometry platforms. We indicate the workflows which are routinely applied and highlight the current limitations which need to be overcome to provide efficient, accurate and robust identification of metabolites in untargeted metabolomic studies. These workflows apply to the identification of metabolites, for which the structure can be assigned based on entries in databases, and for those which are not yet stored in databases and which require a de novo structure elucidation.