An untargeted metabolomics method for archived newborn dried blood spots in epidemiologic studies

Introduction

For pediatric diseases like childhood leukemia, a short latency period points to in-utero exposures as potentially important risk factors. Untargeted metabolomics of small molecules in archived newborn dried blood spots (DBS) offers an avenue for discovering early-life exposures that contribute to disease risks.

Objectives

The purpose of this study was to develop a quantitative method for untargeted analysis of archived newborn DBS for use in an epidemiological study (California Childhood Leukemia Study, CCLS).

Methods

Using experimental DBS from the blood of an adult volunteer, we optimized extraction of small molecules and integrated measurement of potassium as a proxy for blood hematocrit. We then applied this extraction method to 4.7-mm punches from 106 control DBS samples from the CCLS. Sample extracts were analyzed with liquid chromatography—high resolution mass spectrometry (LC-HRMS) and an untargeted workflow was used to screen for metabolites that discriminate population characteristics such as sex, ethnicity, and birth weight.

Results

Thousands of small molecules were measured in extracts of archived DBS. Normalizing for potassium levels removed variability related to varying hematocrit across DBS punches. Of the roughly 1000 prevalent small molecules that were tested, multivariate linear regression detected significant associations with ethnicity (three metabolites) and birth weight (15 metabolites) after adjusting for multiple testing.

Conclusions

This untargeted workflow can be used for analysis of small molecules in archived DBS to discover novel biomarkers, to provide insights into the initiation and progression of diseases, and to provide guidance for disease prevention.

     

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.     

Primase-based whole genome amplification

In vitro DNA amplification methods, such as polymerase chain reaction (PCR), rely on synthetic oligonucleotide primers for initiation of the reaction. In vivo, primers are synthesized on-template by DNA primase. The bacteriophage T7 gene 4 protein (gp4) has both primase and helicase activities. In this study, we report the development of a primase-based Whole Genome Amplification (pWGA) method, which utilizes gp4 primase to synthesize primers, eliminating the requirement of adding synthetic primers. Typical yield of pWGA from 1 ng to 10 ng of human genomic DNA input is in the microgram range, reaching over a thousand-fold amplification after 1 h of incubation at 37 degrees C. The amplification bias on human genomic DNA is 6.3-fold among 20 loci on different chromosomes. In addition to amplifying total genomic DNA, pWGA can also be used for detection and quantification of contaminant DNA in a sample when combined with a fluorescent reporter dye. When circular DNA is used as template in pWGA, 10(8)-fold of amplification is observed from as low as 100 copies of input. The high efficiency of pWGA in amplifying circular DNA makes it a potential tool in diagnosis and genotyping of circular human DNA viruses such as human papillomavirus (HPV).     

PCR-mediated whole genome amplification of phytoplasmas

A method was developed for genome analysis of phytoplasmas, bacterial plant pathogens that cannot be cultivated in vitro in cell-free media. The procedure includes a CsCl-bisbenzimide gradient buoyant centrifugation followed by polymerase chain reaction (PCR)-mediated whole genome amplification. The latter step involves digestion of the DNA by a restriction enzyme with an A/T-rich recognition sequence. Due to the different A/T content in the DNA of the pathogen and its plant host, the fragments originating from phytoplasma are shorter and are preferentially amplified in the PCR reaction. Products obtained were cloned and screened by dot-blot hybridization. Results showed that about 90% of recombinant clones appeared to harbor phytoplasma specific DNA inserts. Sequencing of randomly selected clones was carried out and comparison with the NCBI database confirmed the bacterial origin for the sequences, which have been assigned a putative function. The origin of the recombinant clones was further confirmed by the generation of specific amplicons from the phytoplasma-infected plant and not from the healthy control, using PCR primers devised from the sequences of the recombinant clones. This method could be used for genome-wide comparisons between phytoplasmas.     

Quantification of trace-level DNA by real-time whole genome amplification

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.     

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.     

Decontamination of MDA reagents for single cell whole genome amplification

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells.     

Impact of whole genome amplification on analysis of copy number variants

Large-scale copy number variants (CNVs) have recently been recognized to play a role in human genome variation and disease. Approaches for analysis of CNVs in small samples such as microdissected tissues can be confounded by limited amounts of material. To facilitate analyses of such samples, whole genome amplification (WGA) techniques were developed. In this study, we explored the impact of Phi29 multiple-strand displacement amplification on detection of CNVs using oligonucleotide arrays. We extracted DNA from fresh frozen lymph node samples and used this for amplification and analysis on the Affymetrix Mapping 500k SNP array platform. We demonstrated that the WGA procedure introduces hundreds of potentially confounding CNV artifacts that can obscure detection of bona fide variants. Our analysis indicates that many artifacts are reproducible, and may correlate with proximity to chromosome ends and GC content. Pair-wise comparison of amplified products considerably reduced the number of apparent artifacts and partially restored the ability to detect real CNVs. Our results suggest WGA material may be appropriate for copy number analysis when amplified samples are compared to similarly amplified samples and that only the CNVs with the greatest significance values detected by such comparisons are likely to be representative of the unamplified samples.     

Use of whole genome amplification to rescue DNA from plasma samples

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on phi29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40 degrees C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5' exonuclease (TaqMan) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 microg (standard deviation of 31.2 microg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.     

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.     

Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays

Clinical DNA is often available in limited quantities requiring whole-genome amplification for subsequent genome-wide assessment of copy-number variation (CNV) by array-CGH. In pre-implantation diagnosis and analysis of micrometastases, even merely single cells are available for analysis. However, procedures allowing high-resolution analyses of CNVs from single cells well below resolution limits of conventional cytogenetics are lacking. Here, we applied amplification products of single cells and of cell pools (5 or 10 cells) from patients with developmental delay, cancer cell lines and polar bodies to various oligo tiling array platforms with a median probe spacing as high as 65 bp. Our high-resolution analyses reveal that the low amounts of template DNA do not result in a completely unbiased whole genome amplification but that stochastic amplification artifacts, which become more obvious on array platforms with tiling path resolution, cause significant noise. We implemented a new evaluation algorithm specifically for the identification of small gains and losses in such very noisy ratio profiles. Our data suggest that when assessed with sufficiently sensitive methods high-resolution oligo-arrays allow a reliable identification of CNVs as small as 500 kb in cell pools (5 or 10 cells), and of 2.6–3.0 Mb in single cells.     

Increase of ethanol tolerance of Saccharomyces cerevisiae by error-prone whole genome amplification

Saccharomyces cerevisiae was transformed for higher ethanol tolerance by error-prone whole genome amplification. The resulting PCR products were transformed back to the parental strain for homologous recombination to create a library of mutants with the perturbed genomic networks. A few rounds of transformation led to the isolation of mutants that grew in 9% (v/v) ethanol and 100 g glucose l⁻¹ compared to untransformed yeast which grew only at 6% (v/v) ethanol and 100 g glucose l⁻¹.     

Method for the quantification of underivatized amino acids on dry blood spots from newborn screening by HPLC-ESI-MS/MS

In our study we have developed an HPLC-ESI-MS/MS method for qualitative and quantitative analysis of underivatized amino acids on dry blood spots. The sensitive and specific instrumental performances permitted the chromatographic separation of 40 amino acids and their isomers within 10 min. The method has been set up for cases of suspected metabolic diseases revealed by newborn screening. What is new is that it is applied on the same blood spots used for newborn screening, instead of plasma, in order to avoid involvement of doctors, increased anxiety for parents, stress for patients for plasma collection, long time of waiting and further costs for analysis.     

The use of whole genome amplification in the study of human disease

The availability of large amounts of genomic DNA is of critical importance for many of the molecular biology assays used in the analysis of human disease. However, since the amount of patient tissue available is often limited and as particular foci of interest may consist of only a few hundred cells, the yield of DNA is often insufficient for extensive analysis. To address this problem, several whole genome amplification (WGA) methodologies have been developed. Initial WGA approaches were based on the polymerase chain reaction (PCR). However, recent reports have described the use of non-PCR-based linear amplification protocols for WGA. Using these methods, it is possible to generate microgram quantities of DNA starting with as little as 1mg of genomic DNA. This review will provide an overview of WGA approaches and summarize some of the uses for amplified DNA in various high-throughput genetic applications.     

Brugia malayi: whole genome amplification for genomic characterization of filarial parasites

Genetic characterization of field isolates and clinical specimens of filarial nematodes is often limited by a shortage of DNA; therefore, we evaluated a multiple displacement amplification (MDA) based whole genome amplification method. The quality of amplified DNA was examined by conventional PCR, real-time PCR, and DNA hybridization. MDA of 5.0 ng of adult Brugia malayi DNA and one-fifteenth of the DNA isolated from a single microfilaria resulted in 6.3 and 4.2 μg of amplified DNA, respectively. Amplified DNA was equivalent to native genomic DNA for hybridization to B. malayi BAC library clones or to an oligonucleotide microarray with approximately 18,000 filarial DNA sequences. MDA is useful for whole genome amplification of filarial DNA from very small amounts of starting material. This technology will permit detailed studies of genetic diversity that were not previously feasible.     

Genotyping DNA isolated from UV irradiated human bloodstains using whole genome amplification

Molecular Biology Reports - Analysis of DNA polymorphisms are the primary technique used for personal identification in forensic cases. However, DNA samples collected as evidence from crime scenes...     

Selective whole genome amplification and sequencing of Coxiella burnetii directly from environmental samples

Whole genome sequencing (WGS) is a widely available, inexpensive means of providing a wealth of information about an organism's diversity and evolution. However, WGS for many pathogenic bacteria remain limited because they are difficult, slow and/or dangerous to culture. To avoid culturing, metagenomic sequencing can be performed directly on samples, but the sequencing effort required to characterize low frequency organisms can be expensive. Recently developed methods for selective whole genome amplification (SWGA) can enrich target DNA to provide efficient sequencing. We amplified Coxiella burnetii (a bacterial select agent and human/livestock pathogen) from 3 three environmental samples that were overwhelmed with host DNA. The 68- to 147-fold enrichment of the bacterial sequences provided enough genome coverage for SNP analyses and phylogenetic placement. SWGA is a valuable tool for the study of difficult-to-culture organisms and has the potential to facilitate high-throughput population characterizations as well as targeted epidemiological or forensic investigations.     

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

Background

Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen^® and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR^® Identifiler^® panel) and N = 49 SNP (TaqMan^®) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.

Results

The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.

Conclusion

The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan^® SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR^® Identifiler^® panel from wgaDNA equivalent to that of gDNA.

     

Genotypic analysis of multiple loci in somatic cells by whole genome amplification.

To screen multiple loci in small purified samples of diploid and aneuploid cells a PCR-based technique of whole genome amplification was adapted to the study of somatic lesions. DNA samples from different numbers of flow-sorted diploid and aneuploid cells from biopsies were amplified with a degenerate 15mer primer. Aliquots of these reactions were then used in locus-specific reactions using a single round of PCR cycles with individual sets of primers representing polymorphic markers for different regions. As a result, polymorphic markers for different chromosomal regions, including VNTRs and dinucleotide repeats, can be used to perform up to 30 locus-specific PCR assays with a single sample obtained from fewer than 1000 cells.