Investigating the utility of combining Φ29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray™ genotyping

Background

Sustainable DNA resources and reliable high-throughput genotyping methods are required for large-scale, long-term genetic association studies. In the genetic dissection of common disease it is now recognised that thousands of samples and hundreds of thousands of markers, mostly single nucleotide polymorphisms (SNPs), will have to be analysed. In order to achieve these aims, both an ability to boost quantities of archived DNA and to genotype at low costs are highly desirable. We have investigated Φ29 polymerase Multiple Displacement Amplification (MDA)-generated DNA product (MDA product), in combination with highly multiplexed BeadArray? genotyping technology. As part of a large-scale BeadArray genotyping experiment we made a direct comparison of genotyping data generated from MDA product with that from genomic DNA (gDNA) templates.

Results

Eighty-six MDA product and the corresponding 86 gDNA samples were genotyped at 345 SNPs and a concordance rate of 98.8% was achieved. The BeadArray sample exclusion rate, blind to sample type, was 10.5% for MDA product compared to 5.8% for gDNA.

Conclusions

We conclude that the BeadArray technology successfully produces high quality genotyping data from MDA product. The combination of these technologies improves the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples.     

Epigenetic analysis of laser capture microdissected fetal epithelia

Laser capture microdissection (LCM) is a superior method for nondestructive collection of specific cell populations from tissue sections. Although DNA, RNA, and protein have been analyzed from LCM-procured samples, epigenetic analyses, particularly of fetal, highly hydrated tissue, have not been attempted. A standardized protocol with quality assurance measures was established to procure cells by LCM of the medial edge epithelia (MEE) of the fetal palatal processes for isolation of intact microRNA for expression analyses and genomic DNA (gDNA) for CpG methylation analyses. MicroRNA preparations, obtained using the RNAqueous Micro kit (Life Technologies), exhibited better yields and higher quality than those obtained using the Arcturus PicoPure RNA Isolation kit (Life Technologies). The approach was validated using real-time polymerase chain reaction (PCR) to determine expression of selected microRNAs (miR-99a and miR-200b) and pyrosequencing to determine CpG methylation status of selected genes (Aph1a and Dkk4) in the MEE. These studies describe an optimized approach for employing LCM of epithelial cells from fresh frozen fetal tissue that enables quantitative analyses of microRNA expression levels and CpG methylation.     

Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies.

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g WGA using gDNA from two paired cytobrushes (cytobush 'A' kept in a cell lysis buffer, and 'B' dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.     

Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies

Abstract

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g? WGA using gDNA from two paired cytobrushes (cytobush ‘A’ kept in a cell lysis buffer, and ‘B’ dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.     

Genotyping performance assessment of whole genome amplified DNA with respect to multiplexing level of assay and its period of storage

Whole genome amplification can faithfully amplify genomic DNA (gDNA) with minimal bias and substantial genome coverage. Whole genome amplified DNA (wgaDNA) has been tested to be workable for high-throughput genotyping arrays. However, issues about whether wgaDNA would decrease genotyping performance at increasing multiplexing levels and whether the storage period of wgaDNA would reduce genotyping performance have not been examined. Using the Sequenom MassARRAY iPLEX Gold assays, we investigated 174 single nucleotide polymorphisms for 3 groups of matched samples: group 1 of 20 gDNA samples, group 2 of 20 freshly prepared wgaDNA samples, and group 3 of 20 stored wgaDNA samples that had been kept frozen at -70°C for 18 months. MassARRAY is a medium-throughput genotyping platform with reaction chemistry different from those of high-throughput genotyping arrays. The results showed that genotyping performance (efficiency and accuracy) of freshly prepared wgaDNA was similar to that of gDNA at various multiplexing levels (17-plex, 21-plex, 28-plex and 36-plex) of the MassARRAY assays. However, compared with gDNA or freshly prepared wgaDNA, stored wgaDNA was found to give diminished genotyping performance (efficiency and accuracy) due to potentially inferior quality. Consequently, no matter whether gDNA or wgaDNA was used, better genotyping efficiency would tend to have better genotyping accuracy.     

Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using 29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and “clusters of orthologous groups” (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.     

利用末龄幼虫蜕和蛹壳对苹果蠹蛾基因的无创伤检测方法

苹果蠹蛾Cydia pomonella（L.）是我国重大农业入侵害虫,对我国果业健康发展造成严重威胁。在利用基因编辑等技术对相关基因进行功能研究时,通常采用单对交配策略对纯合突变体进行筛选。因此,在配对前明确个体基因型,避免对虫体造成损伤尤为重要。本研究分别收集末龄幼虫蜕（10个蜕）和蛹壳（1个、5个、10个、15个蛹壳）,通过基因组DNA提取、目的基因PCR扩增、琼脂糖凝胶电泳检测和PCR产物测序,以评估该方法作为苹果蠹蛾基因检测的可行性。结果显示,以苹果蠹蛾末龄幼虫10个蜕及5个以上蛹壳提取的基因组DNA作为模板,扩增精巢特异性丝氨酸/苏氨酸蛋白激酶（Testis specific serine/threonine protein kinase,TSSK1）基因,PCR产物经琼脂糖凝胶电泳检测得到单一、明亮的条带,经测序证实该产物是目的基因序列。基于高效及节约成本的原则,拟推荐利用10个末龄幼虫蜕或10个蛹壳提取基因组DNA、通过PCR扩增和测序进行苹果蠹蛾的无损伤基因型检测。本研究建立了一种利用幼虫蜕和蛹壳进行苹果蠹蛾无损伤、高效的基因检测方法,为提高苹果蠹蛾基因功能研究的效率奠定了基础。     

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.

     

Isolation of genomic DNA suitable for community analysis from mature trees adapted to arid environment

Isolation of intact and pure genomic DNA (gDNA) is essential for many molecular biology applications. It is difficult to isolate pure DNA from mature trees of hot and dry desert regions because of the accumulation of high level of polysaccharides, phenolic compounds, tannins etc. We hereby report the standardized protocol for the isolation and purification of gDNA from seven ecologically and medically important tree species of Combretaceae viz. Anogeissus (Anogeissus sericea var. nummularia, Anogeissus pendula, and Anogeissus latifolia) and Terminalia (Terminalia arjuna, Terminalia bellirica, Terminalia catappa and Terminalia chebula). This method involves (i) washing the sample twice with Triton buffer (2%) then (ii) isolation of gDNA by modified-CTAB (cetyl trimethyl ammonium bromide) method employing a high concentration (4%) of PVP (Polyvinylpyrrolidone) and 50 mM ascorbic acid, and (iii) purification of this CTAB-isolated gDNA by spin-column. gDNA isolated by modified CTAB or spin-column alone were not found suitable for PCR amplification. The Triton washing step is also critical. The quality of DNA was determined by the A₂₆₀/A₂₈₀ absorbance ratio. gDNA was also observed for its intactness by running on 0.8% agarose gel. The suitability of extracted DNA for PCR was tested by amplification with RAPD primers, which was successful. Further, rbcLa (barcoding gene) was amplified and sequenced to check the quality of extracted gDNA for its downstream applications.     

Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues

A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA.     

From the Field to the Lab: Best Practices for Field Preservation of Bat Specimens for Molecular Analyses

Studies in molecular ecology depend on field-collected samples for genetic information, and the tissue sampled and preservation conditions strongly affect the quality of the DNA obtained. DNA yields from different tissue types have seldom been compared, and the relative performance of storage media has never been directly tested, even though these media may influence DNA degradation under field conditions. We analyzed DNA yield from buccal swabs and wing punches harvested from live bats using nucleic acid quantification as well as quantitative PCR for a single-copy nuclear locus. We also compared DNA yields from wing tissue preserved in three media: ethanol, NaCl-saturated dimethyl sulfoxide (DMSO), and silica desiccant. Wing punches yielded more total DNA than did buccal swabs, and wing tissues preserved in silica beads yielded significantly more total and nuclear DNA than those preserved in DMSO or ethanol. These results show that tissue type and preservation media strongly influence the quantity of DNA obtained from non-lethal genetic samples, and based on these effects we provide recommendations for field collection of tissues for genetic analyses.     

Assessment of the Genetic Diversity among Arcobacters Isolated from Poultry Products by Using Two PCR-Based Typing Methods

In this study, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) were optimized for characterization of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. In addition, a simple and rapid DNA extraction method was tested for use in both typing procedures. Both methods had satisfactory typeability and discriminatory power, but the fingerprints generated with ERIC-PCR were more reproducible and complex than those obtained with RAPD-PCR. The use of nondiluted boiled cell suspensions as DNA templates was found to be very useful in ERIC-PCR. Characterization of large numbers of Arcobacter isolates is therefore preferably performed by the ERIC-PCR procedure. Isolates for which almost identical ERIC fingerprints are generated may subsequently be characterized by RAPD-PCR, although adjustment and standardization of the amount of the DNA template are necessary. In the second part of this study, the genotypic diversity of arcobacters present on broiler carcasses was assessed by using both typing methods. A total of 228 cultures from 24 samples were examined after direct isolation and enrichment. The isolates were identified by using a multiplex PCR as A. butzleri (n = 182) and A. cryaerophilus (n = 46). A total of 131 types (91 A. butzleri types and 40 A. cryaerophilus types) were discerned without discordance between the two typing techniques. The analysis of the poultry isolates showed that poultry products may harbor not only more than one species but also multiple genotypes. All genotypes were confined to one poultry sample, and only three genotypes were found after simultaneous enrichment and direct isolation. These results demonstrate that different outcomes can be obtained in epidemiological studies depending on the isolation procedure used and the number of isolates characterized.     

Enhanced sequencing coverage with digital droplet multiple displacement amplification

Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing.     

香蕉枯萎菌基因组DNA提取方法的研究

以香蕉枯萎菌菌株为试验材料,在SDS～CTAB法和高盐沉淀法等基础上加以改进,对两种提纯香蕉枯萎菌基因组DNA的方法进行了比较研究。结果表明：高盐沉淀法是适合于香蕉枯萎菌基因组DNA提取的方法。该方法提取的DNA OD260／OD280的比值为1．841,DNA产量为0．81mgDNA／g菌丝体。基因组DNA经琼脂糖凝胶电泳得到一条带型较宽且清晰的DNA谱带,基本无DNA碎带;将提取的DNA直接用于PCR扩增,得到带多而且清晰、整齐、基本无拖尾的RAPD图谱。     

Novel Cell and Tissue Acquisition System (CTAS): Microdissection of Live and Frozen Brain Tissues

We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS - Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.     

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.     

Increased DNA typing success for feces and feathers of capercaillie (Tetrao urogallus) and black grouse (Tetrao tetrix)

Noninvasive sampling, for example, of droppings or feathers, is a promising approach for molecular genetic studies on endangered and elusive animal species. Yet, such specimens are known for containing only minute amounts of DNA, resulting in lower typing success rates relative to analyses on fresh tissues such as muscle or blood. Furthermore, artefactual signals as well as contamination are more likely to occur when DNA is limited. To increase the reliability of DNA typing from noninvasive samples, optimized DNA extraction and polymerase chain reaction protocols were developed, taking advantage of developments in the forensic field aiming at successful molecular genetic analysis of DNA templates being low in quality and quantity. In the framework of an extensive monitoring project on population dynamics of capercaillie and black grouse in the Tyrolean Alps, feces samples and molted feathers from both species were collected. On a subset comprising about 200 specimens of either species, eight polymorphic short tandem repeat (STR) markers were analyzed to test these improved protocols. Besides optimizing DNA yields, both lowered sample consumption and reduced hands‐on time were achieved, and the rates of informative profiles amounted to 90.7% for capercaillie and 92.4% for black grouse. Similarly, high success rates had not been achieved in earlier studies and demonstrate the benefit of the improved methodology, which should be easily adaptable for use on animal species other than those studied here. The STR genotypes were not only powerful enough to discriminate among unrelated birds but also appeared fit for telling apart closely related animals, as indicated by Pi and Pi_sib values. The software package allelematch aided analysis of genotypes featuring possible dropout and drop‐in effects. Finally, a comparison between molecular genetic and morphology‐based species‐of‐origin determination revealed a high degree of concordance.