ISO 17025 validation of a next-generation sequencing assay for relationship testing.

The HID-Ion AmpliSeq™ Identity Panel is a next-generation sequencing assay with 90 autosomal and 34 Y-chromosome SNPs that are amplified in one PCR step and subsequently sequenced using the Ion Personal Genome Machine (Ion PGM™) System. This assay was validated for relationship testing in our ISO 17025 accredited laboratory in 2015. Here, the essential parts of the validation report submitted to the Danish Accreditation Fund are presented. A total of 100 unrelated Danes were typed in duplicates and the locus balance, heterozygote balance (Hb) and noise levels were analysed in detail. Two loci were disregarded for casework because genotyping was uncertain. Hb for rs7520386 was skewed and high levels of noise were observed in rs576261. Three general acceptance criteria for analysis of single-source samples were defined: (i) sequencing depth > 200 reads, (ii) noise level < 3% and (iii) Hb > 0.3. A Python script named SNPonPGM was developed to assist the analyst by highlighting loci that do not fulfil the general acceptance criteria. Furthermore, SNPonPGM has functions that reduce the hands-on time of the reporting officer to a few minutes per case. Mixtures with DNA from two individuals in a 1:24 ratio were readily identified using the three criteria and the SNPonPGM script.

Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays.

DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions corresponding to the temperature zones of the MTAW. After hybridization with amplified patient material, the slides were mounted in the MTAW, and each subarray was exposed to different temperatures ranging from 22 to 40 degrees C. When processed in the MTAW, probes selected without considering melting temperature resulted in improved genotyping compared with probes selected according to theoretical melting temperature and run under one condition. In conclusion, the MTAW is a versatile tool that can facilitate screening of a large number of probes for genotyping assays and can also enhance the performance of diagnostic arrays.

Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface.

Here, we describe a multi-parametric study of DNA hybridization to probes with 20-70% G + C content. Probes were designed towards 71 different sites/mutations in the phenylalanine hydroxylase gene. Seven probe lengths, three spacer lengths and six stringencies were systematically varied. The three spacer lengths were obtained by placing the gene-specific sequence in discrete steps along the 60-mer probes. The study was performed using Agilent 8 x 15 000 probes custom-made arrays and a home-built array washer providing different stringencies to each of the eight sub-arrays on the slides. Investigation of hybridization signals, specificity and dissociation curves indicated that probes close to the surface were influenced by an additional stringency provided by the microarray surface. Consistent with this, probes close to the surface required 4 x SSC, while probes placed away from the surface required 0.35 x SSC wash buffers in order to give accurate genotyping results. Multiple step dissociation was frequently observed for probes placed furthest away from surface, but not for probes placed proximal to the surface, which is consistent with the hypothesis that there is different stringency along the 60-mer. The results have impact on design of probes for genotyping, gene expression and comparative genome hybridization analysis.

Genotyping of mutation in the beta-globin gene using DNA microarrays.

Genotyping using DNA microarrays is a cost-efficient method compared with real-time PCR and DNA sequencing. Here, a DNA microarray-based method using allele-specific oligo hybridization is demonstrated. This method relies on immobilization of probes that are specific for wild-type sequences or the mutated sequences, respectively. The method makes use of agarose film-coated glass slides, unmodified DNA probes, target preparation using T7 in vitro transcription, labeling of target using biotin labels, and detection using alkaline phosphatase precipitation reaction. Visualization is performed using a desktop computer scanner. Because the biotin/strepavidin chemistry is utilized, the method described here is compatible with many different detection methods. The demonstrated colorimetric detection of mutations has an expected error frequency of about 5 x 10(-7) per mutation or better. Given this low error frequency, an array diagnosing 100 different mutations would misclassify about 1 patient in 100,000.

Target preparation for genotyping specific genes or gene segments.

Generation of single stranded target is of high importance for hybridization reactions on oligonucleotide microarrays. Several methods have been established for production of single stranded DNA and in vitro transcribed RNA. Here we describe three robust methods for target amplification from purified genomic DNA or pre-amplified DNA. The protocols include incorporation of biotin labels in the target molecules and allow for biotin/streptavidin chemistry to be utilized for flexibility in choice of visualization strategy.

Genotyping of mutations in the beta-globin gene using allele specific hybridization.

The use of DNA microarrays for genotyping is economically favorable when compared to real-time PCR and DNA sequencing. Here, we demonstrate a DNA microarray-based assay using allele-specific oligonucleotide (ASO) probes for genotyping mutations in the beta-globin gene. The assay makes use of agarose film coated glass slides as substrate, unmodified melting temperature matched ASO probes, target amplification and labeling using T7 in vitro transcription, mixing during hybridization and finally visualization using a fluorescent scanner. In this chapter we will emphasize on probe design, optimization, and validation of an allele-specific hybridization assay.

Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies.

DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing of arrays under different assay conditions. These new array processors provide researchers and assay developers with novel possibilities to construct highly specific DNA arrays even towards regions of DNA greatly varying in G + C content. These array processors are also a powerful development tool for building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit microarray-based loci selection prior to high-throughput sequencing.

Pinched flow fractionation devices for detection of single nucleotide polymorphisms.

We demonstrate a new and flexible microfluidic based method for genotyping single nucleotide polymorphisms (SNPs). The method relies on size separation of selectively hybridized polystyrene microspheres in a microfluidic pinched flow fractionation (PFF) device. The microfluidic PFF devices with 13 mum deep channels were fabricated by thermal nanoimprint lithography (NIL) in a thin film of cyclic-olefin copolymer (mr-I T85) on a silicon wafer substrate, and the channels were sealed by thermal polymer bonding. Streptavidin coated polystyrene microspheres with a mean diameter of 3.09 microm and 5.6 microm were functionalized with biotin-labeled oligonucleotides for the detection of a mutant (Mt) or wild-type (Wt) DNA sequence in the HBB gene, respectively. Hybridization to functionalized beads was performed with fluorescent targets comprising synthetic DNA oligonucleotides or amplified RNA, synthesized using human DNA samples from individuals with point mutations in the HBB gene. Following a stringent wash, the beads were separated in a PFF device and the fluorescent signal from the beads was analyzed. Patients being wildtypes, heterozygotes or mutated respectively for the investigated mutation could reliably be diagnosed in the PFF device. This indicates that the PFF technique can be used for accurate and fast genotyping of SNPs.

X-linked Menkes disease: first documented report of germ-line mosaicism.

This work investigated a three-generation Menkes disease family, where germ-line mosaicism was suspected in the maternal grandmother of the index patient. She had given birth to 2 boys who died of suspected Menkes disease on the basis of clinical and photographic evidence. Biochemical analysis of the index patient confirmed the diagnosis of Menkes disease, and DNA analysis established a partial gene deletion (EX11_EX23del), involving exons 11-23 and the 3'-untranslated region (UTR) of ATP7A. A junction fragment was detectable by Southern blot analysis, which enabled carrier analysis. The mother was demonstrated to be a carrier, whereas analysis of lymphoblasts and skin fibroblasts from the maternal grandmother gave no indication of a partial gene deletion. No materials were available from the possibly affected maternal uncles. Further genetic analyses, including biochemical testing of the grandmother and haplotype analysis using four intragenic markers on DNA from selected members of the family, corroborated this finding. The combined results from DNA analyses showed that the grandmother had transmitted three different ATP7A haplotypes to her offspring: (1) the at-risk allele (CA(B))-1 and the deletion; (2) the at-risk allele (CA(B))-1 without deletion; and (3) the second allele (CAB)-2 without deletion. In conclusion, our study demonstrated segregation of Menkes disease within the family investigated that can best be explained by extensive germ-line mosaicism in the maternal grandmother. The finding of germ-line mosaicism has obvious implications for genetic counseling of Menkes disease families.

Evaluation of four automated protocols for extraction of DNA from FTA cards.

Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

Typing of 30 insertion/deletions in Danes using the first commercial indel kit--Mentype® DIPplex.

In this study, we tested the first commercial kit with insertion/deletion (indel) polymorphisms, the Mentype(®) DIPplex PCR Amplification Kit (DIPplex kit). A total of 30 biallelic autosomal indels and Amelogenin were amplified with the DIPplex kit. All loci were amplified in one PCR multiplex and all amplicon lengths were shorter than 160 bp. Full indel profiles were generated from as little as 100 pg of DNA. A total of 117 individuals from Danish paternity cases were successfully typed. No deviation from Hardy-Weinberg equilibrium was observed for any of the indels. The combined mean match probability was 3.3 × 10(-13), the mean paternity exclusion probability was 99.7% and the typical paternity indices for trios and duos were 2350 and 165, respectively. Furthermore, we typed five highly degraded DNA samples with the DIPplex kit, the AmpFlSTR(®) SGM Plus kit and the AmpFlSTR(®) SEfiler Plus kit. Full indel profiles were obtained with the DIPplex kit, whereas only partial profiles were obtained with the STR kits. In general, the DIPplex kit performed well and it would be a valuable assay for forensic genetic testing, especially in crime cases with partially degraded DNA or low amounts of template DNA. However, some difficulties with pull-ups were observed at DNA concentrations of 1000 pg. Rearrangement of the allele windows by changing the lengths of some of the PCR primers would greatly improve the assay, and more robustness towards higher amounts of DNA would allow the use of the DIPplex kit without prior quantification of the samples.

Investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays.

BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%). Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.

Typing of Amerindian Kichwas and Mestizos from Ecuador with the SNPforID multiplex.

A total of 119 unrelated individuals from two of the major ethnic groups in Ecuador were typed for 49 of the autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex using the SNapShot(®) assay. Of the above, 42 samples originated from Mestizos (an admixed population) and the remaining 77 were from Native Amerindian Kichwas. We obtained full SNP profiles in all individuals and concordance of duplicated analyses. No deviation from Hardy-Weinberg equilibrium (HWE) was observed for any SNP in the Mestizo and Kichwa populations and only one and four pairs of loci, respectively showed significant linkage disequilibrium. A relatively low genetic diversity and global positive F(IS) value was observed in Kichwas. A statistically significant global F(ST) value was obtained when the two Ecuadorian populations were compared with populations in Spain, Portugal, Argentina, Denmark, Greenland, China, Somalia and Mozambique. All pairwise F(ST) values were statistically significant. A multi-dimensional scaling based on pairwise F(ST) values showed that the Kichwa population differed from all other populations investigated and that the Mestizos had an intermediate position between Kichwas and Europeans. An admixture analysis indicated that the greater contributor to the Mestizo population was the Kichwas (71.2%) compared to the European contribution. The combined mean match probability and mean paternity exclusion probability were 3.3 × 10(-17) and 0.998, respectively, for the Mestizo population and 3.3 × 10(-14) and 0.993, respectively, for the Kichwa population.

Microfludic device for creating ionic strength gradients over DNA microarrays for efficient DNA melting studies and assay development.

The development of DNA microarray assays is hampered by two important aspects: processing of the microarrays is done under a single stringency condition, and characteristics such as melting temperature are difficult to predict for immobilized probes. A technical solution to these limitations is to use a thermal gradient and information from melting curves, for instance to score genotypes. However, application of temperature gradients normally requires complicated equipment, and the size of the arrays that can be investigated is restricted due to heat dissipation. Here we present a simple microfluidic device that creates a gradient comprising zones of defined ionic strength over a glass slide, in which each zone corresponds to a subarray. Using this device, we demonstrated that ionic strength gradients function in a similar fashion as corresponding thermal gradients in assay development. More specifically, we noted that (i) the two stringency modulators generated melting curves that could be compared, (ii) both led to increased assay robustness, and (iii) both were associated with difficulties in genotyping the same mutation. These findings demonstrate that ionic strength stringency buffers can be used instead of thermal gradients. Given the flexibility of design of ionic gradients, these can be created over all types of arrays, and encompass an attractive alternative to temperature gradients, avoiding curtailment of the size or spacing of subarrays on slides associated with temperature gradients.