Expressed sequence tags for the chicken genome from a normalized 10-day-old white leghorn whole-embryo cDNA library. 3. DNA sequence analysis of genetic variation in commercial chicken populations.

Single nucleotide polymorphisms (SNPs) have emerged as a major class of DNA markers with the advantage of permitting the development of high-density genetic maps adequate for quantitative trait loci (QTL) identification by linkage-disequilibrium analysis. Here we describe results of a relatively high-depth survey of chicken broiler and layer populations for SNPs in targeted genomic regions of chicken expressed sequence tag (EST) sites. The sequences scanned, representing the composite sequence of 12 amplified fragments for a total of 6489 bp, were randomly distributed, occurring on six different chromosomes or linkage groups in the chicken genome. Although one of the genomic DNA sequences did not match the reference cDNA sequence, another contained an intron that separated two putative exons. The number of SNPs observed within each of the 12 EST-targeted genomic regions ranged from 0 to 10 for a total of 44 and a frequency of 0.7%. About 70% of the polymorphisms were shared between layer and broiler populations. The average heterozygosity within the populations ranged from 0.15 to 0.48, with the layer populations showing the higher heterozygosity. SNPs and oligonucleotides described will provide a resource for genetic analysis in commercial chicken populations. The data appear to indicate that the relative frequency of SNPs in the targeted regions scanned is higher than the frequency reported for any of the other regions scanned to date in other eukaryotic genomes. Additionally, the results suggest that the use of DNA pools may offer an efficient approach to SNP detection in chickens, as has been shown in other vertebrates.     

Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing

Detection of the rare polymorphisms and causative mutations of genetic diseases in a targeted genomic area has become a major goal in order to understand genomic and phenotypic variability. We have interrogated repeat-masked regions of 8.9 Mb on human chromosomes 21 (7.8 Mb) and 7 (1.1 Mb) from an individual from the International HapMap Project (NA12872). We have optimized a method of genomic selection for high throughput sequencing. Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold. When assaying HapMap SNPs in NA12872, our sequence genotypes are 91.3% concordant in regions with coverage≥4-fold, and 97.9% concordant in regions with coverage≥15-fold. About 81% of the SNPs recovered with both thresholds are listed in dbSNP. We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA. Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.     

Genomic DNA Enrichment Using Sequence Capture Microarrays: a Novel Approach to Discover Sequence Nucleotide Polymorphisms (SNP) in Brassica napus L

Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait loci –QTL– analysis) to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively). Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species.     

Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism

Accurate analysis of DNA sequence variation in not only humans and animals but also other organisms has played a significant role in expanding our knowledge about genetic variety and diversity in a number of different biological areas. The search for an understanding of the causes of genetic variants and mutations has resulted in the development of a simple laboratory technique, known as the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, for the detection of single nucleotide polymorphisms (SNPs). PCR-RFLP allows rapid detection of point mutations after the genomic sequences are amplified by PCR. The mutation is discriminated by digestion with specific restriction endonucleases and is identified by gel electrophoresis after staining with ethidium bromide (EtBr). This convenient and simple method is inexpensive and accurate for SNP genotyping and especially useful in small basic research studies of complex genetic diseases. The whole protocol takes only a day to carry out.     

Accessing single nucleotide polymorphisms in genomic DNA by direct multiplex polymerase chain reaction amplification on oligonucleotide microarrays

This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.     

Application of high-resolution DNA melting for genotyping in lepidopteran non-model species: Ostrinia furnacalis (Crambidae)

Development of an ideal marker system facilitates a better understanding of the genetic diversity in lepidopteran non-model organisms, which have abundant species, but relatively limited genomic resources. Single nucleotide polymorphisms (SNPs) discovered within single-copy genes have proved to be desired markers, but SNP genotyping by current techniques remain laborious and expensive. High resolution melting (HRM) curve analysis represents a simple, rapid and inexpensive genotyping method that is primarily confined to clinical and diagnostic studies. In this study, we evaluated the potential of HRM analysis for SNP genotyping in the lepidopteran non-model species Ostrinia furnacalis (Crambidae). Small amplicon and unlabeled probe assays were developed for the SNPs, which were identified in 30 females of O. furnacalis from 3 different populations by our direct sequencing. Both assays were then applied to genotype 90 unknown female DNA by prior mixing with known wild-type DNA. The genotyping results were compared with those that were obtained using bi-directional sequencing analysis. Our results demonstrated the efficiency and reliability of the HRM assays. HRM has the potential to provide simple, cost-effective genotyping assays and facilitates genotyping studies in any non-model lepidopteran species of interest.     

Microfluidic droplet enrichment for targeted sequencing

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications.     

MALDI mass spectrometry analysis of single nucleotide polymorphisms by photocleavage and charge-tagging

High-throughput procedures are an important requirement for future large-scale genetic studies such as genotyping of single nucleotide polymorphisms (SNPs). Matrix-assisted laser desorption/ ionisation mass spectrometry (MALDI-MS) has revolutionised the analysis of biomolecules and, in particular, provides a very attractive solution for the rapid typing of DNA. The analysis of DNA by MALDI can be significantly facilitated by a procedure termed ‘charge-tagging’. We show here a novel approach for the generation of charge-tagged DNA using a photocleavable linker and its implementation in a molecular biological procedure for SNP genotyping consisting of PCR, primer extension, photocleavage and a chemical reaction prior to MALDI target preparation and analysis. The reaction sequence is amenable to liquid handling automation and requires no stringent purification procedures. We demonstrate this new method on SNPs in two genes involved in complex traits.     

Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers

A system to use bovine EST data in conjunction with human genomic sequence to improve the bovine linkage map over the entire genome or on specific chromosomes was evaluated. Bovine EST sequence was used to provide primer sequences corresponding to bovine genes, while human genomic sequence directed primer design to flank introns and produce amplicons of appropriate size for efficient direct sequencing. The sequence tagged sites (STS) produced in this way from the four sires of the MARC reference families were examined for single nucleotide polymorphisms (SNPs) that could be used to map the corresponding genes. With this approach, along with a primer/extension mass spectrometry SNP genotyping assay, 100 ESTs were placed on the bovine genetic linkage map. The first 70 were chosen at random from bovine EST–human genomic comparisons. An additional 30 ESTs were successfully mapped to bovine Chromosome 19 (BTA19), and comparison of the resulting BTA19 map to the position of the corresponding human orthologs on the HSA17 draft sequences revealed differences in the spacing and order of genes. Over 80% of successful amplicons contained SNPs, indicating that this is an efficient approach to generating EST-associated genetic markers. We have demonstrated the feasibility of constructing a linkage map based on SNPs associated with ESTs and the plausibility of utilizing EST, comparative mapping information, and human sequence data to target regions of the bovine genome for SNP marker development.     

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina''s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.     

SNP analysis to dissect human traits

The analysis of complex human diseases has been spurred by the number of published genomic sequence variants - many identified in the course of sequencing the human genome. But, to be useful for genetic analysis, variants have to be mapped accurately, their frequencies in various populations determined, and automated high-throughput assay techniques developed. Recently proposed methods address these issues: the use of 'reduced representation shotgun' methods for more efficient detection of single nucleotide polymorphisms (SNPs), the employment of high-throughput genotyping techniques, the development of SNP maps that incorporate information about linkage disequilibrium, and the use of SNPs in identifying susceptibility genes for common illnesses.     

Integration of DNA ligation and rolling circle amplification for the homogeneous,end-point detection of single nucleotide polymorphisms

Association studies using common sequence variants or single nucleotide polymorphisms (SNPs) may provide a powerful approach to dissect the genetic inheritance of common complex traits. Such studies necessitate the development of cost-effective, high throughput technologies for scoring SNPs. The method described in this paper for the co-detection of both alleles of a SNP in a single homogeneous reaction combines the specificity of a high fidelity DNA ligation step with the power of rolling circle amplification. The incorporation of Amplifluor™ energy transfer primers enables signal detection in a homogeneous format, making this approach highly amenable to automation. The adaptation of the genotyping method for high throughput screening using conventional liquid handling systems is described.     

Current status of genome-wide association studies in cancer

Genome-wide association studies in cancer have already identified over 150 regions associated with two dozen specific cancers. Already, a handful of multi-cancer susceptibility regions have been uncovered, providing new insights into perhaps common mechanisms of carcinogenesis. For each new susceptibility allele, investigators now face the arduous task of interrogating each region beginning with fine mapping prior to pursuing the biological basis for the direct association of one or more variants. It appears that there may be a significant number of common alleles that contribute to the heritability of a specific cancer. Since each region confers a small contribution to the risk for cancer, it is daunting to consider any single nucleotide polymorphism (SNP) as a clinical test. Since the complex genomic architecture of each cancer differs, additional genotyping and sequence analysis will be required to comprehensively catalog susceptibility alleles followed by the formidable task of understanding the interactions between genetic regions as well as the environment. It will be critical to assess the applicability of genetic tests in specific clinical settings, such as when to perform screening tests with calculable risks (e.g., biopsies or chemoprevention), before incorporating SNPs into clinical practice. To advance the current genomic observations to the clinical venue, new studies will need to be designed to validate the utility of known genetic variants in assessing risk for cancer as well as its outcomes.     

Cheap, accurate and rapid allele frequency estimation of single nucleotide polymorphisms by primer extension and DHPLC in DNA pools

At present, the cost of genotyping single nucleotide polymorphisms (SNPs) in large numbers of subjects poses a formidable problem for molecular genetic approaches to complex diseases. We have tested the possibility of using primer extension and denaturing high performance liquid chromatography to estimate allele frequencies of SNPs in pooled DNA samples. Our data show that this method should allow the accurate estimation of absolute allele frequencies in pooled samples of DNA and also of the difference in allele frequency between different pooled DNA samples. This technique therefore offers an efficient and cheap method for genotyping SNPs in large case-control and family-based association samples.     

Large-scale genotyping of complex DNA

Genetic studies aimed at understanding the molecular basis of complex human phenotypes require the genotyping of many thousands of single-nucleotide polymorphisms (SNPs) across large numbers of individuals. Public efforts have so far identified over two million common human SNPs; however, the scoring of these SNPs is labor-intensive and requires a substantial amount of automation. Here we describe a simple but effective approach, termed whole-genome sampling analysis (WGSA), for genotyping thousands of SNPs simultaneously in a complex DNA sample without locus-specific primers or automation. Our method amplifies highly reproducible fractions of the genome across multiple DNA samples and calls genotypes at >99% accuracy. We rapidly genotyped 14,548 SNPs in three different human populations and identified a subset of them with significant allele frequency differences between groups. We also determined the ancestral allele for 8,386 SNPs by genotyping chimpanzee and gorilla DNA. WGSA is highly scaleable and enables the creation of ultrahigh density SNP maps for use in genetic studies.     

Development of a Genotyping Microarray for Studying the Role of Gene-Environment Interactions in Risk for Lung Cancer

A microarray (LungCaGxE), based on Illumina BeadChip technology, was developed for high-resolution genotyping of genes that are candidates for involvement in environmentally driven aspects of lung cancer oncogenesis and/or tumor growth. The iterative array design process illustrates techniques for managing large panels of candidate genes and optimizing marker selection, aided by a new bioinformatics pipeline component, Tagger Batch Assistant. The LungCaGxE platform targets 298 genes and the proximal genetic regions in which they are located, using ∼13,000 DNA single nucleotide polymorphisms (SNPs), which include haplotype linkage markers with a minimum allele frequency of 1% and additional specifically targeted SNPs, for which published reports have indicated functional consequences or associations with lung cancer or other smoking-related diseases. The overall assay conversion rate was 98.9%; 99.0% of markers with a minimum Illumina design score of 0.6 successfully generated allele calls using genomic DNA from a study population of 1873 lung-cancer patients and controls.     

Genomic DNA sequence comparison between two inbred soybean cyst nematode biotypes facilitated by massively parallel 454 micro-bead sequencing

Heterodera glycines, the soybean cyst nematode (SCN), is a damaging agricultural pest that could be effectively managed if critical phenotypes, such as virulence and host range could be understood. While SCN is amenable to genetic analysis, lack of DNA sequence data prevents the use of such methods to study this pathogen. Fortunately, new methods of DNA sequencing that produced large amounts of data and permit whole genome comparative analyses have become available. In this study, 400 million bases of genomic DNA sequence were collected from two inbred biotypes of SCN using 454 micro-bead DNA sequencing. Comparisons to a BAC, sequenced by Sanger sequencing, showed that the micro-bead sequences could identify low and high copy number regions within the BAC. Potential single nucleotide polymorphisms (SNPs) between the two SCN biotypes were identified by comparing the two sets of sequences. Selected resequencing revealed that up to 84% of the SNPs were correct. We conclude that the quality of the micro-bead sequence data was sufficient for de novo SNP identification and should be applicable to organisms with similar genome sizes and complexities. The SNPs identified will be an important starting point in associating phenotypes with specific regions of the SCN genome.     

SNP discovery in nonmodel organisms: strand bias and base‐substitution errors reduce conversion rates

Single nucleotide polymorphisms (SNPs) have become the marker of choice for genetic studies in organisms of conservation, commercial or biological interest. Most SNP discovery projects in nonmodel organisms apply a strategy for identifying putative SNPs based on filtering rules that account for random sequencing errors. Here, we analyse data used to develop 4723 novel SNPs for the commercially important deep‐sea fish, orange roughy (Hoplostethus atlanticus), to assess the impact of not accounting for systematic sequencing errors when filtering identified polymorphisms when discovering SNPs. We used SAMtools to identify polymorphisms in a velvet assembly of genomic DNA sequence data from seven individuals. The resulting set of polymorphisms were filtered to minimize ‘bycatch’—polymorphisms caused by sequencing or assembly error. An Illumina Infinium SNP chip was used to genotype a final set of 7714 polymorphisms across 1734 individuals. Five predictors were examined for their effect on the probability of obtaining an assayable SNP: depth of coverage, number of reads that support a variant, polymorphism type (e.g. A/C), strand‐bias and Illumina SNP probe design score. Our results indicate that filtering out systematic sequencing errors could substantially improve the efficiency of SNP discovery. We show that BLASTX can be used as an efficient tool to identify single‐copy genomic regions in the absence of a reference genome. The results have implications for research aiming to identify assayable SNPs and build SNP genotyping assays for nonmodel organisms.