Bulk segregant analysis followed by high-throughput sequencing reveals the Neurospora cell cycle gene, ndc-1, to be allelic with the gene for ornithine decarboxylase, spe-1

With the advent of high-throughput DNA sequencing, it is now straightforward and inexpensive to generate high-density small nucleotide polymorphism (SNP) maps. Here we combined high-throughput sequencing with bulk segregant analysis to expedite mutation mapping. The general map location of a mutation can be identified by a single backcross to a strain enriched in SNPs compared to a standard wild-type strain. Bulk segregant analysis simultaneously increases the likelihood of determining the precise nature of the mutation. We present here a high-density SNP map between Neurospora crassa Mauriceville-1-c (FGSC2225) and OR74A (FGSC2489), the strains most typically used by Neurospora researchers to carry out mapping crosses. We further have demonstrated the utility of the Mauriceville sequence and our approach by mapping the mutation responsible for the only existing temperature-sensitive (ts) cell cycle mutation in Neurospora, nuclear division cycle-1 (ndc-1). The single T-to-C point mutation maps to the gene encoding ornithine decarboxylase (ODC), spe-1 (NCU01271), and changes a Phe to a Ser residue within a highly conserved motif next to the catalytic site of the enzyme. By growth on spermidine and complementation with a wild-type spe-1 gene, we showed that the defect in spe-1 is responsible for the ts ndc-1 mutation. Based on our results, we propose changing ndc-1 to spe-1(ndc), which reflects that this mutation results in an ODC with a specific nuclear division defect.     

Whole-genome shotgun optical mapping of Rhodospirillum rubrum

Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.     

Rapid mapping and identification of mutations in Caenorhabditis elegans by restriction site-associated DNA mapping and genomic interval pull-down sequencing

Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the causal mutation by traditional mapping and candidate gene sequencing is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in Caenorhabditis elegans mutants. First, we report our use of restriction site-associated DNA (RAD) polymorphism markers for rapidly mapping mutations after chemical mutagenesis and mutant isolation. Second, we describe our use of genomic interval pull-down sequencing (GIPS) to selectively capture and sequence megabase-sized portions of a mutant genome. Together, these two methods provide a rapid and cost-effective approach for positional cloning of C. elegans mutant loci, and are also applicable to other genetic model systems.     

Homozygosity for multiple contiguous single-nucleotide polymorphisms as an indicator of large heterozygous deletions: identification of a novel heterozygous 8-kb intragenic deletion (IVS7-19 to IVS15-17) in a patient with glycogen storage disease type II

Current methods for detection of mutations by polymerase chain reaction (PCR) and sequence analysis frequently are not able to detect heterozygous large deletions. We report the successful use of a novel approach to identify such deletions, based on detection of apparent homozygosity of contiguous single-nucleotide polymorphisms (SNPs). The sequence analysis of genomic DNA PCR products containing all coding exons and flanking introns identified only a single heterozygous mutation (IVS18+2t-->a) in a patient with classic infantile-onset autosomal recessive glycogen storage disease type II (GSDII). Apparent homozygosity for multiple contiguous SNPs detected by this sequencing suggested presence of a large deletion as the second mutation; primers flanking the region of homozygous SNPs permitted identification and characterization by PCR of a large genomic deletion (8.26 kb) extending from IVS7 to IVS15. The data clearly demonstrate the utility of SNPs as markers for large deletions in autosomal recessive diseases when only a single mutation is found, thus complementing currently standard DNA PCR sequence methods for identifying the molecular basis of disease.     

Whole-Genome Shotgun Optical Mapping of Rhodospirillum rubrum

Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a “molecular cytogenetics” approach to solving problems in genomic analysis.     

High-resolution, high-throughput SNP mapping in Drosophila melanogaster

Single nucleotide polymorphisms (SNPs) are useful markers for genetic mapping experiments in model organisms. Here we report the establishment of a high-density SNP map and high-throughput genotyping assays for Drosophila melanogaster. Our map comprises 27,367 SNPs in common laboratory Drosophila stocks. These SNPs were clustered within 2,238 amplifiable markers at an average density of 1 marker every 50.3 kb, or 6.3 genes. We have also constructed a set of 62 Drosophila stocks, each of which facilitates the generation of recombinants within a defined genetic interval of 1-2 Mb. For flexible, high-throughput SNP genotyping, we used fluorescent tag-array mini-sequencing (TAMS) assays. We designed and validated TAMS assays for 293 SNPs at an average resolution of 391.3 kb, and demonstrated the utility of these tools by rapidly mapping 14 mutations that disrupt embryonic muscle patterning. These resources enable high-resolution high-throughput genetic mapping in Drosophila.     

A High-Density Single Nucleotide Polymorphism Map for Neurospora crassa

We report the discovery and validation of a set of single nucleotide polymorphisms (SNPs) between the reference Neurospora crassa strain Oak Ridge and the Mauriceville strain (FGSC 2555), of sufficient density to allow fine mapping of most loci. Sequencing of Mauriceville cDNAs and alignment to the completed genomic sequence of the Oak Ridge strain identified 19,087 putative SNPs. Of these, a subset was validated by cleaved amplified polymorphic sequence (CAPS), a simple and robust PCR-based assay that reliably distinguishes between SNP alleles. Experimental confirmation resulted in the development of 250 CAPS markers distributed evenly over the genome. To demonstrate the applicability of this map, we used bulked segregant analysis followed by interval mapping to locate the csp-1 mutation to a narrow region on LGI. Subsequently, we refined mapping resolution to 74 kbp by developing additional markers, resequenced the candidate gene, NCU02713.3, in the mutant background, and phenocopied the mutation by gene replacement in the WT strain. Together, these techniques demonstrate a generally applicable and straightforward approach for the isolation of novel genes from existing mutants. Data on both putative and validated SNPs are deposited in a customized public database at the Broad Institute, which encourages augmentation by community users.     

Differential Relationship of DNA Replication Timing to Different Forms of Human Mutation and Variation

Human genetic variation is distributed nonrandomly across the genome, though the principles governing its distribution are only partially known. DNA replication creates opportunities for mutation, and the timing of DNA replication correlates with the density of SNPs across the human genome. To enable deeper investigation of how DNA replication timing relates to human mutation and variation, we generated a high-resolution map of the human genome’s replication timing program and analyzed its relationship to point mutations, copy number variations, and the meiotic recombination hotspots utilized by males and females. DNA replication timing associated with point mutations far more strongly than predicted from earlier analyses and showed a stronger relationship to transversion than transition mutations. Structural mutations arising from recombination-based mechanisms and recombination hotspots used more extensively by females were enriched in early-replicating parts of the genome, though these relationships appeared to relate more strongly to the genomic distribution of causative sequence features. These results indicate differential and sex-specific relationship of DNA replication timing to different forms of mutation and recombination.     

Molecular Genetics of the Caenorhabditis Elegans Heterochronic Gene Lin-14

We describe a general strategy for the genetic mapping in parallel of multiple restriction fragment length polymorphism (RFLP) loci. This approach allows the systematic identification for cloning of physical genetic loci within about 100 kb of any gene in Caenorhabditis elegans. We have used this strategy of parallel RFLP mapping to clone the heterochronic gene lin-14, which controls the timing and sequence of many C. elegans postembryonic developmental events. We found that of about 400 polymorphic loci in the C. elegans genome associated with the Tc1 family of repetitive elements, six are within 0.3 map unit of lin-14. The three closest lin-14-linked Tc1-containing restriction fragments were cloned and used to identify by hybridization an 830-kb region of contiguous cloned DNA fragments assembled from cosmid and yeast artificial chromosome libraries. A lin-14 intragenic recombinant that separated a previously cryptic lin-14 semidominant mutation from a cis-acting lin-14 suppressor mutation was used to map the location of the lin-14 gene to a 25-kb region of this 830-kb contig. DNA probes from this region detected lin-14 allele-specific DNA alterations and a lin-14 mRNA. Two lin-14 semi-dominant alleles, which cause temporally inappropriate lin-14 gene activity and lead to the reiterated expression of specific early developmental events, were shown to delete sequences from the lin-14 gene and mRNA. These deletions may define cis-acting sequences responsible for the temporal regulation of lin-14.     

Whole-genome sequencing and variant discovery in C. elegans

Massively parallel sequencing instruments enable rapid and inexpensive DNA sequence data production. Because these instruments are new, their data require characterization with respect to accuracy and utility. To address this, we sequenced a Caernohabditis elegans N2 Bristol strain isolate using the Solexa Sequence Analyzer, and compared the reads to the reference genome to characterize the data and to evaluate coverage and representation. Massively parallel sequencing facilitates strain-to-reference comparison for genome-wide sequence variant discovery. Owing to the short-read-length sequences produced, we developed a revised approach to determine the regions of the genome to which short reads could be uniquely mapped. We then aligned Solexa reads from C. elegans strain CB4858 to the reference, and screened for single-nucleotide polymorphisms (SNPs) and small indels. This study demonstrates the utility of massively parallel short read sequencing for whole genome resequencing and for accurate discovery of genome-wide polymorphisms.     

Rapid high resolution single nucleotide polymorphism-comparative genome hybridization mapping in Caenorhabditis elegans

We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of ~200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method.     

Fine-mapping of shotgun template-libraries; an efficient strategy for the systematic sequencing of genomic DNA.

To test the effectiveness of ordering shotgun DNA-templates prior to sequence analysis, the 450 kb left arm of yeast chromosome XII was randomly subcloned into a phagemid vector. Clones were ordered by hybridisation to an average map density of one new insert every 125 bp and are currently used for sequencing the chromosomal fragment. An 11.5 kb overlap between the template map and a DNA fragment that had been sequenced earlier allowed an independent evaluation of the strategy's effectiveness. To this end, clones were selected from the map and tag-sequenced from either end, thus comparing the map position with the actual location within the 11.5 kb. Of 65 selected clones, taken mostly at random from a total of 423, 58 mapped on average about a quarter of a clone length around their predicted position, with the other seven being between 0.6 and 1.5 clone length off. 75-86 sequencing reactions on clones selected from the map would have been sufficient for completely sequencing both strands of the 11.5 kb fragment. The results demonstrate the efficacy of such template sorting, considerably assisting sequencing at relatively little cost on the mapping level.     

Development of a 1.4-Mb BAC/PAC contig and physical map within the critical region for complete X-linked congenital stationary night blindness in Xp11.4

A physical map internal to the markers DXS1368 and DXS228 was developed for the p11.4 region of the human X chromosome. Twenty-four BACs and 10 PACs with an average insert size of 149 kb were aligned to form a contig across an estimated 1.4 Mb of DNA. This contig, which has on average fourfold clone coverage, was assembled by STS and EST content analysis using 46 markers, including 8 ESTs, two retinally expressed genes, and 22 new STSs developed from BAC- and PAC-derived DNA sequence. The average intermarker distance was 30 kb. This physical map provides resources for high-resolution mapping as well as suitable clones for large-scale sequencing efforts in Xp11.4, a region known to contain the gene for complete X-linked congenital stationary night blindness.     

Detection and characterization of SNPs useful for identity control and parentage testing in major European dairy breeds

We propose the use of single nucleotide polymorphisms (SNPs) instead of polymorphic microsatellite markers for individual identification and parentage control in cattle. To this end, we present an initial set of 37 SNP markers together with a gender-specific SNP for identity control and parentage testing in the Holstein, Fleckvieh and Braunvieh breeds. To obtain suitable SNPs, a total of 91.13 kb of random genomic DNA was screened yielding 531 SNPs. These, and 43 previously identified SNPs, were subjected to the following selection criteria: (1) the frequency of the minor allele must be larger than 0.1 in at least two of the three examined breeds, and (2) markers should not be linked closely. Allele frequencies were estimated by analysing sequencing traces of pooled DNA or by genotyping individual DNA samples. The selected SNP loci were physically mapped by radiation hybrid mapping or by fluorescence in situ hybridization, and tested against the neutral mutation hypothesis. The presented marker set theoretically allows probabilities of identity less than 10(-13) for individual verification and exclusion powers exceeding 99.99% for parentage testing.     

A sequence-ready map of the human chromosome 17p telomere.

A half-YAC clone derived from human chromosome 17p was mapped at high resolution using cosmid subclone fingerprint analysis. Colinearity of the half-YAC with the telomeric human genomic DNA fragment was ascertained by RecA-assisted restriction endonuclease cleavage mapping. Previously isolated and radiation hybrid-mapped markers TEL17P37, TEL17P49, and TEL17P80 mapped 30-60 kb from the 17p terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 17p, and will provide the cloned DNA required for ascertaining the nucleotide sequence of this subtelomeric region.     

Application of denaturing high-performance liquid chromatography for mapping of single nucleotide polymorphisms in barley (Hordeum vulgare L.).

Recent advances in DNA sequence analysis and the establishment of high-throughput assays have provided the framework for large-scale discovery and analysis of DNA sequence variation. In this context, single nucleotide polymorphisms (SNPs) are of particular interest. To initiate a systematic approach to develop an SNP map of barley (Hordeum vulgare L.), we have employed denaturing high-performance liquid chromatography (DHPLC) to analyse segregating SNP patterns in a doubled-haploid (DH) mapping population. To this end, SNPs between the parental genotypes were identified using a direct sequencing approach. Once a SNP was established between the parents, the optimal melting temperature of the PCR fragment containing the SNP was predicted for its analysis by DHPLC. Following the detection of the optimal temperature, the DH lines were analysed for the presence of either of the alleles. To test the utility of the analysis, data from previously mapped RFLP markers from which these SNPs were derived were compared. Results from these experiments indicate that DHPLC can be efficiently employed in analysing SNPs on a high-throughput scale.     

A new approach to genome mapping and sequencing: slalom libraries

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with ~46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of ~100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5–10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.