SNPSelect: A scalable and flexible targeted sequence-based genotyping solution.

In plant breeding the use of molecular markers has resulted in tremendous improvement of the speed with which new crop varieties are introduced into the market. Single Nucleotide Polymorphism (SNP) genotyping is routinely used for association studies, Linkage Disequilibrium (LD) and Quantitative Trait Locus (QTL) mapping studies, marker-assisted backcrosses and validation of large numbers of novel SNPs. Here we present the KeyGene SNPSelect technology, a scalable and flexible multiplexed, targeted sequence-based, genotyping solution. The multiplex composition of SNPSelect assays can be easily changed between experiments by adding or removing loci, demonstrating their content flexibility. To demonstrate this versatility, we first designed a 1,056-plex maize assay and genotyped a total of 374 samples originating from an F2 and a Recombinant Inbred Line (RIL) population and a maize germplasm collection. Next, subsets of the most informative SNP loci were assembled in 384-plex and 768-plex assays for further genotyping. Indeed, selection of the most informative SNPs allows cost-efficient yet highly informative genotyping in a custom-made fashion, with average call rates between 88.1% (1,056-plex assay) and 99.4% (384-plex assay), and average reproducibility rates between duplicate samples ranging from 98.2% (1056-plex assay) to 99.9% (384-plex assay). The SNPSelect workflow can be completed from a DNA sample to a genotype dataset in less than three days. We propose SNPSelect as an attractive and competitive genotyping solution to meet the targeted genotyping needs in fields such as plant breeding.

Sequence-based SNP genotyping in durum wheat.

Marker development for marker-assisted selection in plant breeding is increasingly based on next-generation sequencing (NGS). However, marker development in crops with highly repetitive, complex genomes is still challenging. Here we applied sequence-based genotyping (SBG), which couples AFLP®-based complexity reduction to NGS, for de novo single nucleotide polymorphisms (SNP) marker discovery in and genotyping of a biparental durum wheat population. We identified 9983 putative SNPs in 6372 contigs between the two parents and used these SNPs for genotyping 91 recombinant inbred lines (RILs). Excluding redundant information from multiple SNPs per contig, 2606 (41%) markers were used for integration in a pre-existing framework map, resulting in the integration of 2365 markers over 2607 cM. Of the 2606 markers available for mapping, 91% were integrated in the pre-existing map, containing 708 SSRs, DArT markers, and SNPs from CRoPS technology, with a map-size increase of 492 cM (23%). These results demonstrate the high quality of the discovered SNP markers. With this methodology, it was possible to saturate the map at a final marker density of 0.8 cM/marker. Looking at the binned marker distribution (Figure 2), 63 of the 268 10-cM bins contained only SBG markers, showing that these markers are filling in gaps in the framework map. As to the markers that could not be used for mapping, the main reason was the low sequencing coverage used for genotyping. We conclude that SBG is a valuable tool for efficient, high-throughput and high-quality marker discovery and genotyping for complex genomes such as that of durum wheat.

Sequence-based genotyping for marker discovery and co-dominant scoring in germplasm and populations.

Conventional marker-based genotyping platforms are widely available, but not without their limitations. In this context, we developed Sequence-Based Genotyping (SBG), a technology for simultaneous marker discovery and co-dominant scoring, using next-generation sequencing. SBG offers users several advantages including a generic sample preparation method, a highly robust genome complexity reduction strategy to facilitate de novo marker discovery across entire genomes, and a uniform bioinformatics workflow strategy to achieve genotyping goals tailored to individual species, regardless of the availability of a reference sequence. The most distinguishing features of this technology are the ability to genotype any population structure, regardless whether parental data is included, and the ability to co-dominantly score SNP markers segregating in populations. To demonstrate the capabilities of SBG, we performed marker discovery and genotyping in Arabidopsis thaliana and lettuce, two plant species of diverse genetic complexity and backgrounds. Initially we obtained 1,409 SNPs for arabidopsis, and 5,583 SNPs for lettuce. Further filtering of the SNP dataset produced over 1,000 high quality SNP markers for each species. We obtained a genotyping rate of 201.2 genotypes/SNP and 58.3 genotypes/SNP for arabidopsis (n = 222 samples) and lettuce (n = 87 samples), respectively. Linkage mapping using these SNPs resulted in stable map configurations. We have therefore shown that the SBG approach presented provides users with the utmost flexibility in garnering high quality markers that can be directly used for genotyping and downstream applications. Until advances and costs will allow for routine whole-genome sequencing of populations, we expect that sequence-based genotyping technologies such as SBG will be essential for genotyping of model and non-model genomes alike.

High-throughput detection of induced mutations and natural variation using KeyPoint technology.

Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variants among mutant or natural populations. Current (pre-) screening methods such as TILLING and EcoTILLING are based on the detection of single base mismatches in heteroduplexes using endonucleases such as CEL 1. However, there are drawbacks in the use of endonucleases due to their relatively poor cleavage efficiency and exonuclease activity. Moreover, pre-screening methods do not reveal information about the nature of sequence changes and their possible impact on gene function. We present KeyPoint technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations. KeyPoint combines multi-dimensional pooling of large numbers of individual DNA samples and the use of sample identification tags ("sample barcoding") with next-generation sequencing technology. We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection. We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel) allelic variation in a high-throughput fashion.

Generation of a 3D indexed Petunia insertion database for reverse genetics.

BLAST searchable databases containing insertion flanking sequences have revolutionized reverse genetics in plant research. The development of such databases has so far been limited to a small number of model species and normally requires extensive labour input. Here we describe a highly efficient and widely applicable method that we adapted to identify unique transposon-flanking genomic sequences in Petunia. The procedure is based on a multi-dimensional pooling strategy for the collection of DNA samples; up to thousands of different templates are amplified from each of the DNA pools separately, and knowledge of their source is safeguarded by the use of pool-specific (sample) identification tags in one of the amplification primers. All products are combined into a single sample that is subsequently used as a template for unidirectional pyrosequencing. Computational analysis of the clustered sequence output allows automatic assignment of sequences to individual DNA sources. We have amplified and analysed transposon-flanking sequences from a Petunia transposon insertion library of 1000 individuals. Using 30 DNA isolations, 70 PCR reactions and two GS20 sequencing runs, we were able to allocate around 10 000 transposon flanking sequences to specific plants in the library. These sequences have been organized in a database that can be BLAST-searched for insertions into genes of interest. As a proof of concept, we have performed an in silico screen for insertions into members of the NAM/NAC transcription factor family. All in silico-predicted transposon insertions into members of this family could be confirmed in planta.

Complexity reduction of polymorphic sequences (CRoPS): a novel approach for large-scale polymorphism discovery in complex genomes.

Application of single nucleotide polymorphisms (SNPs) is revolutionizing human bio-medical research. However, discovery of polymorphisms in low polymorphic species is still a challenging and costly endeavor, despite widespread availability of Sanger sequencing technology. We present CRoPS as a novel approach for polymorphism discovery by combining the power of reproducible genome complexity reduction of AFLP with Genome Sequencer (GS) 20/GS FLX next-generation sequencing technology. With CRoPS, hundreds-of-thousands of sequence reads derived from complexity-reduced genome sequences of two or more samples are processed and mined for SNPs using a fully-automated bioinformatics pipeline. We show that over 75% of putative maize SNPs discovered using CRoPS are successfully converted to SNPWave assays, confirming them to be true SNPs derived from unique (single-copy) genome sequences. By using CRoPS, polymorphism discovery will become affordable in organisms with high levels of repetitive DNA in the genome and/or low levels of polymorphism in the (breeding) germplasm without the need for prior sequence information.

Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations.

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.

Clonally expanded mtDNA point mutations are abundant in individual cells of human tissues.

Using single-cell sequence analysis, we discovered that a high proportion of cells in tissues as diverse as buccal epithelium and heart muscle contain high proportions of clonal mutant mtDNA expanded from single initial mutant mtDNA molecules. We demonstrate that intracellular clonal expansion of somatic point mutations is a common event in normal human tissues. This finding implies efficient homogenization of mitochondrial genomes within individual cells. Significant qualitative differences observed between the spectra of clonally expanded mutations in proliferating epithelial cells and postmitotic cardiomyocytes suggest, however, that either the processes generating these mutations or mechanisms driving them to homoplasmy are likely to be fundamentally different between the two tissues. Furthermore, the ability of somatic mtDNA mutations to expand (required for their phenotypic expression), as well as their apparently high incidence, reinforces the possibility that these mutations may be involved actively in various physiological processes such as aging and degenerative disease. The abundance of clonally expanded point mutations in individual cells of normal tissues also suggests that the recently discovered accumulation of mtDNA mutations in tumors may be explained by processes that are similar or identical to those operating in the normal tissue.

Searching for genetic determinants of human aging and longevity: opportunities and challenges.

One way of testing possible causal relationships between various functional pathways and aging and longevity processes is to comparatively analyze groups of elderly individuals with select phenotypes for sequence variation in all genes participating in these pathways. Such direct association analysis to identify 'candidate pathways' in aging and longevity is theoretically feasible, with the complete sequence of the human genome known and massive gene annotation projects underway. To find all possible sequence variation of a large number of genes in aging populations, efficient genotyping methods are needed. Here, we describe the use of one such method, two-dimensional gene scanning (TDGS), for screening populations of centenarians and controls for polymorphic variation in the large BRCA1 breast cancer susceptibility gene.