SNPing variation from genomes

A report on the fourth International Meeting on Single Nucleotide Polymorphisms and Complex Genome Analysis, Stockholm, Sweden, 10-13 October 2001.     

群体基因组结构变异检测工作流

结构变异作为人类基因组上的一种大规模的变异类型,对分子与细胞进程、调节功能、基因表达调控、个体表型具有重要的影响,检测群体中基因组结构变异有助于绘制群体基因组变异图谱,刻画群体遗传进化特征,为疾病诊治、精准医疗的发展提供支撑。本研究提出一种面向高通量测序的群体基因组结构变异检测工作流,该工作流通过使用多种高性能基因组结构变异检测算法实现全面、精准的结构变异挖掘,使用多层融合与过滤获得高精度群体结构变异候选集合,利用基因型重新校正、变异修剪、类型校对,最终完整绘制群体基因组结构变异图谱。基于该工作流对由267个样本组成的人群进行群体结构变异检测,检测出了96 202个结构变异,其变异种类和频率分布与其他国际基因组计划相符,这些结果证明了本工作流具有良好的群体结构变异检测能力。同时,工作流通过并行的方式在内存可控的基础上显著降低了分析时间,为大规模人群基因组结构变异的高效检测提供了重要支撑。     

SNPing in the human genome

More than a million genetic markers in the form of single nucleotide polymorphisms are now available for use in genotype-phenotype studies in humans. The application of new strategies for representational cloning and sequencing from genomes combined with the mining of high-quality sequence variations in clone overlaps of genomic and/or cDNA sequences has played an important role in generating this new resource. The focus of variation analysis is now shifting from the identification of new markers to their typing in populations, and novel typing strategies are rapidly emerging. Assay readouts on oligonucleotide arrays, in microtiter plates, gels, flow cytometers and mass spectrometers have all been developed, but decreasing cost and increasing throughput of DNA typing remain key if high-density genetic maps are to be applied on a large scale.     

Virus-derived variation in diverse human genomes

Acquisition of genetic material from viruses by their hosts can generate inter-host structural genome variation. We developed computational tools enabling us to study virus-derived structural variants (SVs) in population-scale whole genome sequencing (WGS) datasets and applied them to 3,332 humans. Although SVs had already been cataloged in these subjects, we found previously-overlooked virus-derived SVs. We detected non-germline SVs derived from squirrel monkey retrovirus (SMRV), human immunodeficiency virus 1 (HIV-1), and human T lymphotropic virus (HTLV-1); these variants are attributable to infection of the sequenced lymphoblastoid cell lines (LCLs) or their progenitor cells and may impact gene expression results and the biosafety of experiments using these cells. In addition, we detected new heritable SVs derived from human herpesvirus 6 (HHV-6) and human endogenous retrovirus-K (HERV-K). We report the first solo-direct repeat (DR) HHV-6 likely to reflect DR rearrangement of a known full-length endogenous HHV-6. We used linkage disequilibrium between single nucleotide variants (SNVs) and variants in reads that align to HERV-K, which often cannot be mapped uniquely using conventional short-read sequencing analysis methods, to locate previously-unknown polymorphic HERV-K loci. Some of these loci are tightly linked to trait-associated SNVs, some are in complex genome regions inaccessible by prior methods, and some contain novel HERV-K haplotypes likely derived from gene conversion from an unknown source or introgression. These tools and results broaden our perspective on the coevolution between viruses and humans, including ongoing virus-to-human gene transfer contributing to genetic variation between humans.     

Natural variation in SAR11 marine bacterioplankton genomes inferred from metagenomic data

Background  

One objective of metagenomics is to reconstruct information about specific uncultured organisms from fragmentary environmental DNA sequences. We used the genome of an isolate of the marine alphaproteobacterium SAR11 ('Candidatus Pelagibacter ubique'; strain HTCC1062), obtained from the cold, productive Oregon coast, as a query sequence to study variation in SAR11 metagenome sequence data from the Sargasso Sea, a warm, oligotrophic ocean gyre.     

The generation of variation in bacterial genomes

In the context of a general overview of molecular mechanisms of microbial evolution, several genetic systems known to either promote or restrain the generation of genetic variations are discussed. Particular attention is given to functions involved in DNA rearrangements and DNA acquisition. Sporadic actions by a variety of such systems influencing genetic stability in either way result in a level of genetic plasticity which is tolerable to the overall wealth of microbial populations but which allows for evolutionary change needed for a steady adaptation to variable selective forces. Although these evolutionarily relevant biological functions are encoded by the genome of each individual, their actions are exerted to some degree randomly in rare individuals and are therefore seemingly nondeterministic and become manifest at the population level.     

Cytoplasmic DNA variation and relationships in cereal genomes

Summary Chloroplast (cp) and mitochondrial (mt) DNAs were isolated from four cereal genomes (cultivated wheat, rye, barley and oats) and compared by restriction nuclease analysis. Cleavage of cp and mt DNAs by Sal I, Kpn I, Xho I and EcoR I enzymes indicated that each cereal group contains specific cytoplasmic DNAs. A phylogenetic tree of cereal evolution has been obtained on the basis of cp DNA homologies. It is suggested that wheat and rye diverged after their common ancestor had diverged from the ancestor of barley. This was preceded by the divergence of the common ancestor of wheat, rye and barley and the ancestor of oats.The molecular weight of the different cp DNAs was determined from the Sal I and Kpn I patterns. cp DNAs from wheat, rye, barley and oats appeared to be characterized by a very similar molecular weight of about 80–82.10⁶ d.In the case of the mt DNAs, the great number of restriction fragments obtained with the restriction enzymes used prevented precise comparisons and determination of molecular weights.     

Transformation of natural genetic variation into Haemophilus influenzae genomes

Many bacteria are able to efficiently bind and take up double-stranded DNA fragments, and the resulting natural transformation shapes bacterial genomes, transmits antibiotic resistance, and allows escape from immune surveillance. The genomes of many competent pathogens show evidence of extensive historical recombination between lineages, but the actual recombination events have not been well characterized. We used DNA from a clinical isolate of Haemophilus influenzae to transform competent cells of a laboratory strain. To identify which of the ~40,000 polymorphic differences had recombined into the genomes of four transformed clones, their genomes and their donor and recipient parents were deep sequenced to high coverage. Each clone was found to contain ~1000 donor polymorphisms in 3-6 contiguous runs (8.1±4.5 kb in length) that collectively comprised ~1-3% of each transformed chromosome. Seven donor-specific insertions and deletions were also acquired as parts of larger donor segments, but the presence of other structural variation flanking 12 of 32 recombination breakpoints suggested that these often disrupt the progress of recombination events. This is the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, connecting experimental studies of transformation with the high levels of natural genetic variation found in isolates of the same species.     

Characterizing complex structural variation in germline and somatic genomes

Genome structural variation (SV) is a major source of genetic diversity in mammals and a hallmark of cancer. Although SV is typically defined by its canonical forms (duplication, deletion, insertion, inversion and translocation), recent breakpoint mapping studies have revealed a surprising number of 'complex' variants that evade simple classification. Complex SVs are defined by clustered breakpoints that arose through a single mutation but cannot be explained by one simple end-joining or recombination event. Some complex variants exhibit profoundly complicated rearrangements between distinct loci from multiple chromosomes, whereas others involve more subtle alterations at a single locus. These diverse and unpredictable features present a challenge for SV mapping experiments. Here, we review current knowledge of complex SV in mammals, and outline techniques for identifying and characterizing complex variants using next-generation DNA sequencing.     

Inter‐varietal structural variation in grapevine genomes

Grapevine (Vitis vinifera L.) is one of the world's most important crop plants, which is of large economic value for fruit and wine production. There is much interest in identifying genomic variations and their functional effects on inter‐varietal, phenotypic differences. Using an approach developed for the analysis of human and mammalian genomes, which combines high‐throughput sequencing, array comparative genomic hybridization, fluorescent in situ hybridization and quantitative PCR, we created an inter‐varietal atlas of structural variations and single nucleotide variants (SNVs) for the grapevine genome analyzing four economically and genetically relevant table grapevine varieties. We found 4.8 million SNVs and detected 8% of the grapevine genome to be affected by genomic variations. We identified more than 700 copy number variation (CNV) regions and more than 2000 genes subjected to CNV as potential candidates for phenotypic differences between varieties.     

Detecting microsatellites within genomes: significant variation among algorithms

Background  

Microsatellites are short, tandemly-repeated DNA sequences which are widely distributed among genomes. Their structure, role and evolution can be analyzed based on exhaustive extraction from sequenced genomes. Several dedicated algorithms have been developed for this purpose. Here, we compared the detection efficiency of five of them (TRF, Mreps, Sputnik, STAR, and RepeatMasker).     

Retrotransposon distribution and copy number variation in gymnosperm genomes

Retrotransposable elements (REs) and related sequences form a large proportion of conifer genomes. During genome evolution, some RE sequences are degraded or eliminated, but some are evolutionarily stable, and can be identified even in distantly related species. Use of genome sequence information from loblolly pine (Pinus taeda) enables investigation of divergent non-coding RE sequences in other pine and conifer species, including Scots pine (Pinus sylvestris). Non-specific inter-retrotransposon amplified polymorphism technique (IRAP) as well as the amplification polymorphism of 12 RE families were investigated in 80 gymnosperm species. The obtained results were compared with phylogenetic relationships among gymnosperms. Investigation of distantly related gymnosperm species reveals persistent RE sequences, such as IFG and Pineywoods, distributed among a wide range of plant lineages. RE sequence divergence was observed, reflecting periods of inactivity and degradation during speciation of pine lineages, as demonstrated by the delineation of the main pine subgenera. Intraspecific variation of 10 RE copy numbers (CN) between Scots pine individuals ranged from 8.9 to 26.6% of the overall mean estimates. CN analyses were performed in 16 additional gymnosperm species. The analysed pine species contained a similar complement of RE families; however, CN and genome occupation proportions differ. A decrease in RE CN estimates can reflect sequence divergence, associated with independent transposition events. Transposition of some REs can be induced by stress conditions; therefore, even distantly related species inhabiting extreme environments could have similar patterns or distribution of these elements.     

Anchored pseudo-de novo assembly of human genomes identifies extensive sequence variation from unmapped sequence reads

The human genome reference (HGR) completion marked the genomics era beginning, yet despite its utility universal application is limited by the small number of individuals used in its development. This is highlighted by the presence of high-quality sequence reads failing to map within the HGR. Sequences failing to map generally represent 2–5 % of total reads, which may harbor regions that would enhance our understanding of population variation, evolution, and disease. Alternatively, complete de novo assemblies can be created, but these effectively ignore the groundwork of the HGR. In an effort to find a middle ground, we developed a bioinformatic pipeline that maps paired-end reads to the HGR as separate single reads, exports unmappable reads, de novo assembles these reads per individual and then combines assemblies into a secondary reference assembly used for comparative analysis. Using 45 diverse 1000 Genomes Project individuals, we identified 351,361 contigs covering 195.5 Mb of sequence unincorporated in GRCh38. 30,879 contigs are represented in multiple individuals with ~40 % showing high sequence complexity. Genomic coordinates were generated for 99.9 %, with 52.5 % exhibiting high-quality mapping scores. Comparative genomic analyses with archaic humans and primates revealed significant sequence alignments and comparisons with model organism RefSeq gene datasets identified novel human genes. If incorporated, these sequences will expand the HGR, but more importantly our data highlight that with this method low coverage (~10–20×) next-generation sequencing can still be used to identify novel unmapped sequences to explore biological functions contributing to human phenotypic variation, disease and functionality for personal genomic medicine.     

ENGINES: exploring single nucleotide variation in entire human genomes

Background  

Next generation ultra-sequencing technologies are starting to produce extensive quantities of data from entire human genome or exome sequences, and therefore new software is needed to present and analyse this vast amount of information. The 1000 Genomes project has recently released raw data for 629 complete genomes representing several human populations through their Phase I interim analysis and, although there are certain public tools available that allow exploration of these genomes, to date there is no tool that permits comprehensive population analysis of the variation catalogued by such data.     

Evolution of simple sequence repeat-mediated phase variation in bacterial genomes

Mutability as mechanism for rapid adaptation to environmental challenge is an alluringly simple concept whose apotheosis is realized in simple sequence repeats (SSR). Bacterial genomes of several species contain SSRs with a proven role in adaptation to environmental fluctuations. SSRs are hypermutable and generate reversible mutations in localized regions of bacterial genomes, leading to phase variable ON/OFF switches in gene expression. The application of genetic, bioinformatic, and mathematical/computational modeling approaches are revolutionizing our current understanding of how genomic molecular forces and environmental factors influence SSR-mediated adaptation and led to evolution of this mechanism of localized hypermutation in bacterial genomes.