Developing genomic resources in two Linum species via 454 pyrosequencing and genomic reduction

Recent advances in next-generation DNA sequencing (NGS) have enhanced the development of genomic resources such as contigs or single-nucleotide polymorphisms (SNPs) for evolutionary studies of a nonmodel species with a complex and unsequenced genome. This study presents an application of a NGS technique in combination with genomic reduction and advanced bioinformatics tools to identify contigs and SNPs from multiple samples of two Linum species. A full Roche 454 GS FLX run of 16 diverse Linum samples representing cultivated flax (Linum usitatissimum L.) and its wild progenitor (Linum bienne Mill.) generated approximately 1.6 million sequence reads with a total length of 498 Mbp. Application of the computational pipeline de novo identification of alleles identified 713 contigs and 1067 SNPs. A blast search revealed alignments of all 713 contigs with 491 existing Linum scaffolds and gene annotations associated with 512 contigs. Sanger sequencing confirmed 95% of 79 selected contigs and 94% of 272 SNPs and identified 211 new SNPs and 19 new indels. The scored 454 SNP data were highly imbalanced for assayed samples. These findings not only are useful for evolutionary studies of Linum species but also help to illustrate the utility of NGS technologies in SNP discovery for nonmodel organisms.     

SNP Discovery Using Next Generation Transcriptomic Sequencing in Atlantic Herring (Clupea harengus)

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.     

基于短序列测序数据的四倍体拟南芥转录组研究

为了促进对四倍体拟南芥(A.suecica)的研究,阐明多倍体植物在染色体加倍过程中遗传物质的变化,从而在分子层面上解释多倍体植物的环境适应和进化机制,描述了一套基于第二代测序技术的转录组短序列组装和生物信息学分析方法.通过对23 000 000条来至于Illumina测序平台的序列数据进行SOAPdenovo组装,以...