GReEn: a tool for efficient compression of genome resequencing data

Research in the genomic sciences is confronted with the volume of sequencing and resequencing data increasing at a higher pace than that of data storage and communication resources, shifting a significant part of research budgets from the sequencing component of a project to the computational one. Hence, being able to efficiently store sequencing and resequencing data is a problem of paramount importance. In this article, we describe GReEn (Genome Resequencing Encoding), a tool for compressing genome resequencing data using a reference genome sequence. It overcomes some drawbacks of the recently proposed tool GRS, namely, the possibility of compressing sequences that cannot be handled by GRS, faster running times and compression gains of over 100-fold for some sequences. This tool is freely available for non-commercial use at ftp://ftp.ieeta.pt/~ap/codecs/GReEn1.tar.gz.     

SNPMeta: SNP annotation and SNP metadata collection without a reference genome

The increase in availability of resequencing data is greatly accelerating SNP discovery and has facilitated the development of SNP genotyping assays. This, in turn, is increasing interest in annotation of individual SNPs. Currently, these data are only available through curation, or comparison to a reference genome. Many species lack a reference genome, but are still important genetic models or are significant species in agricultural production or natural ecosystems. For these species, it is possible to annotate SNPs through comparison with cDNA, or data from well‐annotated genes in public repositories. We present SNPMeta, a tool which gathers information about SNPs by comparison with sequences present in GenBank databases. SNPMeta is able to annotate SNPs from contextual sequence in SNP assay designs, and SNPs discovered through genotyping by sequencing (GBS) approaches. However, SNPs discovered through GBS occur throughout the genome, rather than only in gene space, and therefore do not annotate at high rates. SNPMeta can therefore be used to annotate SNPs in nonmodel species or species that lack a reference genome. Annotations generated by SNPMeta are highly concordant with annotations that would be obtained from a reference genome.     

Direct Chloroplast Sequencing: Comparison of Sequencing Platforms and Analysis Tools for Whole Chloroplast Barcoding

Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina) and Ion Torrent (Life Technology) sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genome sequence of a wild rice plant relative to cultivated rice (cv. Nipponbare). Consensus chloroplast sequences were produced by mapping sequence reads to the reference rice chloroplast genome or by de novo assembly and mapping of the resulting contigs to the reference sequence. A total of 122 polymorphisms (SNPs and indels) between the wild and cultivated rice chloroplasts were predicted by these different sequencing and analysis methods. Of these, a total of 102 polymorphisms including 90 SNPs were predicted by both platforms. Indels were more variable with different sequencing methods, with almost all discrepancies found in homopolymers. The Ion Torrent platform gave no apparent false SNP but was less reliable for indels. The methods should be suitable for routine barcoding using appropriate combinations of sequencing platform and data analysis.     

Single nucleotide polymorphism discovery in barley using autoSNPdb

Molecular markers are used to provide the link between genotype and phenotype, for the production of molecular genetic maps and to assess genetic diversity within and between related species. Single nucleotide polymorphisms (SNPs) are the most abundant molecular genetic marker. SNPs can be identified in silico , but care must be taken to ensure that the identified SNPs reflect true genetic variation and are not a result of errors associated with DNA sequencing. The SNP detection method autoSNP has been developed to identify SNPs from sequence data for any species. Confidence in the predicted SNPs is based on sequence redundancy, and haplotype co-segregation scores are calculated for a further independent measure of confidence. We have extended the autoSNP method to produce autoSNPdb, which integrates SNP and gene annotation information with a graphical viewer. We have applied this software to public barley expressed sequences, and the resulting database is available over the Internet. SNPs can be viewed and searched by sequence, functional annotation or predicted synteny with a reference genome, in this case rice. The correlation between SNPs and barley cultivar, expressed tissue type and development stage has been collated for ease of exploration. An average of one SNP per 240 bp was identified, with SNPs more prevalent in the 5' regions and simple sequence repeat (SSR) flanking sequences. Overall, autoSNPdb can provide a wealth of genetic polymorphism information for any species for which sequence data are available.     

Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach

Advancements in next-generation sequencing technology have enabled whole genome re-sequencing in many species providing unprecedented discovery and characterization of molecular polymorphisms. There are limitations, however, to next-generation sequencing approaches for species with large complex genomes such as barley and wheat. Genotyping-by-sequencing (GBS) has been developed as a tool for association studies and genomics-assisted breeding in a range of species including those with complex genomes. GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost. Here we present a GBS approach for species that currently lack a reference genome sequence. We developed a novel two-enzyme GBS protocol and genotyped bi-parental barley and wheat populations to develop a genetically anchored reference map of identified SNPs and tags. We were able to map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map, and 20,000 SNPs and 367,000 tags on the Synthetic W9784 × Opata85 (SynOpDH) wheat reference map. To further evaluate GBS in wheat, we also constructed a de novo genetic map using only SNP markers from the GBS data. The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence. Development of the sequenced reference genome(s) will in turn increase the utility of GBS data enabling physical mapping of genes and haplotype imputation of missing data. Finally, as a result of low per-sample costs, GBS will have broad application in genomics-assisted plant breeding programs.     

DNA-COMPACT: DNA COMpression Based on a Pattern-Aware Contextual Modeling Technique

Genome data are becoming increasingly important for modern medicine. As the rate of increase in DNA sequencing outstrips the rate of increase in disk storage capacity, the storage and data transferring of large genome data are becoming important concerns for biomedical researchers. We propose a two-pass lossless genome compression algorithm, which highlights the synthesis of complementary contextual models, to improve the compression performance. The proposed framework could handle genome compression with and without reference sequences, and demonstrated performance advantages over best existing algorithms. The method for reference-free compression led to bit rates of 1.720 and 1.838 bits per base for bacteria and yeast, which were approximately 3.7% and 2.6% better than the state-of-the-art algorithms. Regarding performance with reference, we tested on the first Korean personal genome sequence data set, and our proposed method demonstrated a 189-fold compression rate, reducing the raw file size from 2986.8 MB to 15.8 MB at a comparable decompression cost with existing algorithms. DNAcompact is freely available at https://sourceforge.net/projects/dnacompact/for research purpose.     

Whole-genome re-sequencing

DNA sequencing can be used to gain important information on genes, genetic variation and gene function for biological and medical studies. The growing collection of publicly available reference genome sequences will underpin a new era of whole genome re-sequencing, but sequencing costs need to fall and throughput needs to rise by several orders of magnitude. Novel technologies are being developed to meet this need by generating massive amounts of sequence that can be aligned to the reference sequence. The challenge is to maintain the high standards of accuracy and completeness that are hallmarks of the previous genome projects. One or more new sequencing technologies are expected to become the mainstay of future research, and to make DNA sequencing centre stage as a routine tool in genetic research in the coming years.     

Construction of Pseudomolecule Sequences of the aus Rice Cultivar Kasalath for Comparative Genomics of Asian Cultivated Rice

Having a deep genetic structure evolved during its domestication and adaptation, the Asian cultivated rice (Oryza sativa) displays considerable physiological and morphological variations. Here, we describe deep whole-genome sequencing of the aus rice cultivar Kasalath by using the advanced next-generation sequencing (NGS) technologies to gain a better understanding of the sequence and structural changes among highly differentiated cultivars. The de novo assembled Kasalath sequences represented 91.1% (330.55 Mb) of the genome and contained 35 139 expressed loci annotated by RNA-Seq analysis. We detected 2 787 250 single-nucleotide polymorphisms (SNPs) and 7393 large insertion/deletion (indel) sites (>100 bp) between Kasalath and Nipponbare, and 2 216 251 SNPs and 3780 large indels between Kasalath and 93-11. Extensive comparison of the gene contents among these cultivars revealed similar rates of gene gain and loss. We detected at least 7.39 Mb of inserted sequences and 40.75 Mb of unmapped sequences in the Kasalath genome in comparison with the Nipponbare reference genome. Mapping of the publicly available NGS short reads from 50 rice accessions proved the necessity and the value of using the Kasalath whole-genome sequence as an additional reference to capture the sequence polymorphisms that cannot be discovered by using the Nipponbare sequence alone.     

Whole genome de novo assemblies of three divergent strains of rice,Oryza sativa,document novel gene space of aus and indica

The use of high throughput genome-sequencing technologies has uncovered a large extent of structural variation in eukaryotic genomes that makes important contributions to genomic diversity and phenotypic variation. When the genomes of different strains of a given organism are compared, whole genome resequencing data are typically aligned to an established reference sequence. However, when the reference differs in significant structural ways from the individuals under study, the analysis is often incomplete or inaccurate. Here, we use rice as a model to demonstrate how improvements in sequencing and assembly technology allow rapid and inexpensive de novo assembly of next generation sequence data into high-quality assemblies that can be directly compared using whole genome alignment to provide an unbiased assessment. Using this approach, we are able to accurately assess the ‘pan-genome’ of three divergent rice varieties and document several megabases of each genome absent in the other two. Many of the genome-specific loci are annotated to contain genes, reflecting the potential for new biological properties that would be missed by standard reference-mapping approaches. We further provide a detailed analysis of several loci associated with agriculturally important traits, including the S5 hybrid sterility locus, the Sub1 submergence tolerance locus, the LRK gene cluster associated with improved yield, and the Pup1 cluster associated with phosphorus deficiency, illustrating the utility of our approach for biological discovery. All of the data and software are openly available to support further breeding and functional studies of rice and other species.     

The design and application of a 50 K SNP chip for a threatened Aotearoa New Zealand passerine,the hihi

Next-generation sequencing has transformed the fields of ecological and evolutionary genetics by allowing for cost-effective identification of genome-wide variation. Single nucleotide polymorphism (SNP) arrays, or “SNP chips”, enable very large numbers of individuals to be consistently genotyped at a selected set of these identified markers, and also offer the advantage of being able to analyse samples of variable DNA quality. We used reduced representation restriction-aided digest sequencing (RAD-seq) of 31 birds of the threatened hihi (Notiomystis cincta; stitchbird) and low-coverage whole genome sequencing (WGS) of 10 of these birds to develop an Affymetrix 50 K SNP chip. We overcame the limitations of having no hihi reference genome and a low quantity of sequence data by separate and pooled de novo assembly of each of the 10 WGS birds. Reads from all individuals were mapped back to these de novo assemblies to identify SNPs. A subset of RAD-seq and WGS SNPs were selected for inclusion on the chip, prioritising SNPs with the highest quality scores whose flanking sequence uniquely aligned to the zebra finch (Taeniopygia guttata) genome. Of the 58,466 SNPs manufactured on the chip, 72% passed filtering metrics and were polymorphic. By genotyping 1,536 hihi on the array, we found that SNPs detected in multiple assemblies were more likely to successfully genotype, representing a cost-effective approach to identify SNPs for genotyping. Here, we demonstrate the utility of the SNP chip by describing the high rates of linkage disequilibrium in the hihi genome, reflecting the history of population bottlenecks in the species.     

SAP-A Sequence Mapping and Analyzing Program for Long Sequence Reads Alignment and Accurate Variants Discovery

The third-generation of sequencing technologies produces sequence reads of 1000 bp or more that may contain high polymorphism information. However, most currently available sequence analysis tools are developed specifically for analyzing short sequence reads. While the traditional Smith-Waterman (SW) algorithm can be used to map long sequence reads, its naive implementation is computationally infeasible. We have developed a new Sequence mapping and Analyzing Program (SAP) that implements a modified version of SW to speed up the alignment process. In benchmarks with simulated and real exon sequencing data and a real E. coli genome sequence data generated by the third-generation sequencing technologies, SAP outperforms currently available tools for mapping short and long sequence reads in both speed and proportion of captured reads. In addition, it achieves high accuracy in detecting SNPs and InDels in the simulated data. SAP is available at https://github.com/davidsun/SAP.     

Crop genome sequencing: lessons and rationales

2010 marks the 10th anniversary of the completion of the first plant genome sequence (Arabidopsis thaliana). Triggered by advancements in sequencing technologies, many crop genome sequences have been produced, with eight published since 2008. To date, however, only the rice (Oryza sativa) genome sequence has been finished to a quality level similar to that of the Arabidopsis sequence. This trend to produce draft genomes could affect the ability of researchers to address biological questions of speciation and recent evolution or to link sequence variation accurately to phenotypes. Here, we review the current crop genome sequencing activities, discuss how variability in sequence quality impacts utility for different studies and provide a perspective for a paradigm shift in selecting crops for sequencing in the future.     

Discovery of single-nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete Ophiognomonia clavigignenti-juglandacearum from 454 sequence data

The benefits from recent improvement in sequencing technologies, such as the Roche GS FLX (454) pyrosequencing, may be even more valuable in non-model organisms, such as many plant pathogenic fungi of economic importance. One application of this new sequencing technology is the rapid generation of genomic information to identify putative single-nucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. The focus of this research was to sequence, assemble, discover and validate SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. Genomic DNA from eight isolates of Ophiognomonia clavigignenti-juglandacearum was pooled in one region of a four-region sequencing run on a Roche 454 GS FLX. This yielded 71 million total bases comprising 217,000 reads, 80% of which collapsed into 16,125,754 bases in 30,339 contigs upon assembly. By aligning reads from multiple isolates, we detected 298 SNPs using Roche's GS Mapper. With no reference sequence available, however, it was difficult to distinguish true polymorphisms from sequencing error. Eagleview software was used to manually examine each contig that contained one or more putative SNPs, enabling us to discard all but 45 of the original 298 putative SNPs. Of those 45 SNPs, 13 were validated using standard Sanger sequencing. This research provides a valuable genetic resource for research into the genus Ophiognomonia, demonstrates a framework for the rapid and cost-effective discovery of SNP markers in non-model organisms and should prove especially useful in the case of asexual or clonal fungi with limited genetic variability.     

How can we deliver the large plant genomes? Strategies and perspectives

The first sequenced plant genome, from the small mustard plant Arabidopsis thaliana, was published at the end of 2000. The sequencing of the rice genome is well under way. The sizes of plant genomes vary by a factor of up to 1000, and many important crop plants have genomes that are several times larger than the human genome. To gain insight into the gene toolbox of plant species, numerous large-scale EST sequencing projects have been launched successfully, and analysis procedures are constantly being refined to add maximum value to the sequence data. In addition, an alternative approach to exclude repetitive noncoding DNA and to enrich sequence libraries for gene-containing genomic regions has been developed. This strategy has the potential to deliver information about both genes and regulatory regions outside the transcribed regions.     

SOP3: a web-based tool for selection of oligonucleotide primers for single nucleotide polymorphism analysis by Pyrosequencing

SOP3 is a web-based software tool for designing oligonucleotide primers for use in the analysis of single nucleotide polymorphisms (SNPs). Accessible via the Internet, the application is optimized for developing the PCR and sequencing primers that are necessary for Pyrosequencing. The application accepts as input gene name, SNP reference sequence number, or chromosomal nucleotide location. Output can be parsed by gene name, SNP reference number, heterozygosity value, location, chromosome, or function. The location of an individual polymorphism, such as an intron, exon, or 5' or 3' untranslated region is indicated, as are whether nucleotide changes in an exon are associated with a change in an amino acid sequence. SOP3 presents for each entry a set of forward and biotinylated reverse PCR primers as well as a sequencing primer for use during the analysis of SNPs by Pyrosequencing. Theoretical pyrograms for each allele are calculated and presented graphically. The method has been tested in the development of Pyrosequencing assays for determining SNPs and for deletion/insertion polymorphisms in the human genome. Of the SOP3-designed primer sets that were tested, a large majority of the primer sets have successfully produced PCR products and Pyrosequencing data.     

From Arabidopsis to rice genomics: a survey of French programmes.

During the last ten years, Arabidopsis thaliana has become the most favoured plant system for the study of many aspects of development and adaptation to adverse conditions and diseases. The sequencing of the Arabidopsis thaliana genome is nearly completed with more than 90% of the sequence being released in public databases. This is the first plant genome to be analysed and it has revealed a tremendous amount of information about the nature of the genes it contains and its largely duplicated organisation. French groups have been involved in Arabidopsis genomics at several steps: EST (expressed sequence tags) sequencing, construction and ordering (physical mapping of chromosomes) of a YAC (yeast artificial chromosomes) library, genomic sequencing. In parallel an extensive programme of functional genomics is being undertaken through the systematic analysis of insertional mutants. This information provides a support for analysing other more economically important plant genomes such as the rice genome and constitutes the beginning of a systematic investigation on plant gene functions and will promote new strategies for plant improvement.