Second-generation Sequencing Platforms Research Articles

A practical comparison of the next-generation sequencing platform and assemblers using yeast genome.

Assembling fragmented whole-genomic information from the sequencing data is an inevitable process for further genome-wide research. However, it is intricate to select the appropriate assembly pipeline for unknown species because of the species-specific genomic properties. Therefore, our study focused on relatively more static proclivities of sequencing platforms and assembly algorithms than the fickle genome sequences. A total of 212 draft and polished de novo assemblies were constructed under the different sequencing platforms and assembly algorithms with the repetitive yeast genome. Our comprehensive data indicated that sequencing reads from Oxford Nanopore with R7.3 flow cells generated more continuous assemblies than those derived from the PacBio Sequel, although the homopolymer-based assembly errors and chimeric contigs exist. In addition, the comparison between two second-generation sequencing platforms showed that Illumina NovaSeq 6000 provides more accurate and continuous assembly in the second-generation-sequencing-first pipeline, but MGI DNBSEQ-T7 provides a cheap and accurate read in the polishing process. Furthermore, our insight into the relationship among the computational time, read length, and coverage depth provided clues to the optimal pipelines of yeast assembly.

Accurate Sequencing and Haplotyping from 10 Cells Using Long Fragment Read (LFR) Technology.

In this chapter, we describe how Long Fragment Read (LFR) technology can be applied to samples consisting of very few cells (5-20) to enable complete genome sequencing and haplotyping with a very low false positive error rate. LFR is a method for processing DNA or cells prior to sequencing on any second-generation DNA sequencing platform (e.g., MGI's DNBSEQ, Illumina sequencers, etc.). First, the LFR process incorporates a low-bias whole genome amplification step allowing accurate sequencing from very low DNA inputs (as low as 32 picograms, the mass contained within 5 diploid human cells). In addition, LFR enables the haplotyping of nearly all genomic variations with N50 contig lengths up to ~1Mb. Furthermore, if data from this method are analyzed with parental genotype data, it is possible to generate phased variants in uninterrupted contigs spanning entire chromosomes. Importantly, the barcoding process utilized in this method allows for the detection and correction of most amplification, sequencing, and mapping errors, yielding false positive error rates as low as 10-9. Finally, the cost of this method is modest and enables extremely high-quality whole genome sequence and haplotype data from as few as 5 cells. We know of few other methods that can achieve this.

Targeted Whole Genome Sequencing (TWG-Seq) of Cucumber Green Mottle Mosaic Virus Using Tiled Amplicon Multiplex PCR and Nanopore Sequencing.

Rapid and reliable detection tools are essential for disease surveillance and outbreak management, and genomic data is essential to determining pathogen origin and monitoring of transmission pathways. Low virus copy number and poor RNA quality can present challenges for genomic sequencing of plant viruses, but this can be overcome by enrichment of target nucleic acid. A targeted whole genome sequencing (TWG-Seq) approach for the detection of cucumber green mottle mosaic virus (CGMMV) has been developed where overlapping amplicons generated using two multiplex RT-PCR assays are then sequenced using the Oxford Nanopore MinION. Near complete coding region sequences were assembled with ≥100× coverage for infected leaf tissue dilution samples with RT-qPCR cycle quantification (Cq) values from 11.8 to 38 and in seed dilution samples with Cq values 13.8 to 27. Consensus sequences assembled using this approach showed greater than 99% nucleotide similarity when compared to genomes produced using metagenomic sequencing. CGMMV could be confidently detected in historical seed isolates with degraded RNA. Whilst limited access to, and costs associated with second-generation sequencing platforms can influence diagnostic outputs, the portable Nanopore technology offers an affordable high throughput sequencing alternative when combined with TWG-Seq for low copy or degraded samples.

Nanopore Sequencing Provides Rapid and Reliable Insight Into Microbial Profiles of Intensive Care Units.

Fast and accurate identification of pathogens is an essential task in healthcare settings. Second-generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopore's minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units of a hospital in Brazil. While our results demonstrate that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.

桥粒斑蛋白基因自发突变致Carvajal综合征一例

Objective To report a case of Carvajai syndrome caused by a spontaneous mutation in the desmoplakin (DSP) gene. Methods Clinical data were collected form a patient with Carvajal syndrome in Department of Dermatology, The First Affiliated Hospital of Zhengzhou University. Peripheral blood samples were obtained from the proband, his parents and 100 unrelated healthy controls, and blood genomic DNA was extracted. The ion torrent PGM second-generation sequencing platform was used to detect sequence variations in coding regions of exons in skin disease-related genes in the proband and his parents, and the pathogenic variation was verified by PCR-Sanger sequencing. Results The proband clinically presented with woolly hair, diffuse palmoplantar keratoderma, onychodysplasia, hypodontia and sinus arrhythmia as shown by electrocardiogram. Gene sequencing revealed a heterozygous missense mutation c.1790C>T (p.Ser597Leu) in exon 14 of the DSP gene in the proband, resulting in the substitution of serine by leucine at amino acid position 597. No mutation was identified in the proband′s parents or the 100 healthy controls, so the mutation in the proband is spontaneous. The patient was finally diagnosed with Carvajal syndrome according to the clinical manifestations, gene detection and auxiliary examination results. Conclusion The heterozygous missense mutation C.1790C>T (p.Ser597Leu) of the DSP gene may be the pathogenic mutation for the clinical phenotype of the patient. Key words: Keratoderma, palmoplantar; Desmoplakins; DNA mutational analysis; Carvajal syndrome; Hypodontia; Woolly hair

Seasonality overwhelms aquacultural activity in determining the composition and assembly of the bacterial community in Lake Taihu, China

Aquaculture accounts for an extremely valuable and rapidly expanding sector of global food production, yet its environmental impacts on aquatic ecosystems have attracted much concern. In this study, we collected water samples from eastern Lake Taihu, China. We targeted sites varying in their intensity of aquacultural activities and sampled them over multiple seasons. For each sample, we measured physicochemical variables, and we sequenced the 16S rRNA gene of the respective bacterial communities using an Illumina second-generation sequencing platform. Marked differences in diversity and bacterial community composition were observed between seasons, whereas we observed relatively weak differences between sites. Remarkable differences in the abundance of the bacterial community were observed at the phylum and genus levels across the different seasons. Stochastic processes dominated the assembly of bacterial communities in the aquaculture-influenced systems, and the assembly processes of bacterial community differed between seasons. Our observations highlight the effect of seasonality on bacterial communities and provide a more complete knowledge base for the proper assessment of the effects of aquacultural activities on freshwater ecosystems.

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

BackgroundSugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.MethodsWe generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.ResultsA total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.DiscussionThe new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

Full-length transcriptome sequences and the identification of putative genes for flavonoid biosynthesis in safflower

BackgroundThe flower of the safflower (Carthamus tinctorius L.) has been widely used in traditional Chinese medicine for the ability to improve cerebral blood flow. Flavonoids are the primary bioactive components in safflower, and their biosynthesis has attracted widespread interest. Previous studies mostly used second-generation sequencing platforms to survey the putative flavonoid biosynthesis genes. For a better understanding of transcription data and the putative genes involved in flavonoid biosynthesis in safflower, we carry our study.ResultsHigh-quality RNA was extracted from six types of safflower tissue. The RNAs of different tissues were mixed equally and used for multiple size-fractionated libraries (1–2, 2–3 and 3-6 k) library construction. Five cells were carried (2 cells for 1–2 and for 2-3 k libraries and 1 cell for 3-6 k libraries). 10.43Gb clean data and 38,302 de-redundant sequences were captured. 44 unique isoforms were annotated as encoding enzymes involved in flavonoid biosynthesis. The full length flavonoid genes were characterized and their evolutional relationship and expressional pattern were analyzed. They can be divided into eight families, with a large differences in the tissue expression. The temporal expressions under MeJA treatment were also measured, 9 genes are significantly up-regulated and 2 genes are significantly down-regulated. The genes involved in flavonoid synthesis in safflower were predicted in our study. Besides, the SSR and lncRNA are also analyzed in our study.ConclusionsFull-length transcriptome sequences were used in our study. The genes involved in flavonoid synthesis in safflower were predicted in our study. Combined the determination of flavonoids, CtC4H2, CtCHS3, CtCHI3, CtF3H3, CtF3H1 are mainly participated in MeJA promoting the synthesis of flavonoids. Our results also provide a valuable resource for further study on safflower.

Hybridization capture reveals microbial diversity missed using current profiling methods

BackgroundMicroorganisms comprise the majority of living organisms on our planet. For many years, exploration of the composition of microbial communities has been performed through the PCR-based study of the small subunit rRNA gene due to its high conservation across the domains of life. The application of this method has resulted in the discovery of many unexpected evolutionary lineages. However, amplicon sequencing is subject to numerous biases, with some taxa being missed, and is limited by the read length of second-generation sequencing platforms, which drastically reduces the phylogenetic resolution.ResultsHere, we describe a hybridization capture strategy that allows the enrichment of 16S rRNA genes from metagenomic samples and enables an exhaustive identification and a complete reconstruction of the biomarker. Applying this approach to a microbial mock community and a soil sample, we demonstrated that hybridization capture is able to reveal greater microbial diversity than 16S rDNA amplicon sequencing and shotgun sequencing. The reconstruction of full-length 16S rRNA genes facilitated the improvement of phylogenetic resolution and the discovery of novel prokaryotic taxa.ConclusionsOur results demonstrate that hybridization capture can lead to major breakthroughs in our understanding of microbial diversity, overcoming the limitations of conventional 16S rRNA gene studies. If applied to a broad range of environmental samples, this innovative approach could reveal the undescribed diversity of the still underexplored microbial communities and could provide a better understanding of ecosystem function.

Targeted metagenomic sequencing data of human gut microbiota associated with Blastocystis colonization

In the past decade, metagenomics studies have become widespread due to the arrival of second-generation sequencing platforms characterized by low costs, high throughput and short read lengths. Today, although benchtop sequencers are considered to be accurate platforms to deliver data for targeted metagenomics studies, the limiting factor has become the analysis of these data. In a previous paper, we performed an Ion Torrent PGM 16S rDNA gene sequencing of faecal DNAs from 48 Blastocystis-colonized patients and 48 Blastocystis-negative subjects, in order to decipher the impact of this widespread protist on gut microbiota composition and diversity. We report here on the Ion Torrent targeted metagenomic sequencing and analysis of these 96 human faecal samples, and the complete datasets from raw to analysed data. We also provide the key steps of the bioinformatic analyses, from library preparation to data filtering and OTUs tables generation. This data represents a valuable resource for the scientific community, enabling re-processing of these targeted metagenomic datasets through various pipelines and a comparative evaluation of microbiota analysis methods.

The heterogeneity of composition and assembly processes of the microbial community between different nutrient loading lake zones in Taihu Lake.

To investigate the differences in the microbial community composition and assembly process in two lake zones (Meiliang Bay (MLB) and Xukou Bay (XKB) in Taihu Lake, China) with different nutrient loadings, water samples were collected. Both the 16S ribosomal RNA (rRNA) gene for the bacterial community and the 18S rRNA gene for the microeukaryote community were investigated using the Illumina second-generation sequencing platform (2×250 paired-end). The results indicated that both the bacterioplankton and microeukaryote community composition derived from the two lake zones were significantly different. Significantly higher operational taxonomic unit (OTU) richness (P<0.01) and phylogenetic diversity (P<0.05) were found for the bacterioplankton community of MLB. However, a comparable alpha diversity was found between the microeukaryote communities of MLB and XKB (P>0.05). Environmental factors significantly affected the community compositions in XKB for both the bacterioplankton and microeukaryotes. However, they did not significantly influence the microbial community composition in MLB, except for a weak correlation between dissolved organic carbon (DOC) and the microeukaryote community. The microbial communities tended to be more phylogenetically clustered than expected by chance in the two lake zones. Moreover, the results of the phylogenetic structure suggest that deterministic processes played overwhelming roles in driving the assembly of both the bacterioplankton and microeukaryote community in XKB.

Targeted genotyping-by-sequencing permits cost-effective identification and discrimination of pasture grass species and cultivars.

A targeted amplicon-based genotyping-by-sequencing approach has permitted cost-effective and accurate discrimination between ryegrass species (perennial, Italian and inter-species hybrid), and identification of cultivars based on bulked samples. Perennial ryegrass and Italian ryegrass are the most important temperate forage species for global agriculture, and are represented in the commercial pasture seed market by numerous cultivars each composed of multiple highly heterozygous individuals. Previous studies have identified difficulties in the use of morphophysiological criteria to discriminate between these two closely related taxa. Recently, a highly multiplexed single nucleotide polymorphism (SNP)-based genotyping assay has been developed that permits accurate differentiation between both species and cultivars of ryegrasses at the genetic level. This assay has since been further developed into an amplicon-based genotyping-by-sequencing (GBS) approach implemented on a second-generation sequencing platform, allowing accelerated throughput and ca. sixfold reduction in cost. Using the GBS approach, 63 cultivars of perennial, Italian and interspecific hybrid ryegrasses, as well as intergeneric Festulolium hybrids, were genotyped. The genetic relationships between cultivars were interpreted in terms of known breeding histories and indistinct species boundaries within the Lolium genus, as well as suitability of current cultivar registration methodologies. An example of applicability to quality assurance and control (QA/QC) of seed purity is also described. Rapid, low-cost genotypic assays provide new opportunities for breeders to more fully explore genetic diversity within breeding programs, allowing the combination of novel unique genetic backgrounds. Such tools also offer the potential to more accurately define cultivar identities, allowing protection of varieties in the commercial market and supporting processes of cultivar accreditation and quality assurance.

Toward Complete Bacterial Genome Sequencing Through the Combined Use of Multiple Next-Generation Sequencing Platforms.

PacBio's long-read sequencing technologies can be successfully used for a complete bacterial genome assembly using recently developed non-hybrid assemblers in the absence of secondgeneration, high-quality short reads. However, standardized procedures that take into account multiple pre-existing second-generation sequencing platforms are scarce. In addition to Illumina HiSeq and Ion Torrent PGM-based genome sequencing results derived from previous studies, we generated further sequencing data, including from the PacBio RS II platform, and applied various bioinformatics tools to obtain complete genome assemblies for five bacterial strains. Our approach revealed that the hierarchical genome assembly process (HGAP) non-hybrid assembler resulted in nearly complete assemblies at a moderate coverage of ~75x, but that different versions produced non-compatible results requiring post processing. The other two platforms further improved the PacBio assembly through scaffolding and a final error correction.

Cap Analysis of Gene Expression (CAGE) Sequencing Reveals Alterations of the Transcript Signatures in Acute Monocytic Leukemia Cells By Fatty Acid Oxidation Inhibition

Adipocytes, differentiated from bone marrow (BM) stroma-resident mesenchymal stem cells (MSC), are the prevalent stromal cell type in adult BM that increase with aging and cause leukemia cell resistance to chemotherapy (Ehsanipour Cancer Res. 2013).We previously reported that MSC-derived adipocytes prominently inhibited the spontaneous apoptosis of co-cultured acute monocytic leukemia (AMoL) cells, a poor-prognosis subtype of AML, and that a pharmacologic fatty acid oxidation (FAO) inhibitor Etomoxir (EX) reversed the prosurvival effects of adipocytes, indicating the importance of FAO dependent metabolic alterations in AMoL survival (Tabe ASH. 2014).To assess the transcription factors responsible for AMoL cell survival in adipocyte co-culture condition and for apoptosis induction by FAO inhibition, the alterations of transcript signatures were examined by the cap analysis of gene expression (CAGE) sequencing utilizing second-generation sequencing platform (Illumina Genome Analyzer). CAGE identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression (Carninci et al. 1996). The TSS of genes altered in U937 and THP1 cells co-cultured with adipocytes in the presence or absence of EX were mapped, and the common alterations observed in both cell types were analyzed. CAGE detected upregulation of 366 genes and downregulation of 219 genes after co-culture with adipocytes (false discovery rate, < 0.05). Ingenuity Pathway Analysis (IPA) revealed that adipocyte co-culture activated the cancer associated transcription factors Myc and FOXM1, and inhibited the p53 transcription regulator IFI16 and FLT1 kinase, an upstream positive regulator of MAPK/ERK and PI3K/AKT signaling. After EX treatment, CAGE-IPA analysis implicated inhibition of the FAO initiation enzyme ACOX1, and activation of the transcription factor ATF4 (Activating Transcription Factor 4), the master coordinator of the integrated stress response (ISR), and of the nuclear receptor PPARG which controls the FAO pathway.To narrow down the specific transcription factors responsible for EX induced apoptosis in AMoL cells co-cultured with adipocytes, CAGE-mapped TTS signature was integrated with the gene expression patterns detected by RNA-seq.CAGE and RNA-seq detected 3 genes consistently upregulated by adipocyte co-culture (KLF9; a transcription factor that activates PPARg2 promoter, FKBP5; HSP90 interacting co-chaperone immunophilin protein, ATP13A2; a member of the P5 subfamily of ATPases) and downregulation of 2 genes (ANPEP; known as CD13 or Alanyl Aminopeptidase, SLC39A10; Zinc transporter which involves in lipid metabolism).EX treatment under adipocyte co-culture condition specifically upregulated 12 genes including ISR mediator ATF4 and its target gene TRIB3. The upregulation of asparagine syntheses gene ASNS, known to be induced by ATF4, was also detected by RNA-seq. Concordant with CAGE-IPA results, EX treatment upregulated lipid accumulation marker PLIN2 and PPARG target of fatty acid binding protein FABP4 likely reflecting the direct feedback of FAO inhibition. Two genes were downregulated by EX treatment (SREBP1; the lipogenic transcription factor, P2RY2; G-protein coupled receptor activated by ATP).Finally, capillary electrophoresis mass spectrometry (CE-MS) detected the upregulation of citric acid by adipocyte co-culture, which was significantly depleted after EX treatment. The EX treatment, however, increased lactic acid along with fructose 6-phosphate and glucose 6-phosphate upregulation, indicating that cellular metabolism shifts from oxidative phosphorylation and FAO to anaerobic glycolysis which is known to activate ISR.Taken together, this study demonstrates that FAO inhibition by EX activates the pro-apoptotic transcriptional program of ISR through the up-regulation of ATF4 in adipocyte co-cultured AMoL cells. The strategies targeting FAO warrant further exploration in AMoL that survives in adipocyte abundant aged adult BM. DisclosuresKonopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Tag jumps illuminated--reducing sequence-to-sample misidentifications in metabarcoding studies.

Metabarcoding of environmental samples on second-generation sequencing platforms has rapidly become a valuable tool for ecological studies. A fundamental assumption of this approach is the reliance on being able to track tagged amplicons back to the samples from which they originated. In this study, we address the problem of sequences in metabarcoding sequencing outputs with false combinations of used tags (tag jumps). Unless these sequences can be identified and excluded from downstream analyses, tag jumps creating sequences with false, but already used tag combinations, can cause incorrect assignment of sequences to samples and artificially inflate diversity. In this study, we document and investigate tag jumping in metabarcoding studies on Illumina sequencing platforms by amplifying mixed-template extracts obtained from bat droppings and leech gut contents with tagged generic arthropod and mammal primers, respectively. We found that an average of 2.6% and 2.1% of sequences had tag combinations, which could be explained by tag jumping in the leech and bat diet study, respectively. We suggest that tag jumping can happen during blunt-ending of pools of tagged amplicons during library build and as a consequence of chimera formation during bulk amplification of tagged amplicons during library index PCR. We argue that tag jumping and contamination between libraries represents a considerable challenge for Illumina-based metabarcoding studies, and suggest measures to avoid false assignment of tag jumping-derived sequences to samples.

Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing.

Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects.

Sequencing of plant genomes – a review

The scientific revolution that started with the human-genome sequencing project, carried out with first-generation sequencing technology, has initiated other sequencing projects, including those for plant species. Different technologies have been developed together with the second- and third-generation sequencing platforms called 1396984945next-generation1396986481 sequencing. This review deals with the most relevant second-generation sequencing platforms, advanced analysis tools, and sequenced plant genomes. To date, a number of plant genomes have been sequenced, with many more projected for the near future. Using the new techniques and developed advanced bioinformatics tools, several studies including both plant genomics and transcriptomics were carried out. Likewise, completion of reference genome sequences and high-throughput resequencing projects presented opportunities to better understand the genomic nature of plants and accelerated the process of crop improvement. Modern sequencing and bioinformatics approaches have led to overcome the challenges that arose mainly in plant genomes with large size, high CG content, heterozygosity, transposable elements, repetitive DNA, and homopolymers or polyploidy, as may be the case with the most important crops. There is no doubt that the rest of the species will also benefit from such breakthroughs, which also include direct RNA sequencing without requiring cDNA synthesis. In fact, we are not in a postgenomic era as is sometimes stated, but rather in the beginning of a genomic revolution.

Mining microsatellites for Gray’s beaked whale from second-generation sequencing data

We used three second-generation sequencing platforms to develop 12 microsatellite markers for Gray's beaked whale (Mesoplodon grayi). There was a strong correlation between the amount of sequence obtained from each platform and the number of microsatellites recovered. In order to design reliable primers we found that it was important in all cases to eliminate low quality reads and trim sequences so that 80 % of each sequence had a PHRED score of (20. We found that tri- and tetranucleo- tide repeats produced less stutter enabling robust peak- calling and consistent genotyping. The microsatellites developed here will be useful for the population genetics and conservation studies of Gray's beaked whales.

Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing.

CAGE (cap analysis gene expression) and RNA-seq are two major technologies used to identify transcript abundances as well as structures. They measure expression by sequencing from either the 5′ end of capped molecules (CAGE) or tags randomly distributed along the length of a transcript (RNA-seq). Library protocols for clonally amplified (Illumina, SOLiD, 454 Life Sciences [Roche], Ion Torrent), second-generation sequencing platforms typically employ PCR preamplification prior to clonal amplification, while third-generation, single-molecule sequencers can sequence unamplified libraries. Although these transcriptome profiling platforms have been demonstrated to be individually reproducible, no systematic comparison has been carried out between them. Here we compare CAGE, using both second- and third-generation sequencers, and RNA-seq, using a second-generation sequencer based on a panel of RNA mixtures from two human cell lines to examine power in the discrimination of biological states, detection of differentially expressed genes, linearity of measurements, and quantification reproducibility. We found that the quantified levels of gene expression are largely comparable across platforms and conclude that CAGE and RNA-seq are complementary technologies that can be used to improve incomplete gene models. We also found systematic bias in the second- and third-generation platforms, which is likely due to steps such as linker ligation, cleavage by restriction enzymes, and PCR amplification. This study provides a perspective on the performance of these platforms, which will be a baseline in the design of further experiments to tackle complex transcriptomes uncovered in a wide range of cell types.

Gene Discovery and Molecular Marker Development, Based on High-Throughput Transcript Sequencing of Paspalum dilatatum Poir

Background Paspalum dilatatum Poir. (common name dallisgrass) is a native grass species of South America, with special relevance to dairy and red meat production. P. dilatatum exhibits higher forage quality than other C4 forage grasses and is tolerant to frost and water stress. This species is predominantly cultivated in an apomictic monoculture, with an inherent high risk that biotic and abiotic stresses could potentially devastate productivity. Therefore, advanced breeding strategies that characterise and use available genetic diversity, or assess germplasm collections effectively are required to deliver advanced cultivars for production systems. However, there are limited genomic resources available for this forage grass species.ResultsTranscriptome sequencing using second-generation sequencing platforms has been employed using pooled RNA from different tissues (stems, roots, leaves and inflorescences) at the final reproductive stage of P. dilatatum cultivar Primo. A total of 324,695 sequence reads were obtained, corresponding to c. 102 Mbp. The sequences were assembled, generating 20,169 contigs of a combined length of 9,336,138 nucleotides. The contigs were BLAST analysed against the fully sequenced grass species of Oryza sativa subsp. japonica, Brachypodium distachyon, the closely related Sorghum bicolor and foxtail millet (Setaria italica) genomes as well as against the UniRef 90 protein database allowing a comprehensive gene ontology analysis to be performed. The contigs generated from the transcript sequencing were also analysed for the presence of simple sequence repeats (SSRs). A total of 2,339 SSR motifs were identified within 1,989 contigs and corresponding primer pairs were designed. Empirical validation of a cohort of 96 SSRs was performed, with 34% being polymorphic between sexual and apomictic biotypes.ConclusionsThe development of genetic and genomic resources for P. dilatatum will contribute to gene discovery and expression studies. Association of gene function with agronomic traits will significantly enable molecular breeding and advance germplasm enhancement.