Transposome-based technologies have enabled the streamlined production of sequencer-ready DNA libraries; however, current methods are highly sensitive to the amount and quality of input nucleic acid.
...We describe a new library preparation technology (Nextera DNA Flex) that utilizes a known concentration of transposomes conjugated directly to beads to bind a fixed amount of DNA, and enables direct input of blood and saliva using an integrated extraction protocol. We further report results from libraries generated outside the standard parameters of the workflow, highlighting novel applications for Nextera DNA Flex, including human genome builds and variant calling from below 1 ng DNA input, customization of insert size, and preparation of libraries from short fragments and severely degraded FFPE samples. Using this bead-linked library preparation method, library yield saturation was observed at an input amount of 100 ng. Preparation of libraries from a range of species with varying GC levels demonstrated uniform coverage of small genomes. For large and complex genomes, coverage across the genome, including difficult regions, was improved compared with other library preparation methods. Libraries were successfully generated from amplicons of varying sizes (from 50 bp to 11 kb), however, a decrease in efficiency was observed for amplicons smaller than 250 bp. This library preparation method was also compatible with poor-quality DNA samples, with sequenceable libraries prepared from formalin-fixed paraffin-embedded samples with varying levels of degradation.
In contrast to solution-based library preparation, this bead-based technology produces a normalized, sequencing-ready library for a wide range of DNA input types and amounts, largely obviating the need for DNA quantitation. The robustness of this bead-based library preparation kit and flexibility of input DNA facilitates application across a wide range of fields.
Globular somatic embryos can be induced from immature cotyledons of soybean (Glycine max L. Merr. cv Jack) placed on high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos ...develop from the adaxial side of the cotyledon, whereas the abaxial side evolves into a callus. Using a 9,280-cDNA clone array, we have compared steady-state RNA from the adaxial side from which embryos develop and from the abaxial callus at five time points over the course of the 4 weeks necessary for the development of globular embryos. In a second set of experiments, we have profiled the expression of each clone in the adaxial side during the same period. A total of 495 genes differentially expressed in at least one of these experiments were grouped according to the similarity of their expression profiles using a nonhierarchical clustering algorithm. Our results indicate that the appearance of somatic embryos is preceded by dedifferentiation of the cotyledon during the first 2 weeks on auxin. Changes in mRNA abundance of genes characteristic of oxidative stress and genes indicative of cell division in the adaxial side of the cotyledons suggest that the arrangement of the new cells into organized structures might depend on a genetically controlled balance between cell proliferation and cell death. Our data also suggest that the formation of somatic globular embryos is accompanied by the transcription of storage proteins and the synthesis of gibberellic acid.
Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to ...accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays.
We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants. The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines.
Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype.
Whole-genome sequencing is fundamental to understanding the genetic composition of an organism. Given the size and complexity of the soybean genome, an alternative approach is targeted random-gene ...sequencing, which provides an immediate and productive method of gene discovery. In this study, more than 120 000 soybean expressed sequence tags (ESTs) generated from more than 50 cDNA libraries were evaluated. These ESTs coalesced into 16 928 contigs and 17 336 singletons. On average, each contig was composed of 6 ESTs and spanned 788 bases. The average sequence length submitted to dbEST was 414 bases. Using only those libraries generating more than 800 ESTs each and only those contigs with 10 or more ESTs each, correlated patterns of gene expression among libraries and genes were discerned. Two-dimensional qualitative representations of contig and library similarities were generated based on expression profiles. Genes with similar expression patterns and, potentially, similar functions were identified. These studies provide a rich source of publicly available gene sequences as well as valuable insight into the structure, function, and evolution of a model crop legume genome.Key words: Glycine max, genome sequencing, functional genomics.
Deep sequencing of cDNA prepared from total RNA (RNA-Seq) or mRNA (mRNA-Seq) has become the method of choice for transcript profiling, discovery of novel transcripts, and identification of ...alternative splicing events. However, standard whole-transcriptome approaches to RNA-Seq face a significant challenge, as the vast majority of reads map to rRNA. One solution—poly(A) enrichment—does not capture several biologically relevant RNA species, such as microRNA and also requires large amounts of Total RNA. Whole blood RNA samples pose an even bigger challenge due to the presence of reticulocyte globin transcripts that can constitute up to 76% of total mRNA and, unless removed along with the ribosomal RNA transcripts, the detection of low-abundance mRNA transcripts is further compromised. To overcome these challenges, Epicentre developed Globin-Zero™ technology for blood derived RNA. The Globin Zero Gold kit removes rRNA and globin mRNA from intact and fragmented blood total RNA samples (human, mouse and rat), and an improved, more user-friendly version of the ScriptSeq™ RNA-Seq library preparation method. Directional cDNA libraries with ∼98% strandedness can be prepared in about 4 hours, in a single-tube workflow, from either intact or fragmented Globin-Zero treated RNA samples, without the need for end-polishing, adaptor ligation, cDNA fragmentation or gel size-selection. The combined Globin-Zero and ScriptSeq workflow generates cluster-ready NGS libraries in ∼6 hours. Globin-Zero-treated samples contain. <2% of rRNA and globin sequences, while maintaining representation of coding and noncoding transcripts independent of polyadenylation. This reduction in rRNA and globin sequence reads improves sequence depth and coverage of mRNA, and increases the percentage of uniquely mapped reads.
RNA seq or Direct cDNA sequencing is an emerging revolutionary tool for whole-transcriptome analysis that provides information about the structure of transcripts and their expression levels. It is ...rapidly gaining momentum for transcript profiling and analysis of novel transcripts, novel isoforms, alternative splice sites, rare transcripts and SNPs, compared to microarrays. Current methods for making sequencer-specific di-tagged DNA fragment libraries for RNA-Seq typically comprise preparing rRNA-depleted RNA and either (i) RNA fragmentation, 5′ and 3′ adaptor-ligation, size selection, cDNA synthesis, and multiple clean-up steps; or (ii) cDNA synthesis followed by cDNA fragmentation, end-polishing, 5′ and 3′ adaptor-ligation, size selection and multiple clean-up steps. These methods are generally time-consuming and require significant hands-on time. Here we introduce an improved, user-friendly version of the ScriptSeq™ RNA-Seq library preparation method. ScriptSeq utilizes a unique Terminal-Tagging technology that simplifies the preparation of directional, paired-end libraries from rRNA-depleted or poly(A)-enriched RNA.
ScripSeq Version 2:
Libraries are prepared in about 4 hours, in a single-tube workflow
Produces directional cDNA libraries with ∼98% strandedness, which is critical for annotation of novel genes
Can be used with both intact and degraded (eg. FFPE) RNA samples
Provides very good 5′/3′ representation of transcripts
When starting from Rib-Zero treated RNA, less than 2 % of the sequence reads from libraries map to rRNA sequences (28S, 18S, 5.8S and 5S). This reduction in rRNA sequence reads improves sequence depth and coverage, and increases the percentage of uniquely mapped reads.
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