Quantitative Transcriptomics using Designed Primer-based Amplification

Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, California, USA.
Scientific Reports (Impact Factor: 5.58). 04/2013; 3:1740. DOI: 10.1038/srep01740
Source: PubMed


We developed a novel Designed Primer-based RNA-sequencing strategy (DP-seq) that uses a defined set of heptamer primers to amplify the majority of expressed transcripts from limiting amounts of mRNA, while preserving their relative abundance. Our strategy reproducibly yielded high levels of amplification from as low as 50 picograms of mRNA while offering a dynamic range of over five orders of magnitude in RNA concentrations. We also demonstrated the potential of DP-seq to selectively suppress the amplification of the highly expressing ribosomal transcripts by more than 70% in our sequencing library. Using lineage segregation in embryonic stem cell cultures as a model of early mammalian embryogenesis, DP-seq revealed novel sets of low abundant transcripts, some corresponding to the identity of cellular progeny before they arise, reflecting the specification of cell fate prior to actual germ layer segregation.

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Available from: Erik Willems, Jun 19, 2014
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    • "However, deep sequencing of low-quantity or low-quality RNA substrates such as samples fixed in paraffin (Ovation RNA-Seq Formalin fixed in paraffin (Formalin Fixed Paraffin Embedded (FFPE)), rare cell populations, or even a single cell is often desired. Amplification protocols [Bhargava et al. 2013; Hashimshony et al. 2012; Ozsolak et al. 2010; Pan et al. 2013] introduced prior to library preparation allow sequencing libraries from low quantities of input to be constructed. The strategy for each amplification method is different in terms of the amount of RNA required, the priming methods utilized, and rRNA minimization [Tariq et al. 2011]. "
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    ABSTRACT: Abstract A significant challenge to the effective application of RNA-seq to the complete transcript analysis of low quantity and/or degraded samples is the amplification of minimal input RNA to enable sequencing library construction. Several strategies have been commercialized in order to facilitate this goal. However, each strategy has its own specific protocols and methodology, and each may introduce unique bias and in some cases show specific preference for a collection of sequences. Our wider investigation of human spermatozoal RNAs was able to reveal their complexity despite being generally characterized by low quantity and high fragmentation. In this study, the following four commercially available RNA-seq amplification and library protocols for the preparation of low quantity/highly fragmented samples, SMARTer™ Ultra Low RNA (SU) for Illumina® Sequencing, SeqPlex RNA Amplification (SP), Ovation® RNA-Seq System V2 (OR), and Ovation® RNA-Seq Formalin Fixed Paraffin Embedded System (FFPES) were assessed using human sperm RNAs. Further investigation analyzed the effects on the end results of two different library preparation methods, Encore NGS Multiplex System I (Enc) and Ovation Ultralow Library Systems (UL), that appeared best suited to this type of RNA, along with other potential confounding factors such as FFPE preservation. Our results indicate that for each library preparation protocol, the differences in the initial amount of input RNA and choice of RNA purification step do not generate marked differences in terms of RNA profiling. However, substantial disparity is introduced by individual amplification methods prior to library construction. These significant differences may be caused by the different priming methods or amplification strategies used in each of the four different protocols examined. The observation of intra-sample variation introduced by the choice of protocol highlights the role that external factors play in planning and subsequent reliable interpretation of results of any RNA-seq experiment.
    Systems Biology in Reproductive Medicine 07/2014; 60(5):1-8. DOI:10.3109/19396368.2014.944318 · 1.60 Impact Factor
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    • "Competence towards this fate emerges progressively and requires the downregulation of the pluripotency factor Nanog. We find no evidence to support the widespread notion that Wnt/β-Catenin signalling suppresses NECT (Aubert et al., 2002; Watanabe et al., 2005; Smith et al., 2008; Patani et al., 2009; Bhargava et al., 2013; Lupo et al., 2013) and instead find that, within the period of competence, it potentiates NECT and mesendoderm in a context-dependent manner. In the presence of high levels of Activin/Nodal and BMP, Wnt/β-Catenin signalling promotes mesendoderm whereas in their absence it produces anterior neurons. "
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    ABSTRACT: Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst, retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analyzed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a “race for fates” at the level of single cells in which the neuroectodermal fate has an advantage.
    Biology Open 03/2014; 3(7). DOI:10.1101/000653 · 2.42 Impact Factor
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    • "In our previous study11, we demonstrated the limitation of Smart-seq to efficiently amplify long transcripts (>4 Kb). DP-seq performs targeted amplification of selected regions of the transcripts; as a consequence, it did not exhibit a transcript length bias. "
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    ABSTRACT: Recent advances in RNA-seq methodologies from limiting amounts of mRNA have facilitated the characterization of rare cell-types in various biological systems. So far, however, technical variations in these methods have not been adequately characterized, vis-à-vis sensitivity, starting with reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, noise in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.
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