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- Klevebring, Daniel, 1981-, et al.
(author)
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Automation of cDNA Synthesis and Labelling Improves Reproducibility
- 2009
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In: Journal of Biomedicine and Biotechnology. - : Hindawi Limited. - 1110-7243 .- 1110-7251. ; 2009, s. 396808-
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Journal article (peer-reviewed)abstract
- Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.
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| 2. |
- Klevebring, Daniel, 1981-, et al.
(author)
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Genome-wide profiling of Populus small RNAs
- 2009
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In: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 10, s. Article number 620-
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Journal article (peer-reviewed)abstract
- Background: Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced Populus trichocarpa using a concatenation strategy in combination with 454 sequencing. Results: The most abundant size class of sRNAs were 24 nt and these were generally associated with a number of classes of retrotransposons and repetitive elements. Some repetitive elements were also associated with 22 nt RNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the sex-determining loci and a major cluster of NBS-LRR genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and NBS-LRR disease resistance genes, classes of genes that have been significantly expanded in Populus. Additional loci enriched for sRNA production were identified. We identified 15 novel predicted microRNAs (miRNAs), including miRNA∗ sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs). Conclusions: The short RNA population of P. trichocarpa is at least as complex as that of Arabidopsis. We provide a first genome-wide view of short RNA production for P. trichocarpa and identify new, non-conserved miRNAs.
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| 3. |
- Klevebring, Daniel, 1981-
(author)
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On Transcriptome Sequencing
- 2009
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Doctoral thesis (other academic/artistic)abstract
- This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci.
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