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      New insights into the blood-stage transcriptome of Plasmodium falciparum using RNA-Seq

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          Abstract

          Recent advances in high-throughput sequencing present a new opportunity to deeply probe an organism's transcriptome. In this study, we used Illumina-based massively parallel sequencing to gain new insight into the transcriptome (RNA-Seq) of the human malaria parasite, Plasmodium falciparum. Using data collected at seven time points during the intraerythrocytic developmental cycle, we (i) detect novel gene transcripts; (ii) correct hundreds of gene models; (iii) propose alternative splicing events; and (iv) predict 5′ and 3′ untranslated regions. Approximately 70% of the unique sequencing reads map to previously annotated protein-coding genes. The RNA-Seq results greatly improve existing annotation of the P. falciparum genome with over 10% of gene models modified. Our data confirm 75% of predicted splice sites and identify 202 new splice sites, including 84 previously uncharacterized alternative splicing events. We also discovered 107 novel transcripts and expression of 38 pseudogenes, with many demonstrating differential expression across the developmental time series. Our RNA-Seq results correlate well with DNA microarray analysis performed in parallel on the same samples, and provide improved resolution over the microarray-based method. These data reveal new features of the P. falciparum transcriptional landscape and significantly advance our understanding of the parasite's red blood cell-stage transcriptome.

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          Most cited references35

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          The transcriptional landscape of the mammalian genome.

          This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.
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            The transcriptional landscape of the yeast genome defined by RNA sequencing.

            The identification of untranslated regions, introns, and coding regions within an organism remains challenging. We developed a quantitative sequencing-based method called RNA-Seq for mapping transcribed regions, in which complementary DNA fragments are subjected to high-throughput sequencing and mapped to the genome. We applied RNA-Seq to generate a high-resolution transcriptome map of the yeast genome and demonstrated that most (74.5%) of the nonrepetitive sequence of the yeast genome is transcribed. We confirmed many known and predicted introns and demonstrated that others are not actively used. Alternative initiation codons and upstream open reading frames also were identified for many yeast genes. We also found unexpected 3'-end heterogeneity and the presence of many overlapping genes. These results indicate that the yeast transcriptome is more complex than previously appreciated.
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              Human malaria parasites in continuous culture.

              Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. The have remained infective to Aotus.
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                Author and article information

                Journal
                Mol Microbiol
                mmi
                Molecular Microbiology
                Blackwell Publishing Ltd
                0950-382X
                1365-2958
                April 2010
                05 February 2010
                : 76
                : 1
                : 12-24
                Affiliations
                [1 ]simpleParasite Genomics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus Hinxton, Cambridge, CB10 1SA, UK
                [2 ]simpleDepartment of Molecular Biology and Lewis-Sigler Institute for Integrative Genomics, Princeton University Princeton, NJ 08544, USA
                [3 ]simpleUniversity of Oxford, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital Headington, Oxford OX3 9DS, UK
                Author notes
                For correspondence. *E-mail cnewbold@ 123456hammer.imm.ox.ac.uk ; Tel. +44 1865 724994
                **E-mail manuel@ 123456genomics.princeton.edu ; Tel. +1 609-258-9391.

                Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/authorresources/onlineopen.html

                Article
                10.1111/j.1365-2958.2009.07026.x
                2859250
                20141604
                6974a57e-89f6-48ff-8272-f44ec9e03aad
                Journal compilation © 2010 Blackwell Publishing

                Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

                History
                : 14 December 2009
                Categories
                Research Articles

                Microbiology & Virology
                Microbiology & Virology

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