Genome annotation by high-throughput 5’ RNA end determination

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    08-Jan-2016

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Genome annotation by high-throughput 5â RNA end determination Byung Joon Hwang, Hans-Michael Müller, and Paul W. Sternberg Paul W. Sternberg B.A. degree from Hampshire…

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Genome annotation by high-throughput 5â RNA end determination Byung Joon Hwang, Hans-Michael Müller, and Paul W. Sternberg Paul W. Sternberg B.A. degree from Hampshire College Ph.D. degree from M. I. T. under Robert Horvitz Postdoctoral research with Ira Herskowitz at the U. C., San Francisco Professor of Biology at the California Institute of Technology and Adjunct Professor of Cell and Neurobiology at the University of Southern California School of Medicine, Los Angeles Sternberg Lab Byung Joon Hwang â Post Doc Hans-Michael Müller - Wormbase Trans-spliced Exon Coupled RNA End Determination (TEC-RED) Identifies 5 â ends of expressed genes Can distinguish coding regions from regulatory regions Useful for identifying genes with alternatively spliced 5 â ends. Developed in nematodes, but can work for any organism which use a spliced leader sequence (Sarcomastigophora, cndarians, nematodes, acoelomate flatworms and ascidians). Trans-splicing 70% of mRNAs have one of two splice-leader sequences (SL1, SL2) trans-spliced onto the 5â end Spliced-leader sequences are transcribed independently as snRNAâs Trans-splicing with splice-leader sequences produces single-gene mRNAs TEC-RED Sequential Concentration Eliminates the large-scale PCR reactions and gel purification of small oligonucleotides steps found in SAGE protocols. Since the 5â tags are directionally concentrated, the 5â end of the 5â tags are found next to the first anchor RE cut site. DNA Sequence Analysis Data 13 525 5â tags (9 401 with SL1 and 4 124 for SL2), obtained from 800 sequencing reactions, were matched to the genome Represents 2 159 different sequences, 1 639 for SL1 and 520 for SL2 90% of tags corresponded to unique sites Of the remaining 10%, 90% matched 2 or 3 sites Cont. To remove false positives they analyzed the just 5â of the 5â tag sites for a conserved splice acceptor consensus sequence 93% of the 5â tag sequences remained true positives â¦. 75% of tag sequences matched know 5â end in WS100 99 new genes identified 32 previously unknown operons identified Conclusion Method for annotation of 5â end of genes. This protocol works for only organisms with a splice leader sequence Sequential concentration method applied to SAGE protocols âThe United States brags about its political system, but the President says one thing during the election, something else when he takes office, something else at midterm and something else when he leaves.â -Deng Xiaoping

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