RadishDB:Sequencing

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Contents

DNA Substrate

Tissues for eight normalized cDNA libraries: four R. sativus cultivars and four R. raphanistrum populations. For details see the Sample section.

We will collect tissue from a variety of plant parts at different developmental stages, focusing particularly on newly-formed flower buds, leaves, pollen, and shoot and root meristems; this will ensure that we get transcripts from developmental genes and the genes affecting the floral traits discussed above, as well as genes involved in domestication of the root crop. Conner’s lab will grow the plant material and isolate total RNA using RNeasy Plant Mini kits (Qiagen); RNA quality will be checked using the Agilent Bioanalyzer at the MSU genomics core facility. We have seeds from all of these populations and experience collecting tissues into liquid nitrogen, isolation of RNA with the RNeasy kits, and quality testing with the Bioanalyzer.

Library construction

Libraries will be constructed by [http:/www.evrogen.com Evrogen]. Evrogen combines the full-length Smart technique to capture full-length sequences ([Zhu, 2001]) with a proprietary normalization strategy using a novel duplex-specific nuclease ([Shagin, 2002 )]. Isolated total RNA will be sent to Evrogen. Normalized double-stranded cDNA generated by Evrogen will be directionally ligated into SfiI A/B sites of pDNR-LIB (Clonetech) and transformed into GC5 High Eff Competent Cells (Gene Choice) at TIGR. The titer of each library will be checked before colony picking and sequencing. Most recently, this strategy in Medicago EST sequencing resulted in 40-60% near full-length cDNAs in various libraries. Therefore, this approach should be able to generate a high yield of novel ESTs including a high percentage of full-length cDNAs from both crop and wild radish cDNA libraries.

Sequencing strategy, sequence quality and quantity

We will use conventional Sanger sequencing. We evaluated the feasibility of an attractive alternative pyrosequencing strategy (8, 9) developed by 454 Life Sciences; JTC is an early access and testing facility. Based on read lengths and quality from our recent Medicago 454 sequencing, we decided against using it for this work. From a normalized Medicago cDNA library, a total of 292,465 reads (252,384 cleaned reads) were generated with an average read length of 92 bp, considerably shorter than the 800+ read lengths that are routinely achieved via Sanger sequencing. After aligning the reads to Medicago Phase III BACs, we also found that hompolymeric tracts of the sequences were sequenced less reliably by this technology. For example, we often saw sequencing errors at the junctions of the hompolymeric tracts, especially at 3’ end, which would be a major obstacle for mining polymorphic sites.

While JTC is working with 454 Life Sciences to develop paired-end read technology, this is a feature not yet available. Together with the short read length, it will be very difficult for us to obtain good EST assemblies, let alone full-length cDNAs, with the 454 system. One key advantage of the 454 technology is bypassing the need for a library. However, radish cDNA clones generated from this project, including many full-length cDNAs, will be a very valuable resource for further functional genomic, ecological or evolutionary studies in the radish research community. Although the lower capital costs of 454 DNA sequencing technology will be effective in revealing the expression of many rare transcripts in normalized cDNA libraries, it is currently not suitable for generating high quality reads for variation studies or assembly. Therefore, considering that there is very limited sequence of any kind available for radish, we chose Sanger sequencing technology to maximize the utility of our results.

Sequencing will be carried out at the TIGR affiliate organization, the J. Craig Venter Science Foundation Joint Technology Center (JTC). JTC has a state-of-the-art facility and is one of the world's leading DNA sequencing organizations in terms of capacity, cost effectiveness and scientific expertise. JTC employs robotics, LIMS tracking and 100 of the most advanced sequencing machines, the Applied Biosystems’ 3730xl automated DNA analyzer. The JTC’s current capacity is greater than 52 million sequence reads (lanes) per year. Current average read lengths are at least 700 bp (sequence quality equivalent to phred 20) or longer and recent EST projects have sequenced with 80% to 90% efficiency.

Approximately 200,000 total sequencing reads with an average read length of at least 700 bp will be generated from both ends of 100,000 cDNA clones from the crop and wild radish libraries. In the first year, the normalized cDNA libraries will be constructed and pilot sequencing of about 5000 clones from each library will be completed in order to assess the quality of both libraries. The production of EST sequences will be accomplished in the rest of the first year and the first half of the second year. Base-callers will be used to provide quality values for each base produced. Our daily QC reports evaluate production success using several summary statistics including number of reads, sequencing success rate, read lengths and average quality values (see Appendix A3 for details). All the sequences will be cleaned, including trimming of vector and adaptor sequences, removal of all low-quality sequence and any contamination, and then will be assembled and clustered to generate a radish gene index or transcript assemblies ([Quackenbush, 2000; Quackenbush, 2001; Lee, 2005]). We estimate based on our experience that the project should produce about 30,000 unique sequences, both tentative consensus sequences (TCs) and singletons. There are currently only 94 EST sequences from radish in GenBank (06/01/2006). Therefore, the immediate outcome of this project will be the significant increase of the numbers of radish ESTs, which will greatly enrich the genomic resources available to the radish research community. The analysis of all the sequences of this project will be finished in the rest of the 2nd year.

References

  1. Karoly, K. and J.K. Conner, Heritable variation in a family-diagnostic trait. Evolution, 2000. 54(4): p. 1433-1438.
  2. Agrawal, A.A., et al., Ecological genetics of an induced plant defense against herbivores: additive genetic variance and costs of phenotypic plasticity. Evolution, 2002. 56(11): p. 2206-2213.
  3. Conner, J.K., Genetic mechanisms of floral trait correlations in a natural population. Nature, 2002. 420: p. 407-410.
  4. Conner, J.K., R. Franks, and C. Stewart, Expression of additive genetic variances and covariances for wild radish floral traits: comparison between field and greenhouse environments. Evolution, 2003. 57(3): p. 487-495.
  5. Conner, J. and S. Via, Patterns of phenotypic and genetic correlations among morphological and life history traits in wild radish, Raphanus raphanistrum. Evolution, 1993. 47(2): p. 704-711.
  6. Agrawal, A.A., J.K. Conner, and J.R. Stinchcombe, Evolution of plant resistance and tolerance to frost damage. Ecology Letters, 2004. 7: p. 1199–1208.
  7. Strauss, S.Y., J.K. Conner, and K.P. Lehtila, Effects of Foliar Herbivory by Insects on the Fitness of Raphanus raphanistrum: Damage Can Increase Male Fitness. American Naturalist, 2001. 158(5): p. 496-504.
  8. Ronaghi, M., Pyrosequencing sheds light on DNA sequencing. Genome Research, 2001. 11: p. 3-11.
  9. Hyman, E.D., A new method of sequencing DNA. Anal. Biochem., 1988. 174: p. 423-436.
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