Call for non-coding mRNA resource


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Genomic projects provided clone resources for coding mRNAs, but non-coding mRNAs are still missing for functional studies. Seeing the growing number of non-coding mRNAs, we hope the community will prepare better resources for studying human non-coding mRNAs.

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Call for non-coding mRNA resource

  1. 1. A call for the creation of a Human long ncRNA Clone Set Matthias Harbers - January 2012Matthias Harbers 1
  2. 2. Long non-coding mRNAs  The dogma that an “mRNA has to encode a Protein” directed the creation of protein coding clone collections  The Mammalian Gene Collection (MGC) and ORFeome Collaboration (OC) focused on the cloning of protein coding transcripts  However, by now it is generally accepted many mRNAs are non-coding transcripts that exercise their functions by other, mostly unknown, mechanisms  All clone collections are incomplete because of the use of oligo-dT priming  However, may be up to 40% or more of the mRNAs could lack polyadenylation and hence are not covered by classical cDNA libraries  Many non-polyadenylated mRNAs could be non-coding mRNAsMatthias Harbers 2
  3. 3. Research is driven by sequencing  High-speed sequencing changed the way genomic research is done  High-speed sequencing has an unmatched power in “discovery”  However, expressed sequences need confirmation by other means  New transcripts have to be analyzed for their functions  Loss-of-function studies became easier with RNAi knock-down experiments  However, gain-of-function experiments are essential to understand mechanisms and functions  We should have the resources needed to study the functions of ncRNAs!Matthias Harbers 3
  4. 4. “Real” versus “predicted”  Public databases like NCBI and others use “reference sequences” for transcripts  This implies that there is only one transcript per gene!  Reference sequences ignore splicing!  Actual cDNA clones in the public domain often do not match reference sequences  High-speed sequencing data provide more “predicted sequences” based on the assembly of short reads into contigs or alike  Predicted sequences are not experimentally verified!  Cloned cDNAs have at least an “experimental origin”!Matthias Harbers 4
  5. 5. The “Knowledge Cycle” Creation of genomic resources and data: Public databasesFunctional Studies:Gene annotation Find the clones you need: Clone Distribution Services  Physical resources are needed to bring “life” to in silico data!Matthias Harbers 5
  6. 6. What is needed?  Define what are long non-coding mRNAs (ongoing in the community)  Description of human non-coding mRNA set  Consent on the features of human non-coding mRNA set  Starting materials available in the community?  New starting materials required?  Build consortium to build human non-coding mRNA clone set  Consider non-coding mRNA collections from other organisms  Small non-coding RNA are not considered here because they are in part already covered by some public collections (e.g. Netherlands Cancer Institute)Matthias Harbers 6
  7. 7. Features of non-coding mRNA set  ORFeome Collaboration committed to Invitrogen Gateway system  Broad Institute also uses Invitrogen Gateway system  Suggestion to stay with Invitrogen Gateway system for ncRNA set?  However, many clone customers do not want Invitrogen Gateway clones!  Addition of restrictions sites could enable sub-cloning without use of the Invitrogen Gateway system  For example Promega offers Flexi® Vectors using SgfI and PmeI  Should the parental clones from cDNA libraries made available?  Should the collection include splice variants?  Are there special requirements we do not know of?Matthias Harbers 7
  8. 8. Available starting materials  Want to use high-quality full-length cDNA clones and libraries where possible!  Human cDNA collections: • RIKEN ~311,000 human end-sequenced human cDNA clones in NCBI? • Other human cDNA clones: e.g. ORIGENE?  Human full-length cDNA libraries in the public domain?  I do not see gene synthesis based on predicted sequences as “general option”  I prefer starting from cDNA libraries using “real transcripts”  Classical cDNA libraries have not been sequenced deep enough to cover rare genes! There is an option to find more unique clones in old libraries  Need new cDNA libraries/pools to cover important biological samples?Matthias Harbers 8
  9. 9. New technologies will help  In the past sequencing cost limiting factor for building clone collections  Many clones in the public domain are not full-length sequenced  Lack of sequence information limits clone annotation  New high-speed sequencing methods can overcome limitation on sequencing cost  Use high-speed sequencing instead of end-sequencing of individual clones to screen cDNA libraries more deeply  Use high-speed sequencing to obtain full-length sequences of all clones within ncRNA collection  Use high-speed sequencing to assure high quality standards of entire collection!Matthias Harbers 9
  10. 10. RNA RNA cDNA Library cDNA Library/cDNA Pool Clone Picking Shotgun sequencing End Sequencing Library Screening Annotated Clone Clones for Targets Collection Limited by sequencing cost Much higher coverage Redundancy in clone collection Focus on new targetsMatthias Harbers 10
  11. 11. New starting materials required  Many mRNAs lack polyadenylation and required new cloning method Total RNA from cell Removal of polyA mRNA Ligation of 3’ adapter to mRNA cDNA synthesis using 3’ adapter Cap selection using Cap Trapper Cloning into cDNA library  Size does matter: Classical cDNA projects had a size cutoffMatthias Harbers 11
  12. 12. Build internet presence  Any clone collection requires an internet presence with a database!  Clone related information can only be provided by a database  Annotation of the clones by reference to other resources is important  Application notes and references could be a great capture for users  Good documentation of the project needed  Provide all clones to community without limitations on the rights to use (follow example of “Good Faith Agreement” of ORFeome Collaboration)  ncRNAs may require “more” for a better understanding on how to study new mechanisms and functions!  Become the “home” for research on ncRNAs!Matthias Harbers 12
  13. 13. Conclusion  MGC and OC set standards for human clone resources!  We should to build on the great record of MGC and OC to move from coding mRNAs to long non-coding mRNAs  After most coding genes have been covered by at least one cDNA clone, we need to work on the non-coding transcripts to move forward  Non-coding genes are essential players in life and we want to provide comprehensive resources for their study and analysis  Starting with human ncRNAs will greatly benefit medical research  Including ncRNAs from other organisms could be an option where those are key model systems to study ncRNA functions (RIKEN FANTOM clone set from mouse includes many long ncRNAs)Matthias Harbers 13
  14. 14. Matthias Harbers 14