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A call for the creation of a
Human long ncRNA Clone Set Matthias Harbers - January 2012
Matthias Harbers 1
Long non-coding mRNAs
Matthias Harbers 2
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 mRNAs
Research is driven by sequencing
Matthias Harbers 3
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!
“Real” versus “predicted”
Matthias Harbers 4
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 5
Creation of genomic resources and data:
Public databases
Functional Studies:
Gene annotation
Find the clones you need:
Clone Distribution Services
The “Knowledge Cycle”
Physical resources are needed to bring “life” to in silico data!
What is needed?
Matthias Harbers 6
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)
Features of non-coding mRNA set
Matthias Harbers 7
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?
Available starting materials
Matthias Harbers 8
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?
New technologies will help
Matthias Harbers 9
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 10
RNA
cDNA Library
Clone Picking
End Sequencing
Annotated Clone Collection
Limited by sequencing cost Redundancy in clone collection
RNA
cDNA Library/cDNA Pool
Shotgun sequencing
Library Screening
Clones for Targets
Much higher coverage Focus on new targets
New starting materials required
Matthias Harbers 11
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 cutoff
Build internet presence
Matthias Harbers 12
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!
Conclusion
Matthias Harbers 13
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 14