Summary of Library Subcommittee Conference Call of 2-25-00
Subcommittee members: Olin Anderson, Tim Close, Jorge Dubcovsky,
Jan Dvorak, Henry Nguyen, Kay Walker-Simmons.
Additional Participants: Vickie Carollo, Shiaoman Chao, Pat McGuire.
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1. LIBRARIES:
A. A total of 40 cDNA libraries will be constructed by Tim Close.
Additional libraries are likely to be constructed by individual
laboratories for specific needs.
B. First priority is given to the five reproductive biology
targets. These targets and relevant laboratories are:
Jorge Dubcovsky - flowering signals
Jan Dvorak, Bikram Gill, and Kulvinder Gill - meiosis
Shahryar Kianian and Perry Gustafson - pollen development
Olin Anderson, Henry Nguyen and Kay Simmons - seed development
Mark Sorrells and Kay Simmons - seed dormancy/germination
C. Chinese Spring to be the preferred cultivar. Exceptions to CS
where necessary or more efficient sources exist.
D. Expansion into other hexaploid cultivars, other Triticeae species,
other tissues, and other conditions that address additional
functional targets will be pursued after the five reproductive targets
are satifactorily supported.
E. The key investigators for each of the five functional genomics targets
should be polled again to ensure that they have reviewed the library status
on the NSF web site and that there is no library needed by them that is not
covered. (Note: This poll was initiated by Tim Close by email on Friday, Feb 25).
F. In additional to the priorities above, selection of libraries and
sequencing will consider the additional larger goal of providing as
comprehensive a coverage of the total set of expressed wheat genes
as possible.
G. The original organization of ITEC included commitments from
laboratories to provide cDNA libraries of various tissues. For the
current project, ITEC libraries will only be requested if they represent a
condition or tissue not available otherwise.
H. Questions on the utility of sequences from other Triticeae species and
additional functional targets when planning microarrays are expected to
be answered as large numbers of sequence become available for comparative
analysis.
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2. LIBRARY CONSTRUCTION PLANS:
A. In answer to Wayne Powell's comments that DuPont had a different cDNA
construction strategy, particularly in regard to their preference of vectors,
Chris Hainey at DuPont was queried on their procedures and rationale. Chris was
very informative. The DuPont group has used both plasmids and Lambda ZAP
vectors. They have developed a preference for the construction of libraries
directly into a pBluescript plasmid vector followed by electroporation of
DH10B, then overnight growth of the transformed culture. This has become
the favored procedure at DuPont principally because not many people who
are handling the libraries at DuPont are familiar with bacteriophage systems,
which makes this method appear to be easier to follow. It has also been favored
at Dupont as a method that yields a large enough quantity of DNA to be used for
other purposes. We note that this method will amplify the transformants about
100,000 fold. The lambda ZAP vector and mass excision method that we are using
generally results in about 10 to 300 fold amplification in our low amplification
mass excision method, so our libraries will not suffer from bias in favor of
faster-growing transformants to the extent that the DuPont libraries will. We
also note that our method gives us ready access to ssDNA from the phagemid
particles, which fits our subtraction and normalization procedures better than
would dsDNA. We also note that our high amplification mass excision method
provides a quantity of ssDNA on par with the quantity of dsDNA obtained
from overnight cultures made by the DuPont method. Since we see no advantages
to the DuPont method but we do see advantages to our method, we will stick
with lambda ZAP and our current procedures.
B. Host cells for library growth are still being evaluated. Several locations
have noted that Stratagene’s SOLR strain does not grow well without vigorous
agitation and aeration, which is not compatible with various high throughput
systems. Since SOLR is the best strain currently available for lambda-ZAP-derived
phagemid plating, because of its F’ element, both lambda and helper phage
resistance, and recombination deficiency, poor growth of SOLR has led some groups
to switch to electroporation of DH10B, which has better growth properties.
Albany has been using SOLR since growth of SOLR is generally compatible with
the Gene Machines shaker. Albany has noted uneven growth of DH10B cultures
produced by electroporation of mass excised DNA molecules.
C. RNA or tissue can be sent to Tim's lab for library construction. He requested
2-5 gm of tissue or at least 1.0 mg of total RNA.
D. Pooling of RNAs collected at different stages, from different treatments,
or in different genotype background, can be used to minimize the number of
libraries to be made, but should be checked with Tim and/or the Library Subcommittee.
The recommended pooling strategy is to isolate RNAs separately for each sample,
quality check the RNA on a gel, and then pool if satisfactory. The rationale
is to avoid having a bad RNA confound the pool.
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3. SEQUENCING:
A. In a change in procedures, aliquots of libraries rather than plates
or DNAs should be sent to Olin's lab for sequencing in cases where the
libraries are not coming through Tim Close.
B. Sequencing priorities will follow the library priorities.
C. To access the quality of a library, the intention is to sequence
384 clones from each library (currently Albany is doing 1000 while the
procedures are being worked out). If the initial sequences are
informative, 3,000 to 4,000 more clones will be sequenced. Further
sequencing could be done on libraries yielding exceptional levels
of new sequence.
D. Libraries from pooling strategy may be sequenced more deeply,
dependent on the same criteria of levels of informative sequence.
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4. SUBTRACTION AND NORMALIZATION:
A. Henry Nguyen's lab will accept materials for subtraction and normalization.
Either tissue samples or lambda ZAP libraries is OK for subtraction.
A root library is currently being normalized, and Albany is sequencing
3000 clones from the unnormalized original library as a basis for
comparison of the efficiency of the normalization.
B. Agreement that subtraction should be pursued as vigorously as possible,
limited by Henry's budget. Still unclear exactly how much subtraction
will be carried out, but this is expected to become clearer as we
proceed.
C. The best methods of subtraction is not clear yet, but Tim suggested
trying both hybridization with high density filters and a kit from
Clontech. Henry's lab is using the Bento Soares's method for normalization
or subtraction of cDNA libraries, based on hybridization of single-stranded
cDNAs. Henry's lab is also testing the Clonetech kit for subtraction.
D. Both Tim and Henry's labs welcome personnel sent from investigators' labs
to learn library techniques. Pat will investigate the use of 'training
funds' to support lab rotation for project postdocs.
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5. COMMUNICATION:
1. The project web page and Bulletin Board is intended to be the main
communication medium for library discussions and postings of summaries
and protocols.