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Listed below is information
on how to initiate a TILLING request, resources for
obtaining sequence data and a description of the programs
that analyze your gene target. All TILLING requests
are submitted by CODDLe, a web-based program that
will store information about your target in our database
and also links to the various analyses for your gene
target that evaluates the quality of the TILLING mutations
we find.
A. Information Required
for CODDLe:
You will need to have
the genomic DNA and either the cDNA (sequence or join
statement) and/or protein sequence for your target.
These sequences need to correspond directly to each
other; if there are discrepancies between them you
will get an error message when you try to CODDLe the
gene.
Many times a discrepancy
type error arises from SNPs when the gDNA and cDNA
are from two different inbreds and can be corrected
by changing the base(s) in one of the sequences. If
there are many discrepancies between the sequences,
we suggest that you make sure that you are not working
with sequences for two related genes. Maize sequence
data can be obtained from many sources (see below)
that are updated on a regular basis.
B. User Profile and Billing
Information:
Each user will need
a "User
Profile" so that we will have the contact
information for each TILLING request. If the contact
person for the TILLING request changes, please update
this form or send an email to maizetilling@purdue.edu.
Invoices for your TILLING request will be sent out
around the 21st of each month. Payment is due upon
receipt and should be directed to Purdue's Centralized
Accounts Receiveables System (CARS) according to the
instructions on the invoice. CARS will accept purchase
orders, wire transfers and credit cards. If you have
questions regarding the invoice or payments please
email tilling-orders@lists.purdue.edu.
C. Maize Sequence Data Resources:
The list below contains
to links to various public databases in case you need
more gDNA and/or cDNA for the target that you wish
to TILL. If you do not find enough maize sequence
for your gene, you should BLAST against Rice
sequences and then work out to other grasses and
plants as needed. With respect to building a gene
model, in most cases the location (position) of introns
within a gene is conserved between maize and rice,
however the size of the intron may vary.
1. PlantGDB
2. TIGR's
Assembled Zea mays release 4.0
3. NCBI's
trace archives and select "Zea mays-WGS"
or "Zea mays-other" as the search database
4. GeneSeqer
if you need help building a gene model based on
your sequence information.
D. Programs to Analyze Your Gene Target
These programs were
developed by the Proweb
bioinformatics group at the Fred Hutchinson Cancer
Research Center and are used by the Maize TILLING
Project to analyze the gene target and any TILLING
mutations we find.
1. BLOCKS
CODDLe uses BLOCKS
to compare your protein sequence to the InterPro 12.0
protein database and find homologous proteins. The
BLOCKS output highlights which regions in your protein
are conserved and also evaluates (along with SIFT)
the TILLING mutations with respect to the predicting
if the mutation will damage the protein.
If the protein is
classified as "unknown" or "hypothetical",
you can submit your own protein alignment for the
BLOCKS output. We suggest that you do this when you
submit your order so that SIFT can accurately assess
the whether the mutation(s) we find would be tolerated
at that position.
2. Submitting an Alignment for BLOCKS
If you have a CLUSTAL
protein alignment that you have assembled for your
gene target, save it as either a .msf file or in FASTA
format. After CODDLE displays the PSI-BLAST results,
leave the results unchecked and click on "proceed
with CODDLE". On the next screen, there will
be a field for "User defined BLOCKS" that
allows you to upload your own alignment.
1) Click on "User defined BLOCKS" to get
to the block maker.
2) If your alignment is saved as .msf (one of the
save as options in CLUSTAL), go to multiple alignment
processor. Your sequence of interest should be the
top one in the alignment.
3) If your alignment is in FASTA, click on the FASTA
format link to double check how your sequence should
be formatted if you are using an alignment in FASTA.
4) Scroll down to the window and cut/paste or upload
your file and click on the "submit" button.
5) After the BLOCKS have been made, click on "BLOCKS
formatted". You should get a text file with the
information arranged in blocks of text according to
regions of similarity.
6) Save as "myfilename.sh.html". This .html
file will be accepted by CODDLE.
3. SIFT
(Sorting Intolerant From Tolerant)
Based on the BLOCKs
output, SIFT evaluates what amino acids are predicted
to be tolerated at each position within the protein.
Amino acid changes with a SIFT score of <0.05 are
considered "Intolerant". If the change has
both a SIFT score of <0.05 and an IC (confidence)
score of < 3.25 the mutationis predicted to be
damaging and likely will affect protein activity.
If your gene target
does not have a BLOCKS output associated with it,
SIFT scores cannot be determined when the mutation
data is assembled. However, you can enter a protein
alignment of the protein and related proteins into
SIFT and determine if the amino acid change would
be tolerated. This option is also useful if you have
a better alignment than the one initially submitted
with your TILLING request.
4. PARSESNPs
(Project Aligned Related Sequences and Evaluate SNPs)
All TILLING mutations
will be analyzed by PARSESNPs to determine if the
mutation creates or destroys a restriction site. The
PARSESNPs data will be included in the mutation summary
that is emailed to you. You also have the option of
entering other sequence variants for your gene target.
We will begin Eco-TILLING
gene targets this fall. Although we will identify SNPs
based on the approximately location in the gene target,
we will not be sequencing the different variants. However,
users can sequence the SNPs of interest to them and
input those changes into both SIFT and PARSESNPs and
get that information for their Eco-TILLING alleles.
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