Frequently Asked Questions
- How long will it take?
Once our queue is filled, about 3-6 months. Depending on the wait list for our HiSeq2000, it could take longer. Sequencing itself takes a full month.
- What is a queue?
We need to have enough orders before we can start processing orders. Our queue is 20-30 orders. In the event where we do not have many orders, we may be able to offer a smaller screening option. The screen size options will range from units of 512 individuals and the prices will be adjusted proportionately.
- Will the seed carrying my mutation be available to everybody?
Yes, but not right away. The facility has been funded by public money. Therefore, we will contribute mutations to the relevant database as they are identified. You will have one year from the time the seed is released to you before the seed will be released to anybody else. A one-year non-disclosure agreement from the seed shipment date will also be in effect.
- What is the reliability of the Illumina sequencing-based calls?
After sequencing the pools of DNA, we obtain a table of mutations that includes the sequence change, the effect, and the probability that such a change would be caused by an error. The False Discovery Rate depends on the probability score, the F(t). In practice, mutations with an F(t) above 5 have reliability comparable to good Sanger calls.
- Can I submit a fragment smaller then 1 kb?
We set a minimum size to 700 bp. A critical requirement in our procedure is the elimination of relatively large primer-dimer type artifacts. Some of these can get as large as 300 bp and if present during library construction result in a large number of primer reads. To ensure the elimination of these artifacts using our current system (magnetic beads), it is necessary for there to be a sufficient size difference between the artifacts and the TILL fragment.
- Can I submit two primer pairs that amplify a similar stretch of my gene to increase the probability of success in your amplification step?
Yes, in fact we recommend this.
- Can I submit two overlapping fragments?
- How many mutations can I expect?
For a 1.5 kb amplicon, we expect rice to have 10-12 in 2,048 individuals and tetraploid arabidopsis to have 20-25 in 524 individuals.
- What type of phenotypic changes will these mutations cause?
If the action of the targeted gene is required for a process that can be scored phenotypically or biochemically, you should detect a change in this process for any mutation that causes loss of function.
- How many of my mutations will result in a loss of function of the protein encoded by the targeted gene?
In average, we expect about 45% of the mutations in a coding region to be missense, i.e. to cause a codon and corresponding amino acid change. About a third of these changes should impact protein function. We also expect 4-5% nonsense and splice site mutations. These mutations result in premature termination of the encoded polypeptide. The above are approximations: the structure and nucleotide composition of your gene will determine the impact of the expected mutations.
- All summed, will TILLING be useful for my analysis?
Based on prior experience, we are guessing that 50-75% of the TILLED genes will produce a useful allele. The chances for a useful allele get better if a deeper search is used or if different amplicons (i.e. more bases) are sequenced for the same gene.