People often ask, "How long will my TILLING request take?". The answer depends on several factors that will be discussed below. The short answer is: if everything works the first time around, then you should get your TILLING mutations in ~ 4 months.

     This graph shows how orders have moved through the Maize TILLING pipeline from February 2005-November 2006. We have been steadily increasing the number of targets that have reached the "mutation delivered" and "screen completed" stages. We have recently added 1408 new lines to our TILLING population (1024 W22 and 384 B73 lines) that should allow us to satisfy the allelic series for most of the TILLING orders that are incomplete. TILLING orders are considered complete when we have delivered an allelic series where there is a 95% confidence level that one of the mutations will be damaging to the protein.

     We are still having difficulty getting GC-rich targets to amplify consistently under our TILLING PCR conditions. We are doing a thorough test of various conditions and chemicals to try and increase the robustness of the PCR reaction with the labeled primers and also TILLING gel quality. GC-rich targets are taking a longer time to get through both the primer testing and the TILLING stages.

     Below is a flowchart listing the steps in the TILLING pipeline and the approximate time that the each step takes. Please remember, that these are estimates and depend in part on how many other TILLING orders are being processed at the same time.

     The primer pre-screening step can take from 2-8 weeks. If the primers do not work, or give us ambiguous results (either at the PCR amplification and/or sequencing step) we will re-test the primers to be sure of the results. If the primers fail, the user will receive an e-mail indicating what the problem was, any relevant sequence information and suggestions on what to try next. At this point, the order is back in the user's hands. Some users will submit a replacement order within a week or two of getting the failure letter; others have put the TILLING request on hold for many months.

     After the primers pass the pre-screen, the IRD (infrared dye)-labeled primers for TILLING are ordered. Synthesis of these primers takes approximately 3 weeks. We will soon be transitioning our TILLING PCR from a 96 well to a 384 well plate format. Once we have made this switch, our goal is to TILL ~ 3- 4 targets/week.

     The TILLING gels are analyzed for putative mutations and then those mutations are put in the sequencing queue and the sequencing reactions prioritized based on the date the TILLING request was submitted. We sequence 96 putative mutations at a time and do one sequencing project every ~ 3 weeks. If we do not get good sequence data the first time around we will repeat the sequencing reaction. Usually, we only sequence the mutation from one direction. However, if the results are ambiguous, we will also sequence from the other direction. The sequencing data is uploaded into the TILLING database and analyzed with the SIFT and PARSESNPs programs. When we have confirmed mutations in your target, you will be sent an email containing links to PARSESNPs and the SIFT results. Please note that if there is not a BLOCKS file for the gene target (either automatically through CODDLE, or manually), the mutations will not have a SIFT or IC (confidence) score. A protein alignment can be manually uploaded into SIFT to determine whether the amino acid change(s) caused by the mutation(s) is predicted to be tolerated or not.