The DNA Sequencing Facility employs both objective and subjective quality controls.

Objective Quality Controls

The facility places 7 controls on each sequencing plate. The controls consist of two negative and 5 positive controls. The negative controls consist of water being added to the sequencing reaction instead of DNA template. This control detects proper sequencing reaction plate setup, purity of the water used in the sequencing reactions, and any cross-contamination between the 96 wells of the reaction plate. The negative controls are spaced so that one of the negative controls is in the first half of the plate and the other is in the second half of the plate.

The positive controls consist of our stock primers being used to sequence either pGEM 3Zf(+) or SK-. The primers T7, SP6, M13F, M13R are all used to sequence pGEM-3Zf(+) and T3 is used to sequence SK-. The controls are dispersed evenly throughout each sequencing reaction plate and are not in the same position on consecutive plates. Both positive and negative control positions are determined by a preset map so that over a set number of sequencing reaction plates each of the 96 wells will have contained a positive control. In this manner the facility can oversee the quality of the ABI 3730 DNA Analyzer’s 96-capillary array and the functioning of the Mulitprobe II liquid handling robot.

For each sequencing reaction plate, all negative controls must be negative and the positive controls must pass certain quality criteria before sequences are released to each investigator. Each positive control is aligned to the known sequence of either pGEM-3Zf(+) or SK- , depending on the primer, using the online program SeqWeb Version 2, http://vector.vanderbilt.edu . With respect to the known control sequence, each positive control is graded on percent identity and read length. The cutoff points for both percent identity and read length were determined by averaging the results over twenty different sequencing reaction plates. Failure of 2 or more positive controls or any of the negative controls being positive will result in a plate failure.

The DNA Sequencing Facility also monitors the quality of the 96-capillary array used in the ABI 3730 DNA Analyzer. The failure/success data for each capillary for each sequencing reaction plate is entered into an Access database. Using the database, the facility can detect abnormal high failure rates of any of the capillaries, quickly correct any problems with the array, and protect against the false failure of any user samples.

Subjective Quality Controls

All sequences are reviewed by trained staff in the DNA Sequencing Facility. A sequence reaction is considered successful if the sequence contains high quality base calls for at least 90% of the first 700 bases. If the sequence fulfills the above criteria and the negative/positive plate controls pass the set quality criteria, the sequence is released to the investigator. If a sequence fails the subjective quality control, the sequence is associated with a specific failure category so as to hopefully correct the problem during “redo” sequencing. Please see our Redo Policy and Troubleshooting section.