Box 3. Sample arraying and pooling • TIMING 6 h per 768 samples.

Arraying individual samples into a multiwell plate before pooling can reduce pipetting errors and the time required to construct a plate of pooled samples. We use standard 96-well microtiter plates for arraying because the spacing of the wells is compatible with multichannel pipettors and larger format pipetting devices.

One-dimensional pooling strategy

1. Array 64 samples into an 8 8 grid on a 96-well plate (leaving the last four columns empty).
2. Pool the samples using an eight-channel pipettor; combine samples from each of the eight rows on the plate into a single column of the pooled plate, so that position A1 of the pool plate contains samples A1–8 of individual plate 1.

Using a one-dimensional pooling strategy, 12 pooled columns are produced from 12 plates of 64 individuals (Fig. 6a). This arraying protocol allows the screening of 768 different individuals in a single assay. Tracking a putative mutation from a pool to an individual sample is straightforward. For instance, if a mutation is found in position B2 of the 96-well pool plate, the eight samples that contributed to this pool are found in row B of the second "individuals" plate of 64 samples used to create the pools.

Two-dimensional pooling strategy

1. Fill the column with one of the pooled plates as described above.
2. Combine all eight samples in a single column of the same individuals plate and deposit those samples into the adjacent column of the 96-well plate of pooled samples. Each individual is then represented in two unique pools.

Two-dimensional eight-fold pooling allows 384 unique samples to be screened per 96-well plate assay (Fig. 6b). When a mutation is discovered, it will appear in two separate gel lanes, the coordinates of which will identify the unique individual harboring the mutation. Additionally, the strategy should reduce false-positive errors, as true signals must replicate in the appropriate lanes. For projects where samples are not pooled, it may be useful to replicate samples within the assay to more easily define false-negative and false-positive signals. Once error rates are clearly defined, sample redundancy can be eliminated and screening throughput increased.

It is advantageous to prepare screening stock plates in a 96-well format. For 384 liquid handling, four 96-well stock plates are combined to create a 384-well assay plate. During Sephadex purification, the 384-well plate array is converted back into four 96-well plate arrays, which facilitates loading the 100 lanes of an LI-COR gel.

Prepare a master stock plate by arraying samples, diluted to the appropriate concentration (see REAGENT SETUP), into 96- or 384-well microtiter plates and pool as desired. Before PCR, transfer 5 l from this plate to the assay plate. Assay plates can be prepared in advance and stored at -20 °C for up to 1 week in a sealed container to limit evaporation. The master stock plate can be stored at 4 °C for years. To limit evaporation, the plate is first sealed with adhesive sealing tape and then vacuum sealed.