PAGE 5 OF 7 time from cultures originally containing 10.0–90.0% insert bearing cells. The use of dual-ﬂuorescence reporting with commercially available cell-sorters is capable of accurately isolating large numbers of insert containing clones from complex libraries. A B C Nucleic Acids Research, 2005, Vol. 33, No. 5 e49 The shotgun sequencing project demonstrates the usefulness of the dual-ﬂuorescence reporter system in real-world applica- tions. Partial assembly of the SU66E20 BAC was achieved after sequencing the BAC to 4.3X coverage. Furthermore, insert size was not biased during the culturing and cell-sorting processes. The demonstration of sequencing capability serves as strong validation of the robustness of this method for selecting clones that are carrying inserts. This reliability is an important factor during any large-scale biological investigation. Modern genomic studies require the processing of large numbers of samples. To date, advantages of scale have been obtained through the increasing use of automation. The development of clone picking robots guided by video recognition technology was essential to the completion of the Human Genome Project. Here, we demonstrate a faster alternative. Clone isolation by cell-sorting is a unique tech- nology that is only limited by the speed at which wells can be transported under the stream of sorted droplets. Assuming a conservative rate of 2 wells/s, it is possible to isolate 7200 clones/h using one cell-sorter; this is faster than the latest generation of colony picking robots. Using one cell-sorting apparatus, 172 800 clones could be isolated in each 24-h per- iod. Assuming 500 bp forward and reverse sequence reads, upwards of 173 Mb of raw sequence data could be generated per day. At this rate, the human genome could be sequenced to greater than 5X coverage in less than 90 days from clones isolated by one cell-sorter. Even further rate increases could be achieved with the use of multiple sort streams or by increasing the well transport rate. With further downstream instrumentation, clone selection by sorting could be a powerful strategy for high-throughput applications. After single cells are cultured, sample handling robots could transfer small culture volumes for further ampliﬁcation. Use of rolling circle ampliﬁcation with sub- microliter volumes of culture can quickly generate sequencing templates in thousands of wells simultaneously. In addition, advances in single cell ampliﬁcation technologies could obviate the need for culturing altogether. Groups have demon- strated ampliﬁcation from single DNA molecules using RCA techniques similar to those utilized here (20). Another method of RCA developed by Tabor and co-workers (21) is nearly able to amplify DNA from single cells (personal communication). The integration of cell sorting, ampliﬁcation technologies and instrumentation represents an efﬁcient and practical method for sequencing large number of clones. Looking further, the rapid handling of samples could be achieved on microfabricated DNA processing chips. Flow cytometers could deposit cells into a landing zone on a chip, and sorted cells are then transported into individual Figure 3. (A) Flow-cytometry bivariate dot plot of E.coli expressing GFPmut3.1 from a constitutive promoter. Cells were picked from a single colony and cultured overnight. Because of the large cell-to-cell variation of fluorescence from a single fluorophore, it is difficult to use a single fluorophore as an indication of the presence or absence of a cloned insert. (B) Dot plot shows a population of E.coli containing native pGRFP. All cells are observed expres- sing both GFP and DsRed. (C) Dot plot showing a S.purpuratus BAC library cloned into the pGRFP vector. A second population of cells that are GFP+/ DsRed appears owing to loss of function of the DsRed half of the fusion protein. This population contains cells with successfully incorporated inserts. This reporter system clearly distinguishes between insert containing and non- insert containing E.coli.