In house computer pipeline for hetDNA mapping
Publication Citation: Gamble D, Hum Y, Jinks-Robertson S. High-throughput Analysis of Heteroduplex DNA in Mitotic Recombination Products. Methods in molecular biology: Homologous Recombination edition [In Review]
Mitotic double-strand breaks (DSBs) are repaired by recombination with a homologous donor template. This process involves the exchange of sequence between the recipient, where the DSB occurs, and the donor, giving rise to regions of heteroduplex DNA (hetDNA). The creation of a defined DSB coupled with the use of a sequence-diverged repair template allows the fine-structure mapping of hetDNA through the sequencing of recombination products. Our lab has designed a high throughput method to analyze the hetDNA in hundreds of recombination products, simultaneously, using PacBio SMRT sequencing. The publication cited above includes detailed step-by-step instructions for this method with all equipment and materials needed.
The computer pipeline described in this publication analyzes SMRT sequencing data by sorting the unique barcode combinations in the pooled library of recombinants and calling the SNPs present at each position in each product. This pipeline is run in the Mac OS X resident application “Terminal” and scripts utilize both perl and R programming languages. Below we have provided all program files for the computer pipeline with the exception of files that must be provided by the user. The first set of program files labeled “ProgramFiles_1SMRTcell” should be used for a single library run on a single SMRT cell. The detailed instructions for these files are included in the publication. The second set of program files labeled “ProgramFiles_2orMoreSMRTcells” should be used for a single library run on two or more SMRT cells. The instructions for these files are linked below on this page as mentioned in the publication.