Analysis of DNA Library and Sequence Quality with the Finch®-Server

     Todd M. Smith, Joe Slagel, Dave Campbell, Sandra Porter
     Geospiza, Inc. 2442 NW Market St. Seattle, WA 98107 USA

Recent years have seen an increased number of laboratories undertake projects in genomic sequencing. However, the technical expertise, background knowledge, and analysis tools required for successfully performing large-scale sequencing, are still largely concentrated among a small number of genome centers. Data management tools such as the Finch®-Server, allow laboratories to analyze many of the steps involved in with genomic sequencing and quickly identify problem areas.

A common strategy for genomic sequencing involves breaking DNA into several random fragments, cloning the fragments, determining the nucleotide sequence of several clones, and using the overlapping regions to assemble a complete genome sequence. In order for this process to work, the clone library must represent sequences that were broken at random points. Often, these libraries are constructed by using physical shearing methods such as sonication to randomly fragment genomic DNA. On occasion, alternative methods are used for library construction, relying instead on digestion with restriction enzymes to generate smaller DNA fragments. The results of one such project are described here. The Finch-Server was used to perform a detailed analysis of library quality by counting the number of vector clones, the number of short clones, and the number clones with poor quality data (less than 100 bases with phred scores > 20). We were also able to determine that the library was not random; by using the Finch-Server to sort clones by size. Morever, many of the sequences represented chimeras, most likely created by ligating non-contiguous restriction fragments, during the cloning step.

 ABRF Conference, 2002