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The Genome Sequencer FLX System from Roche and 454 Life SciencesTM is a versatile sequencing platform suitable for a wide range of applications, including de novo sequencing and assembly of genomic DNA, transcriptome sequencing, metagenomics analysis, and amplicon sequencing. The Genome Sequencer FLX enables long sequence reads separated by kilobase distances of genomic DNA. These Long-Tag Paired End reads enable improved de novo assemblies and genomic structural variation studies. 454 Life Sciences has developed and commercially released a new protocol for generating a library of paired-end fragments to determine the orientation and relative positions of contigs produced by de novo shotgun sequencing and assembly. This 3K Long-Tag Paired End protocol (Figure 1) can also be used to identify genomic structural variations (1) and their associated breakpoints. Structural variation of the genome, involving large, kiloto mega-base-sized deletions, duplications, insertions, inversions, and complex combinations of rearrangements, is widespread in humans and is presumably responsible for a considerable amount of phenotypic variation. The 3K Long-Tag Paired End library DNA fragments contain an approximately 250-bp fragment with a 44-mer adaptor sequence in the middle, flanked by 100-mer sequences, on average. The two flanking 100-bp sequences are segments of DNA that were originally located approximately 3 kb apart in the genome of interest. In addition to the 3-kb paired-end protocol, initial results from an unreleased protocol that generates flanking reads separated by 16 kb are presented (Figure 2). The 16-kb protocol utilizes a different chemistry than the 3-kb protocol described here. Traditional approaches to the sequencing of paired-end reads rely upon inserting a DNA fragment into a vector, such as a BAC or fosmid, cloning into bacteria, and subsequently generating two sequences, one from each end of the vector. These methods entail weeks of laboratory work and could cost several hundred thousand dollars to prepare the libraries needed for Sanger sequencing. The Genome Sequencer FLX method presented here, which requires no cloning, generates up to 200,000 paired-end reads from a single Genome Sequencer FLX instrument run with a total elapsed time — from genomic DNA to result — of less than four days. SAMPLE PREPARATION PROTOCOL

De Novo Assembly and Genomic Structural Variation Analysis with Genome Sequencer FLX 3K Long-Tag Paired End Reads