The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity

Abstract Background The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly. Results RSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3’-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %. Conclusions RSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing templates amenable to DNA sequencing on recently developed platforms. Although our demonstration of the method does not utilize these DNA sequencing platforms directly, our results indicate that the capture of long DNA fragments produce superior coverage of the targeted region

From millions to one: theoretical and concrete approaches to De Novo assembly using short read DNA sequences

One of the most significant advances in biology has been the ability to sequence the DNA of organisms. Even in the shadow of the completion of the human genome, intractable regions of the genome remain incomplete. Next generation high-throughput short read sequencing technologies are now available and have the ability to generate millions of short read DNA sequences per run. Although greater coverage depths are possible, de novo sequence assembly with these shorter sequences is significantly more complex than resequencing; handling them presents new computational problems and opportunities. Identifying repetitive regions, coping with sequencing errors, and manipulating the millions of short reads simultaneously, are some of the difficulties that must be overcome. As a result of these complexities and working with the short read sequences from the Waksman SOLiD sequencing platform, this work explores the problem of de novo assembly. Initially, we develop tools for filtering short read sequence data based on quality scores and find that this procedure is critical for the success of the subsequent de novo assembly. Next, we analyze the key phenomena responsible for producing contigs that are much shorter than the values provided by theoretical estimates. Finally, we explore two different routes to circumventing the difficulty imposed by short contigs. The first involves utilization of information from multiple orthologous genomes in a comparative assembly. In particular, we developed a pipeline for using the reference genome of a close by relative to improve genome assembly. The second approach uses paired read information to build scaffolds that are two orders of magnitude larger than the original contigs. For typical bacterial genomes, less than one hundred of these scaffolds are required to cover the entire genome. The combination of short reads from various platforms, assembly, and recovery pipelines brings mid-sized genomes close to completion. As a result, minimal additional work using conventional sequencing technologies are enough to close the remaining small gaps and return a finished single genome. Current advancements in sequencing technologies leave us hopeful that it would be possible to provide fairly complete assemblies for complex genomes via these technological approaches.Ph.D.Includes bibliographical referencesIncludes vitaby Ariella Syma Sasso

Exome sequencing analysis reveals variants in primary immunodeficiency genes in patients with very early onset inflammatory bowel disease

Very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed ≤5 y of age, frequently presents with a different and more severe phenotype than older-onset IBD. We investigated whether patients with VEO-IBD carry rare or novel variants in genes associated with immunodeficiencies that might contribute to disease development