Mate-pair Libraries Research Articles

SNP discovery performance of two second-generation sequencing platforms in the NOD2 gene region

A potentially important application of second generation sequencing technologies is to identify disease-associated variation. For comparison of the performance in SNP detection, the Crohn's disease (CD)-associated NOD2 gene was subjected to targeted resequencing using two different second-generation sequencing technologies. Eleven CD patients were selected based on their haplotype background at the NOD2 locus. The 40-kb large NOD2 gene region was amplified using long-range PCR (LR-PCR), and sequenced with the Roche 454/FLX system, an Applied Biosystems SOLiD mate-pair library (2 x 25 bp), and a SOLiD fragment (50 bp) library. The entire NOD2 region was also sequenced using conventional Sanger technology. Four-hundred forty-two single nucleotide polymorphisms (SNPs) were discovered with the SOLiD mate-pair library, 454 with the fragment library, and 441 with the 454/FLX. For the homozygous SNPs, 98% were confirmed by Sanger for the mate-pair library, 100% for the fragment library and 99% for the 454/FLX. Ninety-six percent of the heterozygous SNPs detected with the SOLiD mate-pair library, 91% with the fragment library and 96% with the 454/FLX were confirmed by Sanger. In a simulation, the SNP detection performance fell rapidly when the achieved coverage was below 40 x. Due to uneven representation of the target region when using LR-PCR, oversequencing of other regions is necessary.

Abstract 2212: A genomic portrait of tumor progression using next-generation sequencing

Abstract Accumulation of genetic and epigenetic alterations is thought to enable cancers to proliferate and survive more effectively than their neighboring cells, or for aggressive tumors to resist cytotoxic therapies. However, the molecular basis of tumorigenesis and chemoresistance is not fully understood. With the advent of next-generation sequencing technologies, we can determine the whole transcriptome or genome sequence of a tumor and thereby detect all the changes that may result in a particular phenotype such as resistance to cytotoxic therapies. To understand the genetic aberrations underlying the chemoresistance in neuroblastoma, we attempted to catalogue all genetic aberrations in two tumor samples before and after cytotoxic therapies using RNA-seq (including whole transcriptome and miRNA) and genomic DNA sequencing from the same neuroblastoma patient who was refractory to standard chemotherapies. First, we performed whole genome sequencing on patient's constitutional DNA to establish a genetic background. Multiple mate-paired libraries were constructed and subjected to next-generation sequencing. We acquired 53 Gb high-quality mappable data, equivalent to 16X coverage of this patient's genome. Next, whole transcriptome sequencing of the two tumors yielded total 345 million 50bp mappable reads representing 14,128 and 14,484 genes (20875 and 21330 transcripts) respectively. The average coverage for each transcript was >14, while 5297 genes for one sample and 6912 genes for the other had an average coverage of >10. With such deep sequencing, single nucleotide variant (SNV) analysis on the transcriptome sequencing data identified 9103 and 10556 exonic SNVs of total 154871 and 191033 SNVs respectively in two samples. Among the exonic SNVs, 3965 and 4470 SNVs were in the coding regions and about half of them were nonsynonymous. Using SIFT analysis, we predicted 353 and 443 SNVs to be damaging before and after chemotherapies. Of them, 165 SNVs were shared among two samples. Therefore, these shared SNVs and newly acquired SNVs after chemotherapies may contain the genetic alterations that are resistant to the standard neuroblastoma therapies in this patient. In addition, 188 SNVs were present in the original tumor but not in the refractory sample indicating genomic instability of tumors. In parallel, we performed microRNA sequencing on the two samples. While most of 987 mature microRNAs were detected at similar levels after normalized against total reads, 64 microRNAs were significantly differentially expressed before and after chemotherapies using stringent selection criteria (total read counts in two samples>500, >5 fold change, P<0.01, and FDR<0.01). These differentially microRNAs and accumulation of deleterious mutations during therapies are currently being investigated for pathway disruption that may lead to chemo-refractory disease and allow development of targeted therapies to overcome drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2212.

A Scaffold Analysis Tool Using Mate‐Pair Information in Genome Sequencing

We have developed a Windows-based program, ConPath, as a scaffold analyzer. ConPath constructs scaffolds by ordering and orienting separate sequence contigs by exploiting the mate-pair information between contig-pairs. Our algorithm builds directed graphs from link information and traverses them to find the longest acyclic graphs. Using end read pairs of fixed-sized mate-pair libraries, ConPath determines relative orientations of all contigs, estimates the gap size of each adjacent contig pair, and reports wrong assembly information by validating orientations and gap sizes. We have utilized ConPath in more than 10 microbial genome projects, including Mannheimia succiniciproducens and Vibro vulnificus, where we verified contig assembly and identified several erroneous contigs using the four types of error defined in ConPath. Also, ConPath supports some convenient features and viewers that permit investigation of each contig in detail; these include contig viewer, scaffold viewer, edge information list, mate-pair list, and the printing of complex scaffold structures.

Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.