Paige Anderson

Characterization of missing human genome sequences and copy-number polymorphic insertions

The extent of human genomic structural variation suggests that there must be portions of the genome yet to be discovered, annotated and characterized at the sequence level. We present a resource and analysis of 2,363 new insertion sequences corresponding to 720 genomic loci. We found that a substantial fraction of these sequences are either missing, fragmented or misassigned when compared to recent de novo sequence assemblies from short-read next-generation sequence data. We determined that 18–37% of these new insertions are copy-number polymorphic, including loci that show extensive population stratification among Europeans, Asians and Africans. Complete sequencing of 156 of these insertions identified new exons and conserved noncoding sequences not yet represented in the reference genome. We developed a method to accurately genotype these new insertions by mapping next-generation sequencing datasets to the breakpoint, thereby providing a means to characterize copy-number status for regions previously inaccessible to single-nucleotide polymorphism microarrays.

A Method for Multiplex Gene Synthesis Employing Error Correction Based on Expression

Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.

Correction: A method for multiplex gene synthesis employing error correction based on expression.

The fifth author’s name is spelled incorrectly. The correct name is Tobias Strittmatter. The correct citation is: Hsiau TH-C, Sukovich D, Elms P, Prince RN, Strittmatter T, Ruan P, et al. (2015) A Method for Multiplex Gene Synthesis Employing Error Correction Based on Expression. PLoS ONE 10(3): e0119927. doi:10.1371/journal.pone.0119927

Abstract 45: Accurate total and allele-specific copy number measurements in mosaic tumors

For the characterization of genomic copy number changes that occur in the development and progression of cancer, oligonucleotide array comparative genomic hybridization (aCGH) offers high-resolution and precise determination of chromosomal copy number and genome-wide aberrations. We recently reported a new method for making simultaneous measurements of single nucleotide polymorphisms (SNPs) and copy number alterations in the same microarray assay. The combined assay can detect copy-number neutral events, such as acquired loss of heterozygosity (LOH), as well as allelic imbalance in and around amplified regions. Previously reported algorithms for analyzing Agilent CGH+SNP data were able to genotype primarily diploid samples and to detect regions of constitutional LOH. However, tumor heterogeneity, aneuploidy and variable sample quality create significant challenges for both solid and liquid tumors. Our earlier methods were often unable to cope with the high levels of aneuploidy sometimes found in solid tumors, or with admixtures of normal cells and aberrant cells. We now describe new computational methods which can determine total copy number, aneuploid fraction, and allele-specific copy number in many aneuploid tumor samples, even when mixed with up to 80% normal tissue. We report results from genomic DNA isolated from a cancer cell line, a blood sample, and a solid tumor. Each of these sample types presents novel challenges. The cell line we studied, though largely monoclonal, is highly aneuploid. The blood DNA is of high quality, but the fraction of tumor cells in the sample is low. The solid tumor DNA is of relatively low quality, and composed of multiple aberrant clones. We were able to determine the aneuploid fraction of the tumors, and to measure copy number variation in samples with as little as 20% tumor cell content. In addition to detecting copy-neutral LOH regions, we also measured allele-specific copy number, both of the aberrant clone and of admixed normal cells. The new analysis methods allow the extension of the extra allelic information available from the CGH+SNP assay to cancer samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 45. doi:10.1158/1538-7445.AM2011-45