Distinguishing somatic and germline copy number events in cancer patient DNA hybridized to whole-genome SNP genotyping arrays.

Methods Mol Biol. 2013;973:355-72

Authors: Ha G, Shah S

Abstract
Chromosomal aneuploidy and segmental copy number changes are common genomic aberrations in -cancer. Copy number alterations (CNAs) arise from deletions, insertions, or duplications resulting in -chromosomal aberrations and aneuploidy. Genomes of normal cells also exhibit variable copy number called germline copy number variants (CNVs). CNVs in the general population tend to confound interpretation of predictions when attempting to extract relevant driver somatic events in cancer. In large studies of CNAs in cancer patients, it becomes necessary to accurately identify and separate CNAs and CNVs so as to prioritize candidate tumor suppressors and oncogenes. We have developed a probabilistic approach, HMM-Dosage, for segmenting and distinguishing CNAs and CNVs as separate, discrete events in cancer SNP genotyping array data. We outline the steps and computer code for the analysis of whole-genome cancer DNA hybridized to SNP genotyping arrays, focusing on distinguishing somatic CNA and germline CNVs, and describe the combined approach of HMM-Dosage for probabilistic inference and classification of somatic and germline copy number changes.

PMID: 23412801 [PubMed - in process]

Analysis of acquired genomic copy number aberrations and regions of loss of heterozygosity in acute myelogenous leukemia genomes using affymetrix SNP 6.0 arrays and supporting software tools.

Methods Mol Biol. 2013;973:99-119

Authors: Ouillette PD, Shedden KA, Li C, Malek SN

Abstract
The application of SNP array technology to the analysis of cancer genomes has greatly advanced our knowledge of the incidence and functional consequences of acquired genomic copy number aberrations (aCNA) and LOH in various malignancies. The major challenges of using SNP arrays are accurately identifying acquired genomic DNA aberrations in the raw array data with very high sensitivity and specificity and meaningfully assessing the associations between these aberrations and biological characteristics or patient outcomes. Critical to the success and valid interpretation of data derived from SNP array profiling are (1) the purity of cells used as a source of template DNA; (2) the analysis of paired DNA samples (tumor and normal); (3) use of validated software tools for data analysis; (4) access to an acceptable gold standard for aCNA and LOH, including FISH data, cytogenetic results, and Q-PCR data; and (5) statistical support to employ or develop algorithmic approaches to SNP array data analysis. Overcalling of lesions including lack of validation and undercalling of lesions that display low fractional allelic representations are common problems. This guide should help the reader establish this powerful technology in the laboratory and aims to stimulate transition of SNP array profiling into clinical applications.

PMID: 23412786 [PubMed - in process]

Integrated Fluidic Circuits (IFCs) for Digital PCR.

Methods Mol Biol. 2013;949:423-31

Authors: Ramakrishnan R, Qin J, Jones RC, Weaver LS

Abstract
The Fluidigm Digital Array IFC is a nanofluidic biochip where digital PCR reactions can be performed with isolated individual DNA template molecules. This chip is part of a family of integrated fluidic circuits (IFC) and contains a network of fluid lines, NanoFlex™ valves and chambers. NanoFlex™ valves are made of an elastomeric material that deflects under pressure to create a tight seal and are used to regulate the flow of liquids in the IFC. Digital Arrays have enabled a different approach to digital PCR, by partitioning DNA molecules instead of diluting them. Single DNA molecules are randomly distributed into nanoliter volume reaction chambers and then PCR amplified in the presence of a fluorophore-containing probe. Positive fluorescent signal indicates the presence of a DNA molecule in a reaction chamber, while negative chambers are blank. IFC technology enables the delivery of very precise volumes of solutions in a simple, fast procedure, utilizing a minimum of sample and assay reagents. The development of the IFC technology and the Digital Array chip has revolutionized the field of biology, and has been utilized in gene copy number studies, absolute quantitation (molecule counting) of genomic DNA and cDNA, rare mutation detection, and digital haplotyping.

PMID: 23329458 [PubMed - in process]

ABO Genotyping by Capillary Electrophoresis.

Methods Mol Biol. 2013;919:113-20

Authors: Lee JC, Hsieh HM, Teng HF, Tsai LC

Abstract
Single-strand conformation polymorphism by capillary electrophoresis (SSCP-CE) has been developed to detect single nucleotide mutations. This method is used to identify the ABO alleles A(1), A(1v), B, O(1), O(1v), and O(2) in this chapter. Four amplicons (112, 121, 123, and 160 bp) labeled with fluorescence are separately amplified by polymerase chain reaction from exons 6 and 7 of ABO gene. These four fragments are combined into a single tube for SSCP-CE analysis using native gel to identify their single nucleotide polymorphism. This method can fast screen ABO genotypes from unknown samples and will be valuable in clinical transfusion or forensic applications.

PMID: 22976095 [PubMed - in process]