Estimation of DNA contamination and its sources in genotyped samples.

Array genotyping is a cost-effective and widely used tool that enables assessment of up to millions of genetic markers in hundreds of thousands of individuals. Genotyping array data are typically highly accurate but sensitive to mixing of DNA samples from multiple individuals before or during genotyping. Contaminated samples can lead to genotyping errors and consequently cause false positive signals or reduce power of association analyses.

Identification of the Genomic Insertion Site of the Thyroid Peroxidase Promoter-Cre Recombinase Transgene Using a Novel, Efficient, Next-Generation DNA Sequencing Method.

Background: It can be useful to know the transgene insertion site in transgenic mice for a variety of reasons, but determining the insertion site generally is a time consuming, expensive, and laborious task.

Methods: A simple method is presented to determine transgene insertion sites that combines the enrichment of a sequencing library by polymerase chain reaction (PCR) for sequences containing the transgene, followed by next-generation sequencing of the enriched library. This method was applied to determine the site of integration of the thyroid peroxidase promoter-Cre recombinase mouse transgene that is commonly used to create thyroid-specific gene deletions.

Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA.

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Evaluation of portability and cost of a fluorescent PCR ribotyping protocol for Clostridium difficile epidemiology.

Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e.

Rare variant genotype imputation with thousands of study-specific whole-genome sequences: implications for cost-effective study designs.

The utility of genotype imputation in genome-wide association studies is increasing as progressively larger reference panels are improved and expanded through whole-genome sequencing. Developing general guidelines for optimally cost-effective imputation, however, requires evaluation of performance issues that include the relative utility of study-specific compared with general/multipopulation reference panels; genotyping with various array scaffolds; effects of different ethnic backgrounds; and assessment of ranges of allele frequencies. Here we compared the effectiveness of study-specific reference panels to the commonly used 1000 Genomes Project (1000G) reference panels in the isolated Sardinian population and in cohorts of European ancestry including samples from Minnesota (USA).

Next-generation sequencing identifies the Danforth's short tail mouse mutation as a retrotransposon insertion affecting Ptf1a expression.

The semidominant Danforth's short tail (Sd) mutation arose spontaneously in the 1920s. The homozygous Sd phenotype includes severe malformations of the axial skeleton with an absent tail, kidney agenesis, anal atresia, and persistent cloaca. The Sd mutant phenotype mirrors features seen in human caudal malformation syndromes including urorectal septum malformation, caudal regression, VACTERL association, and persistent cloaca.

High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing.

Objective: To identify disease-causing mutations within coding regions of 11 known NPHP genes (NPHP1-NPHP11) in a cohort of 192 patients diagnosed with a nephronophthisis-associated ciliopathy, at low cost.

Methods: Mutation analysis was carried out using PCR-based 48.48 Access Array microfluidic technology (Fluidigm) with consecutive next-generation sequencing.

Exome sequencing reveals cubilin mutation as a single-gene cause of proteinuria.

In two siblings of consanguineous parents with intermittent nephrotic-range proteinuria, we identified a homozygous deleterious frameshift mutation in the gene CUBN, which encodes cubulin, using exome capture and massively parallel re-sequencing. The mutation segregated with affected members of this family and was absent from 92 healthy individuals, thereby identifying a recessive mutation in CUBN as the single-gene cause of proteinuria in this sibship. Cubulin mutations cause a hereditary form of megaloblastic anemia secondary to vitamin B(12) deficiency, and proteinuria occurs in 50% of cases since cubilin is coreceptor for both the intestinal vitamin B(12)-intrinsic factor complex and the tubular reabsorption of protein in the proximal tubule.

Comparison of constitutional and replication stress-induced genome structural variation by SNP array and mate-pair sequencing.

Copy-number variants (CNVs) are a major source of genetic variation in human health and disease. Previous studies have implicated replication stress as a causative factor in CNV formation. However, existing data are technically limited in the quality of comparisons that can be made between human CNVs and experimentally induced variants.

Discovery of mutations in Saccharomyces cerevisiae by pooled linkage analysis and whole-genome sequencing.

Many novel and important mutations arise in model organisms and human patients that can be difficult or impossible to identify using standard genetic approaches, especially for complex traits. Working with a previously uncharacterized dominant Saccharomyces cerevisiae mutant with impaired vacuole inheritance, we developed a pooled linkage strategy based on next-generation DNA sequencing to specifically identify functional mutations from among a large excess of polymorphisms, incidental mutations, and sequencing errors. The VAC6-1 mutation was verified to correspond to PHO81-R701S, the highest priority candidate reported by VAMP, the new software platform developed for these studies.

Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy.

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders that feature dysplasia or degeneration occurring preferentially in the kidney, retina and cerebellum. Here we combined homozygosity mapping with candidate gene analysis by performing 'ciliopathy candidate exome capture' followed by massively parallel sequencing. We identified 12 different truncating mutations of SDCCAG8 (serologically defined colon cancer antigen 8, also known as CCCAP) in 10 families affected by NPHP-RC.

Genotyping of 73 UM-SCC head and neck squamous cell carcinoma cell lines.

Background: We established multiple University of Michigan Squamous Cell Carcinoma (UM-SCC) cell lines. With time, these have been distributed to other labs all over the world. Recent scientific discussions have noted the need to confirm the origin and identity of cell lines in grant proposals and journal articles.

Genetics, gene expression and bioinformatics of the pituitary gland.

Genetic cases of congenital pituitary hormone deficiency are common and many are caused by transcription factor defects. Mouse models with orthologous mutations are invaluable for uncovering the molecular mechanisms that lead to problems in organ development and typical patient characteristics. We are using mutant mice defective in the transcription factors PROP1 and POU1F1 for gene expression profiling to identify target genes for these critical transcription factors and candidates for cases of pituitary hormone deficiency of unknown aetiology.

A systematic approach to mapping recessive disease genes in individuals from outbred populations.

The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays.

Discovery of transcriptional regulators and signaling pathways in the developing pituitary gland by bioinformatic and genomic approaches.

We report a catalog of the mouse embryonic pituitary gland transcriptome consisting of five cDNA libraries including wild type tissue from E12.5 and E14.5, Prop1(df/df) mutant at E14.

Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line.

Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers.

Comparative genomics reveals functional transcriptional control sequences in the Prop1 gene.

Mutations in PROP1 are a common genetic cause of multiple pituitary hormone deficiency (MPHD). We used a comparative genomics approach to predict the transcriptional regulatory domains of Prop1 and tested them in cell culture and mice. A BAC transgene containing Prop1 completely rescues the Prop1 mutant phenotype, demonstrating that the regulatory elements necessary for proper PROP1 transcription are contained within the BAC.

Tissue-preserving approach to extracting DNA from paraffin-embedded specimens using tissue microarray technology.

Background: DNA extracted from tumor cells or normal cells contained in formalin-fixed, paraffin-embedded tissues is widely used in many laboratories. The 2 most common procedures to isolate cells for DNA extraction from paraffin-embedded tissues are scalpel microdissection and laser capture microdissection. A new tissue- and time-conserving method for rapid DNA isolation from small cores taken from paraffin-embedded tissue blocks is described in this report.

A comprehensive mutation analysis of RP2 and RPGR in a North American cohort of families with X-linked retinitis pigmentosa.

X-linked retinitis pigmentosa (XLRP) is a clinically and genetically heterogeneous degenerative disease of the retina. At least five loci have been mapped for XLRP; of these, RP2 and RP3 account for 10%-20% and 70%-90% of genetically identifiable disease, respectively. However, mutations in the respective genes, RP2 and RPGR, were detected in only 10% and 20% of families with XLRP.