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

Raman, Priyadarshini / Grachtchouk, Vladimir / Lyons, Robert H / Koenig, Ronald J

Thyroid : official journal of the American Thyroid Association

2015 Volume 25, Issue 10, Page(s) 1162–1166

Abstract: 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 ... ...

Abstract	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. Results: The insertion site was found to be between bp 12,372,316 and 12,372,324 on mouse chromosome 9, with the nearest characterized genes being Cntn5 and Jrkl, ∼1.5 and 0.9 Mbp from the transgene, respectively. One advantage of knowing a transgene insertion site is that it facilitates distinguishing hemizygous from homozygous transgenic mice. Although this can be accomplished by real-time quantitative PCR, the expected Ct difference is only one cycle, which is challenging to assess accurately. Therefore, the transgene insertion site information was used to develop a 3-primer qualitative PCR assay that readily distinguishes wild type, hemizygous, and homozygous TPO-Cre mice based upon size differences of the wild type and transgenic allele PCR products. Conclusions: Identification of the genomic insertion site of the thyroid peroxidase promoter-Cre mouse transgene should facilitate the use of these mice in studies of thyroid biology.
MeSH term(s)	Animals ; High-Throughput Nucleotide Sequencing ; Integrases/genetics ; Iodide Peroxidase/genetics ; Mice ; Mice, Transgenic ; Promoter Regions, Genetic ; Transgenes
Chemical Substances	Iodide Peroxidase (EC 1.11.1.8) ; Cre recombinase (EC 2.7.7.-) ; Integrases (EC 2.7.7.-)
Language	English
Publishing date	2015-10
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	1086044-7
ISSN	1557-9077 ; 1050-7256
ISSN (online)	1557-9077
ISSN	1050-7256
DOI	10.1089/thy.2015.0215
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Article ; Online: Estimation of DNA contamination and its sources in genotyped samples.

Zajac, Gregory J M / Fritsche, Lars G / Weinstock, Joshua S / Dagenais, Susan L / Lyons, Robert H / Brummett, Chad M / Abecasis, Gonçalo R

Genetic epidemiology

2019 Volume 43, Issue 8, Page(s) 980–995

Abstract: 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 ... ...

Abstract	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. Here, we propose a new method to identify contaminated samples and the sources of contamination within a genotyping batch. Through analysis of array intensity and genotype data from intentionally mixed samples and 22,366 samples of the Michigan Genomics Initiative, an ongoing biobank-based study, we show that our method can reliably estimate contamination. We also show that identifying sources of contamination can implicate problematic sample processing steps and guide process improvements. Compared to existing methods, our approach can estimate the proportion of contaminating DNA more accurately, eliminate the need for external databases of allele frequencies, and provide contamination estimates that are more robust to the ancestral origin of the contaminating sample.
MeSH term(s)	DNA ; DNA Contamination ; Gene Frequency ; Genetic Markers ; Genomics/methods ; Genotype ; Genotyping Techniques/methods ; Humans ; Polymorphism, Single Nucleotide
Chemical Substances	Genetic Markers ; DNA (9007-49-2)
Language	English
Publishing date	2019-08-26
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	605785-8
ISSN	1098-2272 ; 0741-0395
ISSN (online)	1098-2272
ISSN	0741-0395
DOI	10.1002/gepi.22257
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Article: Twenty-one novel microsatellite loci for the endangered Florida salt marsh vole (Microtus pennsylvanicus dukecampbelli)

Austin, James D / Saarinen, Emily V / Arias-Pérez, Alberto / McCleery, Robert A / Lyons, Robert H

Conservation genetics resources. 2014 Sept., v. 6, no. 3

2014

Abstract: We present 21 microsatellite loci developed for Florida salt marsh voles (Microtus pennsylvanicus dukecampbelli). Microsatellites were identified from single molecule real time sequencing (Pacific Biosciences). We screened 30 loci and identified 21 loci ... ...

Abstract	We present 21 microsatellite loci developed for Florida salt marsh voles (Microtus pennsylvanicus dukecampbelli). Microsatellites were identified from single molecule real time sequencing (Pacific Biosciences). We screened 30 loci and identified 21 loci as suitable for genotyping. We screened 17 individuals from Long Cabbage Key, and 3 individuals from an unnamed island. There was no significant departure from Hardy–Weinberg equilibrium or linkage equilibrium. Fifteen of the 21 loci were variable, with overall observed heterozygosity averaging 0.39, and a mean number of alleles of 3.14. Linkage disequilibrium estimate of N ₑ was 10.7 (95� % CI 6.1–20.1). These markers will be useful for conservation genetics studies of this endangered species.
Keywords	Microtus pennsylvanicus ; alleles ; cabbage ; endangered species ; genotyping ; heterozygosity ; linkage disequilibrium ; loci ; microsatellite repeats ; salt marshes ; Florida
Language	English
Dates of publication	2014-09
Size	p. 637-639.
Publishing place	Springer-Verlag
Document type	Article
ZDB-ID	2508018-0
ISSN	1877-7260 ; 1877-7252
ISSN (online)	1877-7260
ISSN	1877-7252
DOI	10.1007/s12686-014-0159-y
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Article ; Online: Next-generation sequencing identifies the Danforth's short tail mouse mutation as a retrotransposon insertion affecting Ptf1a expression.

Vlangos, Christopher N / Siuniak, Amanda N / Robinson, Dan / Chinnaiyan, Arul M / Lyons, Robert H / Cavalcoli, James D / Keegan, Catherine E

PLoS genetics

2013 Volume 9, Issue 2, Page(s) e1003205

Abstract: 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 ... ...

Abstract	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. The Sd mutation was previously mapped to a 0.9 cM region on mouse chromosome 2qA3. We performed Sanger sequencing of exons and intron/exon boundaries mapping to the Sd critical region and did not identify any mutations. We then performed DNA enrichment/capture followed by next-generation sequencing (NGS) of the critical genomic region. Standard bioinformatic analysis of paired-end sequence data did not reveal any causative mutations. Interrogation of reads that had been discarded because only a single end mapped correctly to the Sd locus identified an early transposon (ETn) retroviral insertion at the Sd locus, located 12.5 kb upstream of the Ptf1a gene. We show that Ptf1a expression is significantly upregulated in Sd mutant embryos at E9.5. The identification of the Sd mutation will lead to improved understanding of the developmental pathways that are misregulated in human caudal malformation syndromes.
MeSH term(s)	Animals ; DNA Transposable Elements/genetics ; Embryonic Development ; Exons ; Gene Expression Regulation, Developmental/genetics ; Genome ; Humans ; Mice ; Mutagenesis, Insertional/genetics ; Phenotype ; Sequence Analysis, DNA ; Spinal Cord/abnormalities ; Tail/anatomy & histology ; Transcription Factors/genetics ; Transcription Factors/metabolism
Chemical Substances	DNA Transposable Elements ; Transcription Factors ; transcription factor PTF1
Language	English
Publishing date	2013-02-21
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2186725-2
ISSN	1553-7404 ; 1553-7390
ISSN (online)	1553-7404
ISSN	1553-7390
DOI	10.1371/journal.pgen.1003205
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Article ; Online: Evaluation of portability and cost of a fluorescent PCR ribotyping protocol for Clostridium difficile epidemiology.

Martinson, Jonathan N V / Broadaway, Susan / Lohman, Egan / Johnson, Christina / Alam, M Jahangir / Khaleduzzaman, Mohammed / Garey, Kevin W / Schlackman, Jessica / Young, Vincent B / Santhosh, Kavitha / Rao, Krishna / Lyons, Robert H / Walk, Seth T

Journal of clinical microbiology

2015 Volume 53, Issue 4, Page(s) 1192–1197

Abstract: 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., portable data) to help ... ...

Abstract	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., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified.
MeSH term(s)	Clostridium Infections/epidemiology ; Clostridium Infections/microbiology ; Clostridium difficile/genetics ; Humans ; Molecular Epidemiology ; Polymerase Chain Reaction/methods ; Ribotyping/methods
Language	English
Publishing date	2015-04
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
DOI	10.1128/JCM.03591-14
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Article ; Online: Comparison of constitutional and replication stress-induced genome structural variation by SNP array and mate-pair sequencing.

Arlt, Martin F / Ozdemir, Alev Cagla / Birkeland, Shanda R / Lyons, Robert H / Glover, Thomas W / Wilson, Thomas E

Genetics

2011 Volume 187, Issue 3, Page(s) 675–683

Abstract: 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 ... ...

Abstract	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. Here, we used two high-resolution strategies-single nucleotide polymorphism (SNP) arrays and mate-pair sequencing-to compare CNVs that occur constitutionally to those that arise following aphidicolin-induced DNA replication stress in the same human cells. Although the optimized methods provided complementary information, sequencing was more sensitive to small variants and provided superior structural descriptions. The majority of constitutional and all aphidicolin-induced CNVs appear to be formed via homology-independent mechanisms, while aphidicolin-induced CNVs were of a larger median size than constitutional events even when mate-pair data were considered. Aphidicolin thus appears to stimulate formation of CNVs that closely resemble human pathogenic CNVs and the subset of larger nonhomologous constitutional CNVs.
MeSH term(s)	Aphidicolin/pharmacology ; Cell Line ; DNA Copy Number Variations/drug effects ; DNA Copy Number Variations/genetics ; DNA Damage/genetics ; DNA Replication ; Genome, Human ; Humans ; Oligonucleotide Array Sequence Analysis/methods ; Polymorphism, Single Nucleotide/genetics ; Sequence Analysis, DNA/methods
Chemical Substances	Aphidicolin (38966-21-1)
Language	English
Publishing date	2011-01-06
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2167-2
ISSN	1943-2631 ; 0016-6731
ISSN (online)	1943-2631
ISSN	0016-6731
DOI	10.1534/genetics.110.124776
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Article ; Online: Discovery of mutations in Saccharomyces cerevisiae by pooled linkage analysis and whole-genome sequencing.

Birkeland, Shanda R / Jin, Natsuko / Ozdemir, Alev Cagla / Lyons, Robert H / Weisman, Lois S / Wilson, Thomas E

Genetics

2010 Volume 186, Issue 4, Page(s) 1127–1137

Abstract: 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 ... ...

Abstract	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. Sequence data further revealed the large extent of strain background polymorphisms and structural alterations present in the host strain, which occurred by several mechanisms including a novel Ty insertion. The results provide a snapshot of the ongoing genomic changes that ultimately result in strain divergence and evolution, as well as a general model for the discovery of functional mutations in many organisms.
MeSH term(s)	Biological Evolution ; DNA Mutational Analysis/methods ; Genetic Linkage ; Genetic Speciation ; Genome, Fungal/genetics ; Mutation ; Saccharomyces cerevisiae/genetics ; Sequence Analysis, DNA/methods ; Software
Language	English
Publishing date	2010-10-05
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2167-2
ISSN	1943-2631 ; 0016-6731
ISSN (online)	1943-2631
ISSN	0016-6731
DOI	10.1534/genetics.110.123232
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Article ; Online: Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA.

Shanker, Savita / Paulson, Ariel / Edenberg, Howard J / Peak, Allison / Perera, Anoja / Alekseyev, Yuriy O / Beckloff, Nicholas / Bivens, Nathan J / Donnelly, Robert / Gillaspy, Allison F / Grove, Deborah / Gu, Weikuan / Jafari, Nadereh / Kerley-Hamilton, Joanna S / Lyons, Robert H / Tepper, Clifford / Nicolet, Charles M

Journal of biomolecular techniques : JBT

2015 Volume 26, Issue 1, Page(s) 4–18

Abstract: This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome ... ...

Abstract	This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.
MeSH term(s)	Animals ; Base Sequence ; DNA, Complementary/genetics ; Humans ; Limit of Detection ; Mice ; Nucleic Acid Amplification Techniques/standards ; Polyadenylation ; RNA/genetics ; Rats ; Reference Standards ; Sequence Analysis, RNA/standards
Chemical Substances	DNA, Complementary ; RNA (63231-63-0)
Language	English
Publishing date	2015-04
Publishing country	United States
Document type	Evaluation Studies ; Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1943-4731
ISSN (online)	1943-4731
DOI	10.7171/jbt.15-2601-001
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Article ; Online: Genotyping of 73 UM-SCC head and neck squamous cell carcinoma cell lines.

Brenner, J Chad / Graham, Martin P / Kumar, Bhavna / Saunders, Lindsay M / Kupfer, Robbi / Lyons, Robert H / Bradford, Carol R / Carey, Thomas E

Head & neck

2009 Volume 32, Issue 4, Page(s) 417–426

Abstract: 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 ... ...

Abstract	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. We genotyped the UM-SCC cell lines in our collection to confirm their unique identity. Method: Early-passage UM-SCC cell lines were genotyped and photographed. Results: Thus far, 73 unique head and neck UM-SCC cell lines (from 65 donors, including 21 lines from 17 females) were genotyped. In 7 cases, separate cell lines were established from the same donor. Conclusions: These results will be posted on the UM Head and Neck SPORE Tissue Core website for other investigators to confirm that the UM-SCC cells used in their laboratories have the correct features. Publications using UM-SCC cell lines should confirm the genotype.
MeSH term(s)	Carcinoma, Squamous Cell/genetics ; Cell Line, Tumor ; DNA, Neoplasm/genetics ; Female ; Gene Expression Regulation, Neoplastic ; Genotype ; Head and Neck Neoplasms/genetics ; Humans ; Male ; Microsatellite Repeats/genetics ; Sensitivity and Specificity
Chemical Substances	DNA, Neoplasm
Language	English
Publishing date	2009-09-16
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	645165-2
ISSN	1097-0347 ; 0148-6403 ; 1043-3074
ISSN (online)	1097-0347
ISSN	0148-6403 ; 1043-3074
DOI	10.1002/hed.21198
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Article ; Online: High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing.

Halbritter, Jan / Diaz, Katrina / Chaki, Moumita / Porath, Jonathan D / Tarrier, Brendan / Fu, Clementine / Innis, Jamie L / Allen, Susan J / Lyons, Robert H / Stefanidis, Constantinos J / Omran, Heymut / Soliman, Neveen A / Otto, Edgar A

Journal of medical genetics

2012 Volume 49, Issue 12, Page(s) 756–767

Abstract: 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 ...

Abstract	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. We applied a 10-fold primer multiplexing approach allowing PCR-based amplification of 475 amplicons (251 exons) for 48 DNA samples simultaneously. After four rounds of amplification followed by indexing all of 192 patient-derived products with different barcodes in a subsequent PCR, 2 × 100 paired-end sequencing was performed on one lane of a HiSeq2000 instrument (Illumina). Bioinformatics analysis was performed using 'CLC Genomics Workbench' software. Potential mutations were confirmed by Sanger sequencing and shown to segregate. Results: Bioinformatics analysis revealed sufficient coverage of 30 × for 168/192 (87.5%) DNA samples (median 449 ×) and of 234 out of 251 targeted coding exons (sensitivity: 93.2%). For proof-of-principle, we analysed 20 known mutations and identified 18 of them in the correct zygosity state (90%). Likewise, we identified pathogenic mutations in 34/192 patients (18%) and discovered 23 novel mutations in the genes NPHP3 (7), NPHP4 (3), IQCB1 (4), CEP290 (7), RPGRIP1L (1), and TMEM67 (1). Additionally, we found 40 different single heterozygous missense variants of unknown significance. Conclusions: We conclude that the combined approach of array-based multiplexed PCR-amplification on a Fluidigm Access Array platform followed by next-generation sequencing is highly cost-efficient and strongly facilitates diagnostic mutation analysis in broadly heterogeneous Mendelian disorders.
MeSH term(s)	Base Sequence ; Cilia/pathology ; Computational Biology/methods ; DNA Mutational Analysis ; Exons ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; Kidney Diseases, Cystic/congenital ; Kidney Diseases, Cystic/genetics ; Kidney Diseases, Cystic/pathology ; Multiplex Polymerase Chain Reaction ; Mutation ; Reproducibility of Results
Language	English
Publishing date	2012-12
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	220881-7
ISSN	1468-6244 ; 0022-2593
ISSN (online)	1468-6244
ISSN	0022-2593
DOI	10.1136/jmedgenet-2012-100973
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