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Custom amplicon

Alexander J Brown, Aneesa T Al-Soodani, Miles Saul, Stephanie Her, Juan C Garcia, Dale A Ramsden, Chengtao Her, Steven A Roberts
The mechanistic understanding of how DNA double-strand breaks (DSB) are repaired is rapidly advancing in part due to the advent of inducible site-specific break model systems as well as the employment of next-generation sequencing (NGS) technologies to sequence repair junctions at high depth. Unfortunately, the sheer volume of data produced by these methods makes it difficult to analyze the structure of repair junctions manually or with other general-purpose software. Here, we describe methods to produce amplicon libraries of DSB repair junctions for sequencing, to map the sequencing reads, and then to use a robust, custom python script, Hi-FiBR, to analyze the sequence structure of mapped reads...
2018: Methods in Enzymology
Thomas Schneider, Geoffrey H Smith, Michael R Rossi, Charles E Hill, Linsheng Zhang
Bioinformatic analysis is an integral and critical part of clinical next-generation sequencing. It is especially challenging for some pipelines to consistently identify insertions and deletions. We present the validation of an open source tumor amplicon pipeline (OTA-pipeline) for clinical next-generation sequencing targeting solid tumor-associated variants. Raw data generated from 557 TruSight Tumor 26 samples as well as in silico data were analyzed by the OTA-pipeline and legacy pipeline and compared. Discrepant results were confirmed by orthogonal methods...
February 19, 2018: Journal of Molecular Diagnostics: JMD
Alexander G Litov, Andrey A Deviatkin, Irina A Goptar, Vladimir G Dedkov, Anatoly P Gmyl, Mikhail L Markelov, German A Shipulin, Galina G Karganova
We studied minor variants within two tick-borne encephalitis virus (TBEV) populations with a common ancestor: the mouse brain-adapted variant EK-328c and the tick-adapted variant M. High-throughput sequencing with custom amplicons from RT-PCR viral RNA was performed on Illumina MiSeq 2*250 paired-end v2 chemistry. Using the LowFreq program (default settings) and Sanger-sequenced consensus as a reference, variants with an abundance of 1 % and above within the studied populations were identified. Using the obtained data in the context of our previous studies, we concluded that TBEV variants, which are different from the major population phenotype and can become a major part of the viral population under favourable environmental conditions, can exist at abundances of less than 1 % in the long-term...
February 2018: Journal of General Virology
George S Krasnov, Nataliya V Melnikova, Valentina A Lakunina, Anastasiya V Snezhkina, Anna V Kudryavtseva, Alexey A Dmitriev
We present MethyMer, a Python-based tool aimed at selecting primers for amplification of complete CpG islands. These regions are difficult in terms of selecting appropriate primers because of their low-complexity, high GC content. Moreover, bisulfite treatment, in fact, leads to the reduction of the 4-letter alphabet (ATGC) to 3-letter one (ATG, except for methylated cytosines), and this also reduces region complexity and increases mispriming potential. MethyMer has a flexible scoring system, which optimizes the balance between various characteristics such as nucleotide composition, thermodynamic features (melting temperature, dimers [Formula: see text]G, etc...
January 30, 2018: Journal of Bioinformatics and Computational Biology
B Hirsch, V Endris, S Lassmann, W Weichert, N Pfarr, P Schirmacher, V Kovaleva, M Werner, I Bonzheim, F Fend, J Sperveslage, K Kaulich, A Zacher, G Reifenberger, K Köhrer, S Stepanow, S Lerke, T Mayr, D E Aust, G Baretton, S Weidner, A Jung, T Kirchner, M L Hansmann, L Burbat, E von der Wall, M Dietel, M Hummel
The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series...
January 27, 2018: Virchows Archiv: An International Journal of Pathology
Garold Fuks, Michael Elgart, Amnon Amir, Amit Zeisel, Peter J Turnbaugh, Yoav Soen, Noam Shental
BACKGROUND: Most of our knowledge about the remarkable microbial diversity on Earth comes from sequencing the 16S rRNA gene. The use of next-generation sequencing methods has increased sample number and sequencing depth, but the read length of the most widely used sequencing platforms today is quite short, requiring the researcher to choose a subset of the gene to sequence (typically 16-33% of the total length). Thus, many bacteria may share the same amplified region, and the resolution of profiling is inherently limited...
January 26, 2018: Microbiome
Silvia Pulignani, Cecilia Vecoli, Andrea Borghini, Ilenia Foffa, Lamia Ait-Alì, Maria Grazia Andreassi
Congenital heart disease (CHD) is a genetically heterogeneous disease. Targeted next-generation sequencing (NGS) offers a unique opportunity to sequence multiple genes at lower cost and effort compared to Sanger sequencing. We tested a targeted NGS of a specific gene panel in a relatively large population of non-syndromic CHD patients. The patient cohort comprised 68 CHD patients (45 males; 8.3 ± 1.7 years). Amplicon libraries for 16 CHD-strictly related genes were generated using a TruSeq® Custom Amplicon kit (Illumina, CA) and sequenced using the Illumina MiSeq platform...
January 13, 2018: Pediatric Cardiology
H Kiyomi Komori, Sarah A LaMere, Traver Hart, Steven R Head, Ali Torkamani, Daniel R Salomon
Many methods exist for examining CpG DNA methylation. However, many of these are qualitative, laborious to apply to a large number of genes simultaneously, or are not easy to target to specific regions of interest. Microdroplet PCR-based bisulfite sequencing allows for quantitative single base resolution analysis of investigator selected regions of interest. Following bisulfite conversion of genomic DNA, targeted microdroplet PCR is conducted with custom primer libraries. Samples are then fragmented, concatenated, and sequenced by high-throughput sequencing...
2018: Methods in Molecular Biology
R S Zimmerman, X Tao, D Marin, M D Werner, K H Hong, A Lonczak, J Landis, D Taylor, Y Zhan, R T Scott, N R Treff
STUDY QUESTION: Can a novel targeted next generation sequencing (tNGS) platform accurately detect whole chromosome aneuploidy in a trophectoderm biopsy and provide additional information to improve testing? SUMMARY ANSWER: Karyotypes obtained by tNGS were concordant with other validated platforms and single nucleotide polymorphism genotyping information obtained can be used for improved detection and quality control. WHAT IS KNOWN ALREADY: qPCR-based whole chromosome aneuploidy screening is highly accurate in comparison to other common methods and has been shown to improve IVF success in two randomized clinical trials...
January 1, 2018: Molecular Human Reproduction
Moses Okee, Alice Bayiyana, Carol Musubika, Moses L Joloba, Fred Ashaba-Katabazi, Bernard Bagaya, Misaki Wayengera
Efficiency of artificial restriction enzymes toward curing HIV has only been separately examined, using differing delivery vehicles. We compared the in vitro transduction and target-mutagenesis efficiency of consortium plasmid and adenoviral vector delivered HIV-1 pol gene targeting zinc finger nuclease (ZFN) with CRISPR/Cas, Custom-ZFN, CRISPR-Cas-9, and plasmids and vectors (murCTSD_pZFN, pGS-U-gRNA, pCMV-Cas-D01A, Ad5-RGD); cell lines (TZM-bl and ACH-2/J-Lat cells); and the latency reversing agents prostratin, suberoylanilide hydroxamic acid, and phorbol myristate acetate...
January 2018: AIDS Research and Human Retroviruses
Linda S Pescatello, Elizabeth D Schifano, Garrett I Ash, Gregory A Panza, Lauren M L Corso, Ming-Hui Chen, Ved Deshpande, Amanda Zaleski, Burak Cilhoroz, Paulo Farinatti, Beth A Taylor, Rachel J O'Neill, Paul D Thompson
In previous studies, we found an endothelial nitric oxide synthase gene ( NOS3 ) variant rs2070744 associated with the ambulatory blood pressure (BP) response following bouts of moderate and vigorous intensity acute exercise, termed post-exercise hypotension (PEH). In a validation cohort, we sequenced NOS3 exons for associations with PEH Obese (30.9 ± 3.6 kg. m-2 ) African American ( n  = 14) [AF] and Caucasian ( n  = 9) adults 42.0 ± 9.8 years with hypertension (139.8 ± 10.4/84.6 ± 6.2 mmHg) performed three random experiments: bouts of vigorous and moderate intensity cycling and control...
November 2017: Physiological Reports
Joanne W Chiu, Monika K Krzyzanowska, Stefano Serra, Jennifer J Knox, Neesha C Dhani, Helen Mackay, David Hedley, Malcolm Moore, Geoffrey Liu, Ronald L Burkes, Christine Brezden-Masley, Michael H Roehrl, Kenneth J Craddock, Ming-Sound Tsao, Tong Zhang, Celeste Yu, Suzanne Kamel-Reid, Lillian L Siu, Philippe L Bedard, Eric X Chen
BACKGROUND: Molecular aberrations in KRAS, NRAS, BRAF, and PIK3CA have been well-described in advanced colorectal cancer. The incidences of other mutations are less known. We report results of molecular profiling of advanced colorectal cancer in an academic cancer center. PATIENTS AND METHODS: Patients with advanced colorectal were enrolled in an institution-wide molecular profiling program. Profiling was performed on formalin-fixed paraffin embedded archival tissues using a customized MassArray panel (23 genes, 279 mutations) or the Illumina MiSeq TruSeq Cancer Panel (48 genes, 212 amplicons, ≥ 500× coverage) in a Clinical Laboratory Improvement Amendments-certified laboratory...
October 23, 2017: Clinical Colorectal Cancer
Daniel E Jasso-Selles, Francesca De Martini, Katalina D Freeman, Mikaela D Garcia, Trevor L Merrell, Rudolf H Scheffrahn, Gillian H Gile
The subterranean termite Heterotermes aureus is endemic to arid regions of southwestern USA and northern Mexico. Like other termites in the family Rhinotermitidae, it harbors a community of protists (Phylum Parabasalia) in its hindgut that aid in cellulose digestion. We investigated the hindgut community of H. aureus using light microscopy, single cell isolation, and high throughput amplicon sequencing. Here we describe four new parabasalid species from the classes Trichonymphea and Spirotrichonymphea. Three of the new species include Pseudotrichonympha aurea (Trichonymphea), Holomastigotoides aureus, and Holomastigotoides oxyrhynchus (Spirotrichonymphea)...
October 2017: European Journal of Protistology
Ann Arfken, Bongkeun Song, Jeff S Bowman, Michael Piehler
The eastern oyster (Crassostrea virginica) is a foundation species providing significant ecosystem services. However, the roles of oyster microbiomes have not been integrated into any of the services, particularly nitrogen removal through denitrification. We investigated the composition and denitrification potential of oyster microbiomes with an approach that combined 16S rRNA gene analysis, metabolic inference, qPCR of the nitrous oxide reductase gene (nosZ), and N2 flux measurements. Microbiomes of the oyster digestive gland, the oyster shell, and sediments adjacent to the oyster reef were examined based on next generation sequencing (NGS) of 16S rRNA gene amplicons...
2017: PloS One
Katherine Angela Benson, Sourabh Chand, Alexander Peter Maxwell, Laura Jane Smyth, Jill Kilner, Richard Borrows, Amy Jayne McKnight
BACKGROUND: Biologically active vitamin D has an important regulatory role within the genome. It binds the vitamin D receptor (VDR) in order to control the expression of a wide range of genes as well as interacting with the epigenome to modify chromatin and methylation status. Vitamin D deficiency is associated with several human diseases including end-stage renal disease. METHODS: This article describes the design and testing of a custom, targeted next generation sequencing (NGS) panel for selected vitamin D associated genes...
July 28, 2017: BMC Research Notes
Eric Konnick, Christina M Lockwood, David Wu
Mutation profiling of acute leukemias is a valuable tool for identifying genetic mutations with prognostic, predictive, therapeutic, and diagnostic utility. Technological advances, such as massively parallel sequencing, have allowed laboratories to assess for variation across dozens or hundreds of genes simultaneously with relatively low cost per target.Here, we describe a procedure for designing and using a TruSeq Custom Amplicon assay targeting genes involved in acute leukemias. This method is a fully customizable, amplicon-based assay for targeted resequencing, allowing interrogation of selected genomic regions of interest...
2017: Methods in Molecular Biology
Eric J de Muinck, Pål Trosvik, Gregor D Gilfillan, Johannes R Hov, Arvind Y M Sundaram
BACKGROUND: Advances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized. METHODS: We present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods...
July 6, 2017: Microbiome
Dario Kringel, Marco Sisignano, Sebastian Zinn, Jörn Lötsch
BACKGROUND: Transient receptor potential cation channel subfamily V member 1 (TRPV1) are sensitive to heat, capsaicin, pungent chemicals and other noxious stimuli. They play important roles in the pain pathway where in concert with proinflammatory factors such as leukotrienes they mediate sensitization and hyperalgesia. TRPV1 is the target of several novel analgesics drugs under development and therefore, TRPV1 genetic variants might represent promising candidates for pharmacogenetic modulators of drug effects...
2017: PloS One
Jacquelyn A DuVall, Delphine Le Roux, Brandon L Thompson, Christopher Birch, Daniel A Nelson, Jingyi Li, Daniel L Mills, An-Chi Tsuei, Martin G Ensenberger, Cindy Sprecher, Douglas R Storts, Brian E Root, James P Landers
Forensic DNA analysis requires several steps, including DNA extraction, PCR amplification, and separation of PCR fragments. Intuitively, there are numerous situations where it would be beneficial to speed up the overall DNA analysis process; in this work, we focus on the most time-consuming component in the analysis pipeline, namely the polymerase chain reaction (PCR). Primers were specially designed to target 10 human genomic loci, all yielding amplicons shorter than 350 bases, for ease of downstream integration with on-board microchip electrophoresis...
August 8, 2017: Analytica Chimica Acta
Sten Anslan, Mohammad Bahram, Indrek Hiiesalu, Leho Tedersoo
High-throughput sequencing methods have become a routine analysis tool in environmental sciences as well as in public and private sector. These methods provide vast amount of data, which need to be analysed in several steps. Although the bioinformatics may be applied using several public tools, many analytical pipelines allow too few options for the optimal analysis for more complicated or customized designs. Here, we introduce PipeCraft, a flexible and handy bioinformatics pipeline with a user-friendly graphical interface that links several public tools for analysing amplicon sequencing data...
May 25, 2017: Molecular Ecology Resources
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