Bioflorida Chapter Meeting Jupiter 2008v2
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EcoArray provides various services related to gene discovery, microarray development, and bioinformatics including: 1) cDNA library construction for identifying expressed genes and differentially expressed genes between populations. 2) Custom gene array construction and hybridizations for species without commercially available arrays. 3) RT-PCR for validating gene expression changes between controls and treated samples. 4) Bioinformatics services such as gene sequencing, annotation, and statistical analysis of microarray data.
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Automated fluorescent dye-terminator DNA Sequencing using capillary electrophoresis (also known as CE or Sanger sequencing) has been instrumental in the detailed characterization of the human genome and is now widely used as gold standard method for verification of mutation findings, notably in tumor samples. The primary information of the DNA sequencing process is the identification of the nucleotides and of possible sequence variants. A largely unexplored feature of fluorescent Sanger sequencing traces is the quantitative information embedded therein. With the growing need for quantifying somatic mutations in tumor tissue it is desirable to exploit the potential of the quantitative information obtained from sequencing traces.
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To this end, we have developed a software tool that converts a Sanger sequencing trace file into a .comma separated value (.csv) file containing numerical data of peak data characteristics that can be explored and analyzed using conventional spreadsheet software. The web-based tool can be accessed at: http://apps.lifetechnologies.com/ab1peakreporter .
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Sanger sequencing using fluorescent BigDye® terminator chemistry and semi‐automated capillary electrophoresis (CE) has long been considered the gold standard for identifying sequence variations such as disease‐causing mutations. The robustness, low error rate, ease of use, human interpretable visual displays of the signals generated by the instruments, and low cost per sample and target have all contributed to this reputation. Homozygous and heterozygous germ line mutations are reliably detected and reported using established DNA sequencing analysis software such as the Applied Biosystems Variant Reporter™ software. However, somatic variants with an allelic proportion of 25% or less are often undetected (i.e. not "called") by the software and thus escape awareness if not detected by careful visual inspection of the electropherograms. With the rapid adoption of next generation sequencing technology (NGS) and its use for characterization of specific and discrete mutations in tumor samples, an urgent need has emerged to establish an orthogonal technology for reliable and sensitive detection of somatic mutations which may occur at proportions of 10% or lower compared to the normal allele.
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Materials and Methods
To this end, we have developed a software tool that converts a Sanger sequencing trace file into a .comma separated value (.csv) file containing numerical data of peak data characteristics that can be explored and analyzed using conventional spreadsheet software. The web-based tool can be accessed at: http://apps.lifetechnologies.com/ab1peakreporter .
The output file contains the peak height and quality values for each nucleotide and peak height ratios for all 4 bases at any given locus allowing the detection and assessment of subtle changes at any given allele.
Results and Discussion
We demonstrate the utility of this tool by analyzing mixed DNA samples with known amounts of spiked in variant alleles from the human TP53 gene ranging from 2.5%, 5%, 7.5%, 10%, 15% and 25% and show that the minor alleles could be readily detected below the 10% level.
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What is the impact of assay failure in your laboratory and how do you monitor for it?
The most heavily degraded samples are not suitable for standard exome coverage: sometimes it’s not even a matter of getting bad sequencing, you might get nothing at all!
FFPE artifacts increase with storage time
Artifacts go against the statistical power of your variant calling analysis
Molecular reference standards help filter out bad mappings and spurious variants
Bioinformatics pipelines allow adding Molecular Reference Standards in your joint variant calling pipeline
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NIST and its collaborators shared datasets created with most NGS technologies
Horizon Diagnostics shared annotated, merged variant calls from NIST for the Ashkenazim Trio
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GM24385 (Ashkenazim Son) includes 352 small variants with high/moderate impact which are absent in Father and Mother
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SteilLinkedInResume2015
Benjamin P. Steil has over 15 years of experience in virology research. He received his Ph.D. in Microbiology and Immunology from the University of Colorado Denver in 2008, and has since worked as a postdoctoral fellow and staff scientist developing viral vaccine adjuvants. His skills include viral particle production, nucleic acid techniques, mouse models, and immunological assays. Currently he is seeking a position where he can apply his expertise in vaccine design and development.
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Horizon is the only source of rAAV expertise and is uniquely capable of exploiting multiple platforms: CRISPR, ZFNs and rAAV singularly or combined. Horizon’s scientists are experts at all forms of gene editing and so have the experience to help guide customers towards the approach that best suits their project
Next generation sequencing & microarray-- Genotypic Technology
Greetings from Genotypic Technology, Bangalore (www.genotypic.co.in). We are a 13 year old genomics and bioinformatics company ( 65+ employees, Service. Products and R & D) based in Bangalore, India, primarily working on applications of Microarrays and Next Generation Sequencing in Human Health and Disease, including in Molecular Diagnostics, Prognosis, Therapeutics, Vaccine Research, Basic Science Research, Veterinary Science, Agriculture, Industrial Biotechnology, Microbial Genetics and more.
Our major strength is in customized genomics solutions, particularly in your field, we can develop panel of markers for specific diseases, optimize, validate and help commercialize on open platforms or specific instrument platforms- in microarrays and sequencing based methods/ assays. We can also use genomic markers to aid in treatment of specific disease using personalized medicine approaches. All this can be done on a comprehensive end-to-end manner in our company as we have a very good blend of molecular biology and bioinformatics with totally 6 Ph.Ds. We work closely with Agilent's R &D as their partner.
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Mason abrf single_cell_2017
1. Single-cell RNA sequencing was performed on hematopoietic stem cells isolated from myelodysplastic syndrome patients and normal individuals to characterize heterogeneity. Cells were collected before and after treatment with decitabine from responders and non-responders.
2. Differential expression analysis identified genes dysregulated in MDS compared to normal, including pathways involved in hematopoiesis. Clusters of patients were identified based on expression of hematopoietic stem cell signature genes.
3. The study aims to understand heterogeneity in MDS, factors influencing response to therapy, and disease progression by characterizing gene expression profiles at the single-cell level. This may help identify new therapeutic targets.
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2013 oct 2 rna sequencing
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Enabling RNA-Seq With Limited RNA Using Whole Transcriptome Amplification
RNA-Seq was developed to perform transcriptome profiling and provides a highly precise measurement of expression levels of transcripts and their isoforms. Normally, RNA-Seq analysis requires at least 500 ng –1 μg of total RNA. When working with small biopsies, single cells (such as circulating tumor cells), or other limited material, whole transcriptome amplification (WTA) is normally required. Various WTA methods overcome limited RNA availability and enable transcriptome analysis from limited material or even single cells. In standard PCR-based WTA procedures, however, bias from uneven coverage of cDNA regions with high GC or AT content or amplification errors can lead to the loss of transcripts and wrong variant calling. Here, we compare a standard RNA-Seq library preparation method and the REPLI-g RNA library protocol. The REPLI-g procedure is a PCR-free protocol to efficiently generate RNA-Seq libraries from small amounts of RNA or a single cell in 6.5–7 hours. The REPLI-g protocol uses whole transcriptome amplification based on multiple displacement amplification (MDA), combined with an efficient library adaptor ligation procedure, to prepare RNA-Seq libraries from small RNA amounts. The procedure demonstrates high fidelity, minimal bias and retention of sample‘s transcriptional profile. Compared to standard RNA-Seq library prep, the REPLI-g protocol demonstrates similar reproducibility and sensitivity in transcript detection.
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WHAT IS DNA SEQUENCER ?
DNA SEQUENCING.
HISTORY
MANUFACTURERES OF DNA SEQUENCER.
OTHER APPLICATIONS
CONCLUSION
REFERENCES
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