Critical Role for the DNA Sensor AIM2 in Stem Cell Proliferation and Cancer

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Researchers from StJude, the UTHSC and Gannan Medical University recently published research in Cell describing how therapeutic modulation of the expression of AIM2, an innate immune sensor frequently identified in patients with colorectal cancer, affects microbiota and has the potential to prevent colorectal cancer (Man, 2015).

Read more to learn how they used the NEXTflex™ 16S V4 Amplicon-Seq Kit to analyze the effect of AIM2 expression of microbiota and how this in turn affects susceptibility to tumor development.

Man, S. M. et al. (2015) Critical Role for the DNA Sensor AIM2 in Stem Cell Proliferation and Cancer. Cell. doi:10.1016/j.cell.2015.06.001.

Read more here: http://www.cell.com/cell/abstract/S0092-8674(15)00684-4

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Maximize Your Library Diversity with Enhanced Adapter Ligation Technology

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Highly efficient adapter ligation is the key to success for most NGS library preps. Robust, unbiased ligation is necessary to minimize loss in overall coverage during sequencing. Only “doubly-ligated” target DNA fragments (i.e. those containing adapters ligated at both the 5’ and 3’ ends) can serve as templates for Illumina sequencing, so maximizing the yield of doubly-ligated product is essential for maximizing efficiency of library production.

Increasing ligation efficiency maximizes library diversity in libraries constructed with both low and high amounts of sample input. Highly diverse libraries require less amplification for sequencing, resulting in lower duplication rates and improved coverage.

The scientists at Bioo Scientific have carefully optimized the ligation reaction step in NGS library prep to allow for the construction of the most diverse libraries possible with both low and high amounts of sample input.

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A comparison of the NEXTflex Rapid DNA-Seq Kit to a competitor’s kit by analysis of proportions of ligation products. Red portions of the graph indicate non-ligated product proportion; yellow, singly-ligated; green, the desired doubly-ligated amplicon.

All of the NEXTflex Library Prep Kits incorporate Bioo Scientific’s proprietary Enhanced Adapter Ligation Technology into the adapter ligation reactions to improve library diversity.

Other tips for increasing ligation efficiency:

  • Do not heat Illumina-compatible adapters above room temperature. They are constructed from two oligos which have been annealed together. Heating can cause the adapter to denature.
  • Use the recommended adapter concentration dilutions for various input amounts. Using an excessive amount of adapters can result in an undesired increase in adapter dimers which will generate clusters that can be sequenced, and will reduce the total proportion of desired sequence obtained from a run.

Contact us at nexgen@biooscientific.com if you have any questions about how improved ligation efficiency can improve your NGS analysis.

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Featured Publication – CGGBP1 Mitigates Cytosine Methylation at Repetitive DNA Sequences

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Dr. Singh and others from Uppsala University and EMBL recently reported that CGGBP1 is important for regulation of DNA methylation. CGGBP1 is a repetitive DNA-binding transcription regulator with target sites at CpG-rich sequences such as CGG repeats and Alu-SINEs and L1-LINEs.

Dr. Singh et al. used the NEXTflex Bisulfite-Seq Kit and NEXTflex Bisulfite Barcodes to construct Illumina-compatible libraries for genome-wide CpG methylation analysis of DNA isolated from 1064Sk normal human foreskin fibroblasts with and without CGGBP1-depletion. With this analysis they identified CGGBP1 to be a negative regulator of CpG methylation at repetitive DNA sequences.

Apart from histone-modifying proteins HDACs and HMTs and pRB, CGGBP1 is the first factor described to have negative effects on cytosine methylation, and is important for regulation of DNA methylation. This has implications on silencing of Alu and LINE-1 repeats, heterochromatin formation on simple and satellite repeats, and hence on genome integrity and function.

Read the entire publication: Agarwal, P., et al. (2015) CGGBP1 mitigates cytosine methylation at repetitive DNA sequences. BMC Genomics, 16:390. doi:10.1186/s12864-015-1593-2.

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Automate NEXTflex™ DNA-Seq and RNA-Seq Library Prep on the Sciclone® NGS and NGSx Workstations

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Optimized automation protocols for the NEXTflex™ library preparation kits are now available for the Sciclone® NGS and NGSx Workstations. These protocols are designed to facilitate higher sample throughput, less hands-on time, greater reproducibility and improved process control.

The NEXTflex™ DNA and RNA Library Preparation Kits are optimized for automated, high-throughput NGS library construction and offer up to 96 single-index barcodes for deep multiplexing. In addition to automation-friendly protocols, these library preparation kits contain the necessary overages required for automated liquid handling platforms.

Download Sciclone NGS and NGSx Workstation Automation Guides today:
NEXTflex Rapid Directional qRNA-Seq Kit
NEXTflex Rapid Directional RNA-Seq Kit
NEXTflex Rapid RNA-Seq Kit
NEXTflex Rapid DNA-Seq Kit

If you have any questions about automating these NEXTflex Library Prep Kits or any of the other NGS kits we offer on your Sciclone NGS or NGSx Workstation, contact a Bioo Scientific automation specialist today.

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Additional methods for other kits and platforms are continually under development.

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Tech Tips: Installing Barcode Indices in Illumina Experiment Manager and Generating Sample Sheets for Sequencing Runs with More Than 96 Samples in a Single Sheet

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Since the NEXTflex™ adapter indices are not included by default with Illumina Experiment Manager, specific index information needs to be added to the Illumina Experiment Manager data source. To simplify your Illumina NGS sequencing, Bioo Scientific has posted instructions describing how to install these indices. Files containing the index sequences needed for installation are also included.

Read more here.

Now that Illumina Experiment Manager (IEM) v1.9 supports sample sheet generation for sequencing runs with more than 96 samples in a single sheet, Bioo Scientific has also created instructions describing how to easily generate sample sheets with more than 96 samples.

Read more here

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Featured Publication – Activating MET Kinase Rearrangements in Melanoma and Spitz Tumours

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Dr. Yeh from the University of California, San Francisco, along with others from this university and Centre Léon Bérard, recently published research in Nature Communications, in which they identified six different melanocytic tumors with genomic rearrangements of MET, fusing the kinase domain of MET in-frame to six different N-terminal partners, using the NEXTflex™ Pre-Capture Combo Kit to make DNA-Seq libraries for upstream target capture using a SeqCap EZ Choice Custom Exome Bait Panel.

Read the entire article here: http://www.nature.com/ncomms/2015/150527/ncomms8174/full/ncomms8174.html

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Featured Publication – Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement

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Dr. Goodarzi and others from Rockefeller University recently published their research in Cell describing a novel class of tRFs derived from tRNAGlu, tRNAAsp, tRNAGly, and tRNATyr that, upon induction, suppress the stability of multiple oncogenic transcripts in breast cancer cells by displacing their 3′ untranslated regions (UTRs) from the RNA-binding protein YBX1. Using the NEXTflex™ Small RNA Sequencing Kit v2 to measure small RNA levels under normal and hypoxic stress conditions, they identified a group of tRFs that were upregulated under hypoxia in breast cancer cells as well as in non-transformed mammary epithelial cells.

Goodarzi, H., et al. (2015) Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement. Cell. 161:4, p790–802.

The entire article can be read here: http://www.cell.com/cms/attachment/2031254416/2048399252/mmc1.pdf

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Featured Publication – Plasma Genetic and Genomic Abnormalities Predict Treatment Response and Clinical Outcome in Advanced Prostate Cancer

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Liquid biopsies have shown promise for predicting clinical outcomes. Shu Xia, from the Huazhong University of Science and Technology and the Medical College of Wisconsin, and others recently published their research evaluating tumor-associated genomic and genetic variations in plasma cell-free DNA (cfDNA) and their associations with treatment response and overall patient survival.

Drs. Xia and others used the NEXTflex DNA Sequencing Kit and NEXTflex DNA Barcodes to construct libraries from cell free DNA obtained from 20 patients with advanced prostate cancer for whole genome and targeted sequencing. Read our latest featured publication to learn more about their research.

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Slideshow – Simplify and Reduce Cost of mtDNA Isolation and Library Prep

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Mutations in mitochondrial DNA (mtDNA) have been implicated in various human disorders and in aging, making NGS analysis of mtDNA a priority for a number of labs. However, accurately determining the diversity of mtDNA has been difficult for a number of reasons. The standard methods for mitochondrial DNA extraction have a number of limitations making them inferior solutions for NGS library preparation. Bioo Scientific has commercialized a kit which overcomes these limitations of mtDNA isolation by selectively digesting linear nuclear DNA (nDNA) while leaving circular mtDNA intact. This technology has been incorporated into the NEXTflex mtDNA-Seq Kit which includes optimized reagents for the isolation of mtDNA and for the construction of Illumina mtDNA libraries. Libraries constructed using the NEXTflex mtDNA-Seq Kit are ideal for many NGS applications including heteroplasmy analysis.


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Simplify and Reduce Cost of mtDNA Sequencing

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The NEXTflex™ mtDNA-Seq Kit includes optimized reagents for the isolation of mitochondrial (mtDNA) and construction of Illumina-compatible paired-end or single read libraries from genomic DNA. The NEXTflex mtDNA-Seq Kit enables selective enrichment of mitochondrial DNA from total genomic DNA samples by selectively digesting linear nuclear DNA (nDNA) while leaving circular mtDNA intact; enriching the mitochondrial genome 100 – 350 times. After enrichment libraries prepared using the NEXTflex mtDNA-Seq Kit produce a greater number of unique reads reducing the number of sequencing reads required per sample.

 

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Figure 1. Enrichment of mtDNA reads in samples prepared using the NEXTflex mtDNA-Seq Kit. Triplicate libraries were made from mtDNA isolated using the NEXTflex mtDNA-Seq Kit (red) or untreated gDNA (purple). mtDNA was isolated from 4 µg blood or A549 gDNA. The bars represent mean and standard deviation.

Learn more about the NEXTflex mtDNA-Seq Kit.

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