Diet Dependent Microbiome Changes Protect Against Disease

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Lukens, Gurung, Vogel, et. al. have recently uncovered diet-associated changes in the intestinal microbiome of mouse models as a significant factor regulating IL-1β maturation and the development of osteomyelitis. The researchers used the NEXTflex™ 16S V4 Amplicon-Seq Kit to characterize the organisms comprising the microbiomes of Pstpip2cmo mice that were fed a diet rich in fat and cholesterol. This diet was found to reduce the population of Prevotella bacteria in the intestines of the mice, and reduce pro-IL-1β expression in neutrophils. Pstpip2cmo mice treated with antibiotics, and wild-type mice raised in germ-free conditions also experienced protection from IL-1β-dependent osteomyelitis.

Read the article at Nature, here.

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Visit Bioo Scientific at ASHG 2014

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Stop by Bioo Scientific’s booth (#939) at the ASHG meeting in San Diego,CA to learn about the latest advances in NGS library prep. Learn how to reduce bias in small RNA-seq library prep, how to improve your Illumina RNA-Seq and ChIP-Seq quantitation and how to increase your library prep multiplexing capabilities.

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Optimized Library Prep for Cell-Free DNA from Human Plasma

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Researchers and clinicians are increasingly interested in using Next Generation Sequencing (NGS) analysis of cell-free DNA (cfDNA) found in plasma (the cell-free fraction of anticoagulated blood) for biomarker discovery and diagnostic applications. Two areas of specific interest are non-invasive prenatal diagnostics (1-6) and monitoring efficacy of treatment in cancer patients (7-20). Many recent studies have shown the feasibility of detecting fetal aneuploidies such as Chromosome 21 trisomy (the cause of Down’s Syndrome) by shotgun sequencing of DNA-Seq libraries produced from cell-free DNA isolated from maternal blood. Other aneuploidies including trisomies of Chromosomes 13 and 18 have also been detected (3). This approach is attractive since it avoids the risk of miscarriage associated with invasive tests (amniocentesis and chorionic villi sampling) and may offer cost benefits for prenatal diagnosis. Also, NGS- based assessment has the potential to uncover defects arising from more subtle genetic alterations including point mutations, insertion/ deletion mutations, translocations, etc. Although intact fetal cells can be detected in maternal blood, they are vastly outnumbered by the mother’s blood cells, and the proportional concentration of informative fetal DNA sequences has been reported to be higher in cell-free circulating DNA in plasma compared to DNA recovered from whole blood. The same findings hold true in the case of cancer diagnostics, where the genetic signal from rare circulating tumor cells is harder to discern against the background of non-malignant cells in whole blood, compared to circulating cell-free DNA. Assessing cancer-related genetic alterations in cell-free DNA can also avoid positional bias inherent in direct sampling of tumors, where the spectrum of mutations observed can differ for different biopsy locations within the malignant tissue. The concept of “liquid biopsy” refers to using readily obtainable body fluids, primarily blood and blood fractions, as surrogate tissue for monitoring levels of malignancy-associated mutations that arise from tumor cells and cell-free DNA shed from tumors (18 – 20). Monitoring levels of cancer-associated genetic alterations in cell-free DNA in blood plasma is a promising new approach to assess the benefits of chemotherapy and other types of cancer treatment. Liquid biopsy of circulating cell-free DNA also has potential for early detection of cancer, and for stratifying cancer patients for treatment decisions. To realize the full potential of these promising opportunities, robust and standardized methods are needed for creating DNA-Seq libraries from cell-free DNA extracted from plasma. To meet this need, Bioo Scientific has developed the NEXTflex™ Cell Free DNA-Seq Kit designed for making NGS libraries from low-input samples. Below, we report results demonstrating the use of this kit to produce high-quality informative libraries from cell-free DNA extracted from plasma. Continue reading…

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Simplified Demultiplexing of Molecular Indexed Reads

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Typically PCR duplicates are eliminated from RNA-Seq data using unique start and stop (USS) site analysis. However, many original fragments have identical start and stop sites and can incorrectly eliminate unique molecules from NGS data using USS analysis alone. To overcome this issue and offer more accurate mRNA analysis, Bioo Scientific incorporated Molecular Indexes™ into the NEXTflex™ qRNA-Seq™ and NEXTflex™ Rapid Directional qRNA-Seq™ library prep kits. Molecular Indexes independently and randomly ligate to each end of a DNA fragment prior to the PCR step of library prep. This allows clonal duplicates made during PCR amplification to be identified during sequence analysis. These Molecular Indexes can be applied either in combination with the USS method or alone, to eliminate duplicates and to correctly detect somatic mutations. Bioo Scientific now has a free computer program available to help researchers simplify this analysis.

Learn more …

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Best Practices for Magnetic Bead Based Cleanups

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The use of magnetic beads for DNA purification and size-selection offers a quick and high yield alternative compared to using column-based cleanups or gel purifications. We have compiled a number of suggestions to improve results for all four parts of magnetic bead based cleanups: adding beads, washing, resuspending and elution.

Adding Beads to Your Sample

  • Add the exact volume of beads that is listed in your protocol. Magnetic beads have high DNA binding capacity and adding more beads does not lead to better performance.
  • The magnetic bead solution is viscous, so care must be taken to avoid beads sticking to the outside of the pipette tip, which will alter the volume that is transferred to your sample.
  • If beads remain on the outside of the pipette tip, drag it against the inside edge of the bead container to remove them before adding the beads to the sample.
  • Use low-retention pipette tips when possible.
  • After the addition of beads, the solution should be mixed until it is completely homogenous.

Washing

  • This step is performed with 70 to 80% ethanol. Using ethanol that is too dilute or too concentrated can negatively affect the yield.
  • When adding and removing ethanol, be careful to not disturb the bead pellet.
  • Make sure that all residual ethanol is removed before proceeding to the next step. To help accomplish this, after the first removal, allow residual ethanol to pool in the bottom of the well and remove this ethanol as well.

Resuspending 

  • When resuspending in low volumes it may be necessary to pipette the resuspension solution directly on to the bead pellet.
  • Over-drying the bead pellet can cause difficulty for resuspension.

Elution

  • Take care to avoid any bead carryover when transferring the sample.
  • When eluting a sample, avoid touching the bead pellet with the pipette tip by resting the tip on the opposite side of the well from the bead.
  • Pull up the sample slowly to avoid disturbing the bead pellet
  • If using a PCR plate, the used row of the plate can be cut off in order to allow the new well to be in front. When cutting the plate, prevent cross-contamination by sealing the rows containing the samples.

Do you have any questions about magnetic bead based cleanups? If so, email us at BiooNGS@biooscientific.com and we would be happy to help you.

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Use of RF and TRAP-RF Ribo-seq libraries for Precise Mapping of Ribosome Footprints to Study Translational Regulation in Arabidopsis thaliana

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Piyada Juntawong, Thomas Girke, Jérémie Bazin, and Julia Bailey-Serres mapped ribosome footprints on Arabidopsis thaliana mRNAs to examine translational regulation under hypoxic stress conditions. RF and TRAP-RF Ribo-seq libraries were used to determine ribosome footprint number and position, in order to characterize posttranscriptional and translational control under normoxic and hypoxic conditions. Hypoxic conditions were found to produce a global decline in initiation of translation. In polysome complexes, hypoxia-up-regulated gene transcripts increased, but the number of ribosomes per transcript was not increased. Upstream Open Reading Frames (ORFs) were also found to have inhibitory effects on the translation of downstream protein-coding regions under both conditions. The researchers used the NEXTflex Small RNA Sequencing Kit and NEXTflex Small RNA Barcodes to prepare RF and TRAP-RF Ribo-seq libraries for this experiment. Read the full paper here.

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Installing the NEXTflex™ Barcode Indices in Illumina Experiment Manager

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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 indexes sequences needed for installation are also included. Read more here: http://bit.ly/1ohw5fg

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Increase Your Bisulfite-Seq Multiplexing Capabilities

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NEXTflex™ Bisulfite-Seq Barcodes are now available in 24 barcodes for higher throughput multiplexing of Illumina-compatible libraries, an improvement over the previous multiplexing capability of 12 barcodes. This represents the largest set of methylated adapters available separately for reduced representation bisulfite sequencing (RRBS) and whole-genome bisulfite sequencing (WGBS).

Additionally, all of the NEXTflex Bisulfite-Seq Barcodes now offer greater flexibility in low-plex multiplexing.

The NEXTflex Bisulfite-Seq Barcodes were developed to be used in conjunction with the NEXTflex Bisulfite-Seq Kit, which offers a complete and versatile kit designed to facilitate assessment of the methylation state of the genome and simplify library prep workflow by reducing the number of time-consuming steps in library preparation. However, NEXTflex Bisulfite-Seq Barcodes can be used with other Illumina-compatible library preparation protocols.

Increase your bisulfite-seq multiplexing capabilities with the NEXTflex Bisulfite-Seq Barcodes.

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Fighting to Reduce Small RNA-Seq Bias

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Today Bioo Scientific launched the NEXTflex™ Small RNA Sequencing Kit v2 which provides an easy, flexible, cost-effective solution for generating libraries with reduced bias from small RNA using Illumina sequencing platforms.

More Accurate Results with Randomized Adapters

Bioo Scientific’s proprietary approach to overcoming ligation bias in small RNA-seq libraries involves using a pool of adapters having randomized sequences at the ligation site, where each adapter sequence is present in vast molar excess over any given small RNA in the sample. Most of the bias in adapter ligation is due to the sequence of the nucleotides adjacent to the target junction. No single adapter sequence is able to efficiently ligate to all small RNAs, indicating that the target sequence, as well as the adapter sequence, is a source of bias. The new NEXTflex Small RNA Sequencing Kit v2 uses a randomized adapter strategy to allow small RNAs of any sequence to “find” their corresponding optimal adapters, resulting in small RNA-seq libraries that show a dramatic reduction in bias.

Learn More

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The Power of Intelligent Barcoding

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The ability to multiplex samples accurately is a critical aspect of next-generation sequencing. However, polymerases used during sequencing can introduce errors which can prevent proper binning, wasting the associated sequencing reads. To prevent this, it is necessary to build error correction into the barcodes.  Bioo Scientific’s NEXTflex™ 16S V1 – V3 Amplicon-Seq Kit includes barcoded adapters containing a 12 nt index that allows for up to two error corrections and has multiple low-diversity pooling options to prevent barcode read errors.

Continue reading…

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