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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Designing Your ChIP-seq Experiments: The Data is in the Details

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One question that many people have when transitioning to NGS for their chromatin immunoprecipitation (ChIP) experiments is what parameters need to be considered in the experimental design phase in order to produce optimal data quality. The good news is that ChIP-seq has become a widely used assay and multiple resources are available to assist in nearly every step of the process. Here we will discuss several frequently asked questions and important details that should be considered in the experimental design, sequencing, and analysis of ChIP-seq projects. Continue reading…

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16S rRNA Amplicon Sequencing Offers Enhanced Metagenomic Detection

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Using 16S ribosomal RNA (rRNA) gene sequencing to identify and characterize microbial populations has greatly increased our understanding of the role microbes play in environmental, agricultural and health-related settings. The 16S rRNA gene is comprised of highly conserved regions flanking nine hyper-variable regions, which are ubiquitous in bacterial species. The following data illustrates how the NEXTflex™ 16S V1-V3 Amplicon-Seq Kit, combined with the Illumina MiSeq, provides users with a time-efficient and robust method to study bacterial metagenomics, using any sample from which DNA can be isolated.

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Optimized Library Prep for 16S V1 – V3 rRNA Sequencing for Improved Bacterial Metagenomics Studies

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Bioo Scientific recently launched the NEXTflex™ 16S V1 – V3 Amplicon-Seq Kit for bacterial metagenomics analysis. This kit facilitates the preparation of multiplexed amplicon libraries that span the V1-V3 hypervariable regions of bacterial 16S ribosomal RNA (rRNA) genes, producing multiplexed libraries compatible with paired-end sequencing on the MiSeq Illumina® sequencing platform. The NEXTflex 16S V1 – V3 Amplicon-Seq Kit is ideal for bacterial diversity studies, studies of interactions between host species and bacterial communities, identification of non-culturable bacteria, detection of adventitious agents and enzyme discovery and production. With the ability to multiplex up to 384 samples, the NEXTflex 16S V1 – V3 Amplicon-Seq Kit is optimized to simplify and improve your bacterial metagenomics analysis.

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The Role of Circulating Tumor DNA Analysis in Early Detection and Treatment of Cancer

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The existence of extracellular cell free DNA (cfDNA) was detected in blood as early as 1948. In 1977 it was documented for the first time that levels of cell free tumor DNA (cftDNA) were significantly higher in cancer patients than in healthy persons. In the last decade, human whole genome sequencing has revealed the relationship between somatic mutations in oncogenes, tumor suppression genes, and cancer development. This led to the rediscovery of cftDNA as a valuable resource for diagnosis, prognosis, treatment decisions, and follow-up monitoring of cancer patients. Next generation sequencing has led to a number of interesting findings that could pave the way for doctors to use less invasive techniques for cancer detection, offering actionable diagnostic information at an early stage of cancer development.

In February of 2014, a group from Johns Hopkins presented a comprehensive report in Science Translational Medicine which moved the application of ctDNA analysis as a cancer diagnostic to a new level (1). This study analyzed ctDNA from 640 patients with 18 cancer types at four clinical stages, and was able to present several interesting conclusions:

 

  • ctDNA could be detected in more than 50% of localized tumors and more than 75% of advanced tumors. In most tumor types, levels of ctDNA correlated with the stage of cancer.
  • Detection of cancer based on ctDNA analysis is more sensitive than analysis of circulating tumor cells (CTC). ctDNA and associated mutations were always detected at higher levels in blood of cancer patients, and could even be detected when CTC were absent. This observation suggests that ctDNA does not originate from CTC.
  • ctDNA and localized tumors had the same mutations and/or rearrangements, confirming that most ctDNA is shed to the bloodstream by tumor cells.
  • It is possible to predict the resistance to EGFR blockage treatment of colon cancer by acquisition of new mutations in KRAS, BRAF, NRAS and EGFR, which could help tailor personal treatment during disease progression.
  • 47% of patients with stage I disease had detectable ctDNA in plasma.

 

The most promising finding from this research was that mutations found in ctDNA could be used to detect and treat colon cancer during stage I growth. However, many problems have to be solved before a ctDNA test can be used for routine clinical diagnosis. Currently, it is difficult to determine the origin and location of a tumor from analysis of ctDNA in the blood of an asymptomatic person, because the majority of known mutations occur in many different types of tumors. Moreover, as few as 50 million malignant cells can produce detectable amounts of cftDNA in blood (2), which is far below the detection limit of current imaging methods. Hopefully, the results presented will encourage more prospective studies using analysis of cftDNA by next generation sequencing to produce highly informative data, which will help narrow the suspected origin of mutated ctDNA and allow intervention prior to the progression of disease.

In conclusion, this report validates ctDNA analysis for tumor diagnosis as a promising, non-invasive method for screening, diagnosis, treatment decisions and monitoring of human cancer. The spread of cftDNA analysis promises to generate further information for broad implementation into clinical applications.

Bioo Scientific developed the NEXTflex™ Cell Free DNA-Seq Kit to facilitate the analysis of cfDNA for studies such as these. The NEXTflex Cell Free DNA-Seq Kit has been optimized for the construction of DNA-seq libraries from cell-free fetal or circulating tumor DNA. With a short, two hour DNA library prep protocol, this kit can be used to prepare single, paired-end and multiplexed DNA libraries for sequencing using Illumina® platforms. The NEXTflex™ 1-step End-Repair and Adenylation protocol simplifies workflow and shortens hands-on library construction time. In addition, the availability of up to 192 unique adapter barcodes facilitates high-throughput applications.

 

(1)    Bettegowda C. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 2014; 6: 224a24.

(2)    Diaz LA Jr. et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancer. Nature 2012; 486:537-540.

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