Gel-Free or Low Input Small RNA Library Prep

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The new NEXTflex™ Small RNA-Seq Kit v3 is improving small RNA analysis by making library prep completely gel-free or allowing for low-input small RNA library preparation. This kit also reduces the biases associated with small RNA sequencing by using patent pending randomized adapter technology, resulting in small RNA libraries with more accurate sequencing data and a dramatic reduction in bias.

Read more to learn about how the NEXTflex™ Small RNA-Seq Kit v3 can improve your small RNA library prep.


Featured Publication: The Genetic Evolution of Melanoma from Precursor Lesions

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Dr. Shain and others from the University of California, San Francisco, Cleveland Clinic, Cleveland, Orlando Health, University Hospital of Zurich, Dorset County Hospital and St. John’s Institute of Dermatology recently published research entitled The Genetic Evolution of Melanoma from Precursor Lesions in The New England Journal of Medicine (Shain, 2015).

These researchers used the NEXTflex™ Rapid DNA-Seq Kit and NEXTflex™ DNA Barcodes to prepare libraries for Illumina sequencing from 25 to 250 ng of DNA isolated from 37 FFPE samples of primary melanomas and their adjacent precursor lesions to determine the order of the occurrence of pathogenic mutations in melanoma. Using targeted sequencing, 293 cancer-relevant genes in 150 areas were sequenced from each melanoma and lesion. The histopathological spectrum of the melanomas and their adjacent lesions included unequivocally benign lesions, intermediate lesions, and intraepidermal or invasive melanomas.

This sequencing revealed that precursor lesions were initiated by mutations of genes that are known to activate the mitogen-activated protein kinase pathway. Unequivocally benign lesions harbored BRAF V600E mutations exclusively, whereas those categorized as intermediate were enriched for NRAS mutations and additional driver mutations. A total of 77% of areas of intermediate lesions and melanomas in situ harbored TERT promoter mutations, a finding that indicates that these mutations are selected at an unexpectedly early stage of the neoplastic progression. Biallelic inactivation of CDKN2A emerged exclusively in invasive melanomas. PTEN and TP53 mutations were found only in advanced primary melanomas. The point-mutation burden increased from benign through intermediate lesions to melanoma, with a strong signature of the effects of ultraviolet radiation detectable at all evolutionary stages. Copy-number alterations became prevalent only in invasive melanomas. Tumor heterogeneity became apparent in the form of genetically distinct subpopulations as melanomas progressed.

This study defined the succession of genetic alterations during melanoma progression, showing distinct evolutionary trajectories for different melanoma subtypes. It identified an intermediate category of melanocytic neoplasia, characterized by the presence of more than one pathogenic genetic alteration and distinctive histopathological features. The study also implicated ultraviolet radiation as a major factor in both the initiation and progression of melanoma.

Shain, A. H., et al. (2015) The Genetic Evolution of Melanoma from Precursor Lesions. The New England Journal of Medicine. 373:1926-1936. doi: 10.1056/NEJMoa1502583.

Read more of Bioo Scientific’s NGS Featured Publications.


Featured Publication: Identifying RBP Targets with RIP-Seq

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Drs. Wessels, Hirsekorn, Ohler and Mukherjee from the Max Delbrück Center for Molecular Medicine recently published a RIP-Seq protocol which can be used to uncover genome-wide RNA transcripts that interact with a specific protein or protein complex. In this assay, ribonucleoprotein (RNP) complexes are immunoprecipitated (RIP) from cell lysates. Associated RNAs are then isolated from these RNP complexes. After the associated RNAs were isolated, these researchers used the NEXTflex™ Rapid Directional qRNA-Seq™ Kit for library preparation. Resulting libraries are suitable for sequencing on any Illumina sequencing platform.

Read the featured publication here:

Wessels, H.-H., Hirsekorn, A., Ohler, U. and Mukherjee, N. (2015) Identifying RBP Targets with RIP-seq. Post-Transcriptional Gene Regulation. Methods in Molecular Biology. 1358. 141-152.


Low-Input, High Multiplexing Library Prep Solution for SeqCap Target Capture

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The NEXTflex™ Rapid Pre-Capture Combo Kit (NimbleGen SeqCap Compatible) is a complete low-input library prep solution with the ability to multiplex up to 96 samples, working seamlessly with Roche NimbleGen’s SeqCap EZ solution-based capture system. This kit contains the library prep reagents, barcodes, blockers and post-capture amplification reagents required for SeqCap target capture.

With an optimized workflow, libraries can be constructed from as little as 10 ng of input DNA in only 2 hours. 96 barcodes and blockers are available, offering deep multiplexing of target capture reactions and greatly decreasing sequencing costs.

Visit the NEXTflex Rapid Pre-Capture Combo Kit product page today to learn how this kit can improve your NimbleGen SeqCap target capture.


Improved SureSelectXT Target Capture Library Prep Solutions

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The NEXTflex™ Pre- and Post- Capture Combo Kit (Agilent SureSelectXT Compatible) reduces the time required for library prep and incorporates index-specific blocking oligos which offer a higher percentage of on-target reads than traditional blocking oligos. The NEXTflex Pre- and Post- Capture Combo Kit (Agilent SureSelectXT Compatible) is a complete Illumina-compatible library prep solution designed for DNA library preparation, multiplexing, barcode blocking, hybridization and post-capture amplification upstream of Agilent SureSelectXT target enrichment systems. Additionally flexible barcoding options allow up to 96 libraries to be multiplexed simultaneously. Options are also available for multiplex and single-plex captures. Additionally, the NEXTflex Pre- and Post- Capture Combo Kit offers a significant cost savings over the Agilent library prep solution.

Visit the NEXTflex Pre- and Post- Capture Combo Kit (Agilent SureSelectXT Compatible) product page to learn how this kit can improve your SureSelect XT target capture today.


High-Speed, Multiplexed 16S V4 Microbial Sequencing on the MiSeq

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The new NEXTflex™ 16S V4 Amplicon-Seq Kit 2.0 simplifies the construction of 16S V4 libraries from environmental samples. This complete kit simplifies your workload by combining all reagents necessary for 16S V4 microbial sequencing, including up to 384 barcodes for deep multiplexing. Custom sequencing primers are not required, further simplifying the protocol while reducing the potential for error.

Read more to learn how to simplify your 16S V4 microbial sequencing today.


Automated Sciclone Protocol for Small RNA-Seq Library Prep Now Available

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A protocol is now available which automates the NEXTflex™ Small RNA-Seq Kit v2 on the Sciclone Workstation, to simplify small RNA-Seq while offering the most accurate small RNA-Seq profiling available. The patent pending NEXTflex Small RNA-Seq Kit v2 increases accuracy by incorporating randomized adapter technology to reduce ligation bias in small RNA-Seq libraries. The new Sciclone automation guide can also be used with a Pippen Prep protocol to automate the gel purification step in this protocol.

Download the NEXTflex Small RNA-Seq Sciclone NGS and NGSx Workstation Automation Guide to simplify your small RNA library prep.


Bioo Scientific Honored for Increasing Austin’s Ability to Compete in the Global Marketplace

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Bioo Scientific received the British Airways International Trade, Investment & Expansion Award, recognizing the worldwide economic growth fueled by its international sales team and network of distributors. The British Airways International Trade, Investment & Expansion Award recognizes investments by international companies in Austin that create jobs locally, as well as Austin-based companies who have a global impact through products and services provided worldwide. Bioo Scientific is an Austin-based biotech company which manufactures food and feed safety test kits and products supporting life science research.

The awards, presented by the Austin Chamber, recognize local firms and organizations for achievements, community contributions and milestones. It is one of the largest business events in Central Texas, bringing together 1,000 business leaders, entrepreneurs, organizations, government officials and regional chambers.

About 300 companies were nominated for the Chamber’s Business Awards. Out of this group, Bioo Scientific was recognized for being a top exporter in the Austin area. All of Bioo Scientific’s products are manufactured in the United States; however, international sales make up over three quarters of Bioo Scientific’s business.  Bioo Scientific exports food and feed safety test kits and life science research products to more than 100 countries across Asia, Europe, the Middle East, Africa and Latin America.

Currently Bioo Scientific is focusing its attention on increasing its global presence, as exporting is essential to our future growth. The 2015 Greater Austin Business Export Awards recognizes the success of these efforts.


Rapid Whole-Genome Sequencing of Bacteria

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Clinical personnel and health officials must rapidly identify potential pathogens in order to respond to suspected disease outbreaks to prevent public disturbances. Next-generation sequencing has the potential to complement conventional diagnostics in many instances, and whole genome sequencing is ideal for use as a typing tool to identify the causative agent behind diseases.

One recent example of NGS being used in a clinical setting is illustrated in the publication Rapid Metagenomic Diagnostics for Suspected Outbreak of Severe Pneumonia, a letter to Emerging Infectious Diseases, in which researchers describe the methodology their group used to identify the pathogen responsible for acute respiratory distress syndrome in their patients, using a NEXTflex™ Kit for library construction. This sequencing methodology was used to identify the etiologic agent of a suspected pneumonia outbreak among German police officers.

With a robust two-hour library prep protocol and up to 384 single-index barcodes, which facilitate high level multiplexing while reducing concerns about run-to-run sample carryover and sample crosstalk, the NEXTflex™ Rapid DNA-Seq Kit is ideal for rapid identification of disease-causing pathogens.

To demonstrate the efficacy of next generation sequencing for identification of pathogens, we present below Bioanalyzer traces of libraries constructed from varying inputs of DNA isolated from E. coli, Campylobacter and Salmonella bacteria.


Figure 1. Bioanalyzer trace of a library constructed using the NEXTflex Rapid DNA-Seq Kit from 100 ng of E. coli bacteria in water.


Figure 2. Bioanalyzer trace of a library constructed using the NEXTflex Rapid DNA-Seq Kit from 1 µg of Campylobacter bacteria in water.


Figure 3. Bioanalyzer trace of a library constructed using the NEXTflex Rapid DNA-Seq Kit from 100 ng Salmonella bacteria in water.

Fischer, N., et al. (2014) Rapid metagenomic diagnostics for suspected outbreak of severe pneumonia [letter]. Emerg Infect Dis. doi: 10.3201/eid2006.131526.


Featured Publication: Transcriptomic Analysis of Degraded Forensic Body Fluids

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Researchers Meng-Han Lin, Daniel F. Jones, and Rachel Fleming determined the NGS analysis of RNA obtained from various fluid samples is a reliable method for body fluid identification from the degraded samples frequently encountered at crime scenes, which can be difficult to analyze using the current detection methods, RT-PCR and qPCR.

These researchers have made significant advances in analysis of degraded RNA from forensically relevant fluids, generating significant high quality sequencing output from fresh, two and six week aged samples of oral mucosa/saliva (buccal samples), circulatory blood, menstrual blood and vaginal fluid. Libraries for sequencing were generated using the NEXTflex™ Rapid Directional RNA-Seq Kit, from total RNA which was not subjected to ribosomal RNA depletion. Due to the low concentration and degraded nature of some samples, 13 µL total RNA input was used for library preparation irrespective of concentration.

In these studies, body fluid-specific markers were detected across different body fluid types and aging intervals and FPKM values for body fluid-specific markers corresponded to the target body fluid type. The availability of this type of is contextual information may have significant implications for the investigation of crimes.

Read the entire publication here:

Lin, M.-H., Jones, D. F. and Fleming, R. (2015) Transcriptomic analysis of degraded forensic body fluids. Forensic Science International: Genetics, Volume 17.  35-42. dio: 10.1016/j.fsigen.2015.03.005.

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