Improving Agrigenomics with NEXTflex™ Library Prep : PAG XXIV Poster Session

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This month Bioo Scientific will be attending PAG XXIV, the largest ag-genomics meeting in the world. Over 3,000 leading genetic scientists and researchers in plant and animal research will be attending the meeting. PAG, the Plant and Animal Genome Conference, provides a forum for the discussion of recent developments and future directions of genomic projects, featuring technical presentations, poster sessions, exhibits and workshops.

Bioo Scientific will present two posters at PAG XXIV. The first poster, P1234, Using NEXTflex Amplicon Studio for SNPs Detection in the Flax (Linum usitatissimum) Genome by Illumina-based Targeted Sequencing, describes the use of NGS targeted sequencing of specific genes to identify desired mutations introduced by mutagen exposure in flax plants. The abstract can be seen here.

The second, P0215, Advances in Small RNA Library Preparation Allow Combination of Bias Reduction with Gel-free or Low Input Protocols, describes new methods of reducing ligation bias and adapter-dimer formation in small RNA-seq library prep, allowing gel-free final selection or preparation of libraries from low starting amounts of small RNA. The abstract is available here.

Also presented at the meeting will be the poster Highway to Heaven: Sequencing the Genome of the Waterfall Climbing Hawaiian Goby (Sicyopterus stimpsoni), P0447, a collaboration between the US Army ERDC, Mississippi State University, and St. Cloud State University. Their research uses genomic sequencing to explore the evolution of different strategies in closely related species of gobies to overcome extreme conditions, such as climbing high waterfalls using repurposed mouthparts or walking out of water using modified fins. The abstract can be viewed here.

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New Amplicon Panel Offering Complete BRCA1 and BRCA2 Coverage for Illumina Sequencing

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The NEXTflex™ BRCA1 and BRCA2 Amplicon Panel for Illumina Sequencing is a cost-effective, optimized solution for revealing clinically relevant mutations in BRCA1 and BRCA2 genes by detecting and screening DNA isolated from fresh or frozen tissue. The NEXTflex™ BRCA1 and BRCA2 Amplicon Panels offers best-in-class performance for on-target percentage and coverage uniformity, with 100% uniformity of all targeted coding exons and exon-intron boundaries, facilitating variant discovery and confirmation.

  • Ready-to-sequence libraries in 2 hours
  • Inputs as low as 20 ng
  • On-target specificity > 99%
  • Coverage uniformity 100% of amplicons detected at ≥0.2X mean coverage
  • Complete library generation in a single kit

The NEXTflex™ BRCA1 and BRCA2 Amplicon Panel for Illumina Sequencing is a complete kit that includes all components necessary for generating ready-to-sequence libraries, including primer pairs, library prep reagents, and indexed barcodes.

To learn how you can improve your BRCA1 and BRCA2 analysis, visit the NEXTflex BRCA1 and BRCA2 Amplicon Panel webpage.

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Advantages of Using Amplicon Panels for Targeted Resequencing

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Targeted sequencing is an invaluable tool in clinical and research applications methods to sequence region(s) of interest. Two principally different technologies exist for enrichment of library pools for targeted sequencing – target capture by hybridization and amplicon sequencing (Samorodnitski et al., 2015). These methods differ in their preparation time, input, optimal target range, specificity and cost (Table 1). Target capture by hybridization requires construction of a barcoded library, followed by hybridization to long labeled RNA or DNA probes. Hybridization protocols take days and yield 60% – 80% on-target reads. This method has high input requirements and volume-sensitive reactions. In contrast, targeting by amplicon sequencing, which is based on direct construction of libraries from multiplexed PCR products, could be finished in hours, can handle lower input down to the nanogram range, produces greater than 95% on-target reads, and is substantially less expensive.

Target capture is a viable solution when data is needed from large targets, such as a whole exome. When analyzing targets which are hundreds of kb in length (rather than Mb), amplicon targeted sequencing excels in speed, input requirements, specificity, and cost. With the advancement in knowledge about mutations important in specific diseases, researchers and physicians are selecting smaller targets instead of whole exomes for screening of critical samples. These researchers are increasingly taking advantage of amplicon sequencing for economical screening of large numbers of samples, often with a limited amount of starting material available, and limited time to make treatment decisions. Additionally, for academic researchers, amplicon sequencing offers the ability to screen thousands of samples simultaneously, providing enough data to observe statistically significant differences to drive experimental conclusions.

Table 1: Comparing amplicon-based targeting with targeting by hybridization

custom-panel-table-1

Bioo Scientific now offers custom amplicon-based targeting solutions, the NEXTflex™ Custom Amplicon Panels which are fully customizable, amplicon-based assays for targeted resequencing on the Illumina or Ion Torrent sequencing platforms. The NEXTflex Amplicon Panels are validated, optimized, and verified panels offering 100% coverage of targeted regions and high quality primer design. These amplicon panels allow researchers to sequence up to 2,000 amplicons using a simple workflow, covering regions as small as 1 kb and as large as 500 kb. This highly targeted approach offers unparalleled efficiency for discovering, validating, and screening genetic variants. Custom DNA and RNA panels can be designed for use with fresh or frozen tissue, FFPE, and cell-free samples.

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Slideshow – Reduced Bias Small RNA Library Prep with Gel-Free or Low-Input Options

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microRNAs (miRNAs) may provide useful markers for the development of disease diagnostic and prognostic assays. NGS brings sensitivity, specificity, and the ability to maximize data acquisition and minimize costs of miRNA sequencing by using multiplex strategies to allow many samples to be sequenced simultaneously with small RNA analysis. However, small RNA sequencing has typically suffered from three major drawbacks: severe bias, such that sequencing data does not reflect original miRNA abundances, the need to gel purify final libraries, and lack of low-input protocols. The NEXTflex™ Small RNA-Seq Kit v3 addresses these drawbacks by using two strategies: randomized adapters to reduce ligation-associated bias, and a dual approach to adapter-dimer reduction, thereby allowing gel-free or low-input small RNA library preparation.


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Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers microRNAs that Evade Capture by Current Methods

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Researchers from the University of North Carolina at Chapel Hill, the National Human Genome Research Institute, and the NIH Intramural Sequencing Center, recently published their research, “Addressing bias in small RNA library preparation for sequencing: a new protocol recovers microRNAs that evade capture by current methods,” in the journal Frontiers in Genetics. Their research supports previously published works indicating that the adapter ligation steps of library prep introduce a significant but widely unappreciated bias into the results of high-throughput small RNA sequencing.

Baran-Gale and others isolated total RNA from mouse insulinoma (MIN6) cells and constructed small RNA libraries using the Illumina v1.5 protocol and the Illumina TruSeq protocol. Results obtained with the two widely used Illumina library preparation protocols produced strikingly different microRNA (miRNA) expression profiles in the same batch of cells. There are 102 highly expressed miRNAs that were >5-fold differentially detected and some miRNAs, such as miR-24-3p, were over 30-fold differentially detected. While some level of bias in library preparation was not surprising to the researchers, the apparent massive differential bias between these two widely used adapter sets was not well appreciated.

The researchers then used the NEXTflex Small RNA-Seq Kit v2, which utilizes adapters with random nucleotides at the ligation boundary, to prepare libraries from the same total RNA. Their results indicated that the NEXTflex Small RNA-Seq Kit v2 protocol was able to robustly detect several miRNAs that partially or completely evaded capture by the Illumina-based methods. These results also correlated best with RT-qPCR data.

Bioo Scientific recently launched the NEXTflex Small RNA-Seq Kit v3, which like the NEXTflex Small RNA-Seq Kit v2 also incorporates randomized adapters to reduce the ligation bias described in this research. The NEXTflex Small RNA-Seq Kit v3 additionally incorporates a dual adapter-dimer reduction approach, allowing for either a completely gel-free small RNA library prep when starting with ≥ 200 ng of total RNA, or low-input library prep from as little as 5 ng of total RNA.

To learn more about this bias-reducing technology and how it can be used to increase the efficiency and reduce the cost of small RNA sequencing, read our recent whitepaper or view our Slideshare presentation.

Baran-Gale, J., Kurtz, L. C., Erdos, Sison, M. C., Young, A., Fannin, E. E., Chines, P. S. and Sethupathy, P. (2015) Addressing bias in small RNA library preparation for sequencing: a new protocol recovers microRNAs that evade capture by current methods. Frontiers in Genetics. doi: 10.3389/fgene.2015.00352.

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Reduced-Bias Small RNA Library Preparation with Gel-Free or Low-Input Options

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Several issues have historically reduced the utility of small RNA sequencing, including bias introduced during ligation, tedious gel purification requirements, and high input requirements. Bioo Scientific’s newest whitepaper, Reduced-bias small RNA library preparation with gel-free or low-input options, illustrates how the NEXTflex Small RNA-Seq Kit v3 solves these issues, offering sensitivity, specificity, and the ability to maximize data acquisition and minimize costs.

Read the whitepaper to learn how the NEXTflex Small RNA-Seq Kit v3 can improve your small RNA analysis.

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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.

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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.

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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: http://www.biooscientific.com/Identifying-RBP-Targets-with-RIP-Seq

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.

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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.

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