Analysis Recommendations for DNA-Seq

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The advancements and decreased cost of high-throughput sequencing provide researchers with access to a multitude of genetic data. Based on specific experimental goals, methods, and pipeline tools, the extent of data analysis required can be highly variable. Some bioinformatics packages integrate easily into central analysis pipelines, while other tools are more optimized for use as standalone packages.

At the most basic level of analysis, the sequencing instrument itself performs initial data processing. This hardware-generated data provides base calling results, the quality of every base call, and various other machine-generated statistics about the entire sequencing run. FASTQ files are a typical representation of this data. FastQC software for primary analysis provides information regarding the quality of sequencing reads and is useful in determining how to proceed with data. Most researchers are interested in more than just “raw” sequences; after monitoring data quality, FASTQ files are used in secondary analysis.

Experimental goals, sequencing depth, and sample type impact the type of secondary analysis required. FASTQ files, representing DNA reads, first need to be reassembled in order to gain biological insight about samples. Depending on the sample type, quality filtered reads will be either de novo assembled or mapped to a well characterized reference genome. If performing a de novo assembly, read length plays an important role in determining the most appropriate software. In the more common situation of mapping to a reference, the most familiar aligners for DNA are BWA and Bowtie. Mapping statistics can be gathered through the SAMtools package (flagstat or idxstats features), providing valuable information about the quality of the sample compared to a reference. After alignment and gathering mapping statistics, variant detection is a potential next step in analysis. For mutation calling, SAMtools has a feature known as mpileup, followed by bcftools to produce binary bcf files, and converted to vcf files (variant call formatted files). Alternatively, GATK Unified Genotyper of the GATK pipeline could be used. The above tools can either be used individually, or linked together by an informatics specialist to form a custom DNA-Seq pipeline for analysis. Galaxy offers a user-friendly interface alternative because it is server-based and allows for the simple linking of these tools into workflows. Once files are uploaded to the Galaxy server, a workflow can be initiated, and all tools making up the workflow will execute on the input provided. Galaxy also allows for parallel processing of samples.

Once multiple samples have been processed this far, researchers can continue in tertiary analysis by comparing samples to one another, or relative frequencies of samples can be compared to populations through efforts of the HapMap and the 1000 Genomes Project. Deciphering the data, a very complex task, remains essential in determining biological significance of samples.

As illustrated, sequence analysis can be highly variable and an abundance of sequence analysis tools exist to handle high throughput data. Depending on experimental design and requirements, an analysis pipeline can have several stopping points at any level of analysis. As the NGS field continues to grow, best practices utilizing these tools will become more standardized, in an effort to provide analysis capabilities to more users.

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Detecting Germline Mutations

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Mendelian diseases, also known as monogenic diseases, represent a class of disorders associated with genetic variation in one gene. Their inheritance follows classic Mendelian laws, and the phenotype could be expressed in either a dominant or recessive way. Currently, over three thousands genes have been associated with Mendelian diseases including cystic fibrosis, cancer predisposition, epilepsy, and deafness among others.

From the discovery of the linkage between genetic variations and inherited diseases, scientists and clinicians have striven to identify them in routine ways to confirm diagnosis, provide better genetic counselling, and select proper treatment. Originally, Sanger sequencing was used, but advances in next-generation sequencing (NGS) have offered excellent improvement to the speed and efficiency for molecular diagnostic of monogenic diseases. There are three different qualitative and quantitative NGS approaches for the screening of genetic variations: whole genome sequencing (WGS), whole exome sequencing (WES) and comprehensive gene panel sequencing.  Each of these methods has distinctive advantages and disadvantages which define their applications in research and diagnostics of monogenic diseases. WGS and WES are expensive, laborious, and lack complete and deep coverage of target(s) of interest. However, these methods provide the most complex information. On the other hand, when phenotype or family history of the patient is known, the using of comprehensive gene panels targeting only selected genes is the most economical and straightforward solution.

Comprehensive gene panels composed of candidate disease genes provide complete and deep coverage of a target region in a quick, simple assay suitable for both research and diagnostic laboratory settings.

Bioo Scientific introduced the first in a series of comprehensive panels for Mendelian diseases by launching the NEXTflex™ BRCA1 & BRCA2 Amplicon Panel for identification of variations in BRCA1 and BRCA2 breast cancer predisposition genes. This quick and easy-to-perform panel with 100% coverage and uniformity will be followed by a series of twenty other panels for detection of variations associated with the most common inherited clinical conditions.

 

Jamuar SS, Tan EC. 2015 Clinical application of next-generation sequencing for Mendelian diseases. Hum Genomics.

Saudi Mendeliome Group. Comprehensive gene panels provide advantages over clinical exome sequencing for Mendelian diseases. 2015. Genome Biology 16:134

Easton, D. F. 2015. Gene-Panel sequencing and the prediction of breast-cancer risk. 2015. N. Engl. J. Med. 372: 372:23

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Bioo Scientific’s Cell Free DNA-Seq Tech Now Manufactured under ISO 13485

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To meet the needs of pre-clinical and clinical scientists and to improve traceability and quality control, Bioo Scientific’s NEXTflex™ Cell Free DNA-Seq Kits for the construction of libraries for cell free DNA sequencing are now manufactured under ISO 13485 certification.

The analysis of cell free DNA has many applications; circulating tumor DNA (ctDNA) provides clinically useful information for the diagnosis and monitoring of treatment for cancer patients and circulating fetal DNA (cffDNA) is facilitating noninvasive prenatal testing. Bioo Scientific offers a complete pipeline for cell free DNA analysis from the isolation of cell free DNA to the construction of libraries for whole genome sequencing or targeted sequencing.

The NEXTflex Cell Free DNA-Seq Kit, optimized for whole genome sequencing of cell free DNA, and the NEXTflex™ BRCA1 & BRCA2 Amplicon Panels are both now manufactured under ISO 13485 certification.

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Simply Your Cell-Free DNA Isolation and Obtain Higher Quality Libraries for NGS Sequencing

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The NextPrep-Mag™ cfDNA Isolation Kit is designed for extracting cell-free DNA (cfDNA) from human blood plasma using an ultra-rapid magnetic bead-based format. The kit can be scaled for use with plasma volumes ranging from less than 1 mL to more than 5 mL. The cfDNA recovered is ideal for constructing libraries for NGS sequencing and for other common applications. This easily automatable kit includes novel magnetic beads that attract almost instantaneously, dramatically reducing the time needed for cfDNA isolation. No vacuum pumps, column extenders, or specialized equipment is required.

The cell-free DNA isolated using this kit produces highly concentrated libraries, with fewer PCR cycles needed for amplification compared to libraries constructed using cell-free DNA isolated using silica filter isolation methods.

Bioo Scientific also offers NEXTflex™ Cell Free DNA-Seq Kit for Illumina library preparation, which is optimized for library construction from low input amounts of cfDNA in only two hours. This kit delivers high coverage and reduced bias, along with flexible multiplexing options.

Read here how you can simplify your cell-free DNA isolation and construct higher quality libraries with the NextPrep-Mag cfDNA Isolation Kit and the NEXTflex Cell Free DNA-Seq Kit.

Are you Isolating Cell-Free DNA from Urine?

If you are interested in a fast, automatable solution to improve your yield of cell-free DNA from urine, sign up now to beta test the new mag-bead based kit Bioo Scientific is developing.

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Designing Amplicon Panels

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A recent OMICS Tutorial published in Genetic Engineering News describes a number of factors which need to be taken into consideration when designing custom panels for the detection of germline and somatic mutations with amplicon sequencing. These factors include required including coverage, uniformity, and multiplexing requirements. Read the article, Designing Amplicon Panels, for more information

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To Pool, or Not to Pool

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To pool, or not to pool, that is the question:
Whether ’tis Nobler in the cramping hand to suffer
The XP Beads and Buffer Swaps of outrageous Fortune.

Target capture allows scientists to enrich sample libraries for a targeted subsection of the genome; for example, sequencing all of the exons. Targeted sequencing allows for the multiplexing of many samples in a single sequencing run. These samples can be pooled either prior to, or post-capture. Bioo Scientific’s latest Tech Tip, Pooling Recommendations for SureSelect Target Capture, offers guidelines to help determine which option is best for your needs when using Agilent’s SureSelect target capture system. Learn more at Bioo Scientific.

 

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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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