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Jun
4

A high-throughput method for Illumina RNA-Seq library preparation

Filed Under Information, Publications | Leave a Comment With the introduction of cost effective, rapid and superior quality next generation sequencing (NGS) techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-Sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. Presented here is a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. A set of 96 unique barcodes have been designed for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. This protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. The authors also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation. (read more ) Kumar R, Ichihashi Y, Kimura S, Chitwood DH, Headland LR, Peng J, Maloof JN, Sinha NR. (2012) A high-throughput method for Illumina RNA-Seq library preparation. Front in Plant Genet and Genom [Epub ahead of print]. [abstract] Incoming search terms: a high-throughput method for illumina rna-seq library preparation 2 boxing drawing ngs rna-seq rna-seq illumina hiseq coverage rna-seq illumina coverage next generation sequencing ppt 2011 rna seq paired end illumina library insert size rna-seq blog obtain

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pair-end tag sequencing expression quantification illumina hi-seq adapter

May
23

2012 Next-Gen Sequencing Conferences

Filed Under Events | 1 Comment 2nd Next Generation Sequencing Boston, MA May 30th 31st GTCs 2nd Next Generation Sequencing Conference is a 2-day meeting that will bring together leading technology and diagnostic companies, national laboratories and academic institutions from across the globe to discuss applications of NGS platforms, novel sequencing strategies, developments in nanopore and single molecule sequencing and the implementation of NGS in the clinical space. (read more) Next Generation Sequencing Prague June 12th 13th Informa Life Sciences Our Next Generation Sequencing conference provides you with the opportunity to hear the latest and most exciting developments in NGS technological advances (including 3rd and 4th generation platforms), sample preparation strategies for difficult samples, and how research, drug/diagnostic discovery and development are utilising next generation sequencing. (read more) Next-Gen Sequencing Applications & Translational Technologies Summit San Francisco, CA August 6th 8th ibc Life Sciences How are others leveraging the power of next-generation sequencing in everyday research, and for clinical and diagnostic applications? Are you looking to accelerate your discovery and development efforts using translational technologies? (read more) NGX: Applying Next-Generation Sequencing Providence, RI Aug 13th 15th Cambridge Healthtech Institutes NGX: Applying Next-Generation Sequencing investigates the expanding applications of NGS. Learn from experienced researchers from large sequencing centers, core laboratories, and specialized groups as they share their practical knowledge, real-world experiences and solutions. (read more) Next- Generation Sequencing Summit London, September 17th 18th SMi A data interpretation and analytical perspective. Identifying potential therapeutic drug targets and validating their suitability is a complex process involving numerous experimental platforms, including DNA sequence analysis. (read more) Beyond the Genome 2012 Boston, MA September 27th 29th Genome Medicine and Genome Biology The aim of the conference is to discuss next-generation sequencing and other new technologies, informatic tools, and how these are being used to identify common and rare disease-causing mutations in the research laboratory and toward the clinic. Themes will include cancer
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genomics from discovery sequencing to genome guided therapy, epigenomic technologies and applications, and inherited disease beyond the candidate gene approach. There will also be an informatics workshop. (read more) Next Generation Sequencing Congress London November 15th-16th Oxford Global 2 day conference showcasing cutting edge next generation sequencing platforms, technologies, applications and computational tools for the analysis of NGS sequencing data. (read more) Incoming search terms: next generation sequencing ppt 454 sequencing congress rna-seq next generation sequencing next generation rna sequencing DIAGRAM comparing RNA seq 454 seq rna-seq microbial pathways rna-seq conference 2012 list of next generation sequencers in india paraffin microarray sequencing hawaii next gen sequencing india workshop 2012

Apr
9

Global Next Generation Sequencing (NGS) Market Worth $2,343 Million by 2016
Filed Under News, Press Release | Leave a Comment From: Next Generation Sequencing (NGS) Market Global Trends and Forecasts (2011-2016), marketsandmarkets.com, April 2012 The global NGS market was valued at $842.5 million in the year 2011, growing at a CAGR of 22.7% from 2012 to 2016. North America commanded the largest share of 51.8% of the overall NGS technology market in 2011. The major factors driving the NGS Market is the decrease in the cost of sequencing with increase in the number of applications of sequencing in various fields of science such as cancer research, bio-fuels, marine sciences, live stock research, agricultural, and veterinary research. Thus, there is more demand for the equipment to understand the genome sequencing and utilize it further for human and environmental benefits. The high pace of NGS research is likely to boost the market for personal genome sequencing in the coming years. Manufacturers predominantly have an inclination towards diagnostic applications both as a supplier and manufacturer. Key players in this market include Illumina (U.S.), Life Technologies (U.S.), 454 Roche (Switzerland), Oxford Nanopore Technologies (U.K.), and Pacific Biosciences (U.S.). (read the press release )
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Incoming search terms: next generation sequencing market size ngs market size next generation sequencing markets growth rna ngs market next gen sequencing market RNA discovery mRNA lncRNA ngs microarray market ngs rna-seq market ngs rna-seq methods review ngs sequencing forecast

Aug
17

Study Finds Array and Sequencing Combo Yields Novel Info on Gene Expression
Filed Under Information | Leave a Comment from In Sequence By Monica Heger Microarray technology and next-generation sequencing, when used in conjunction, could improve on using either method alone to evaluate gene expression, according to researchers from the University of Barcelona. The team combined three different microarray platforms from Agilent, Operon, and Illumina with digital gene-expression profiling on the Illumina Genome Analyzer to study genes related to the epidermal growth factor, a regulatory growth factor related to cell proliferation and survival. They found that the combination of the techniques was able to establish a validated gene set including novel genes previously unrelated to EGF as well as help piece together the network of how those genes interact. The study, published in BMC Genomics this month, suggests that using the two different technologies could help generate more reliable data sets than when either method is used alone, and should help improve the confidence of functional analysis. (read the entire story )

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Llorens F, Hummel M, Pastor X, Ferrer A, Pluvinet R, Vivancos A, Castillo E, Iraola S, Mosquera AM, Gonzlez E, Lozano J, Ingham M, Dohm JC, Noguera M, Kofler R, Del Ro JA, Bays M, Himmelbauer H, Sumoy L. (2011) Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis. BMC Genomics 12, 326. [article]

May
24

FusionMap detecting fusion genes from next-generation sequencing data at base-pair resolution
Filed Under Splicing and Junction Mapping | Leave a Comment A novel method, FusionMap, is presented which aligns fusion reads directly to the genome without prior knowledge of potential fusion regions. FusionMap can detect fusion events in both single- and paired-end datasets from either RNA-Seq or gDNA-Seq studies and characterize fusion junctions at base-pair resolution. FusionMap achieved high sensitivity and specificity in fusion detection on two simulated RNA-Seq datasets which contained 75nt paired-end reads. FusionMap achieved substantially higher sensitivity and specificity than the paired-end approach when the inner distance between read pairs was small. Using FusionMap to characterize fusion genes in K562 chronic myeloid leukemia cell line, its accuracy was further demonstrated in fusion detection in both single-end RNA-Seq and gDNA-Seq datasets. These combined results show that FusionMap provides an accurate and systematic solution to detecting fusion events through junction-spanning reads. AVAILABILITY: FusionMap includes reference indexing, read filtering, fusion alignment and reporting in one package. The software is free for non-commercial use at (http://www.omicsoft.com/fusionmap). CONTACT:

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ge@amgen.com SUPPLEMENTARY INFORMATION: Supplementary data are available at the journals web site. Ge H, Liu K, Juan T, Fang F, Newman M, Hoeck W (2011) FusionMap: detecting fusion genes from next-generation sequencing data at base-pair resolution. Bioinformatics [Epub ahead of print]. [abstract] Incoming search terms: isoform ngs fusion genes ngs gene detection rnaseq NGS detecting fusion targeted gene fusion next generation sequencing

Apr
28

RNA-Seq Analysis Software Commercial Packages

Filed Under Data Analysis | 1 Comment A list of the RNA-Seq data analysis software packages available from commercial providers. Again, if Ive missed something, please send me a note and I will be happy to add to this list.

Avidis NGS http://www.avadis-ngs.com/applications/rna_seq Find fused genes via spliced and paired reads spanning the fused genes. Analyze coverage patterns to detect novel genes and exons not present in NCBI, UCSC, and Ensembl annotations. Seamlessly create and manipulate genomic region lists. Filter region lists based on different attributes. Determine gene and deconvoluted transcript expression profiles; identify alternative splicing patterns. Identify differential gene profiles via statitistical tests, cluster genes with similar profiles. Carry out GO analysis to identify significant GO terms.

CLC Genomics Workbench http://www.clcbio.com/index.php?id=1240 Our RNA-seq analysis now supports the use of paired-end data for RNA-seq. A combination of single reads and paired reads can also be used, and expression values can now be stratified into transcript level expression values, both for single and paired reads, allowing users to compare two different samples across transcripts. Another important new feature is our batching functionality of all our high-throughput sequencing tools, enabling researchers to perform the same analysis on several elements in one batch, which is an easy way to analyze multiple datasets in one go and thereby save time for setting up and running the same type of analysis multiple times.

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DNAnexus - https://dnanexus.com/features/researcher/analyses tag-based applications. RNA-seq is now a cost-effective way to measure gene expression, discover alternative splicing, and identify previously unassayed transcriptional activity. ChIP-seq provides a general way to study interactions between DNA and other molecules. With DNAnexus, these and other tag-based applications can be analyzed through our cloud-based infrastructure with the click of a button. Visualize the results in the DNAnexus browser, or export the results in tabular format and integrate your data across multiple experiment types, for example combining RNA-seq expression data with ChIP-seq binding affinity on the same genes.

DNASTAR QSeq http://www.dnastar.com/t-products-qseq.aspx QSeq is DNASTARs Next-Gen application for RNA-Seq, ChIP-Seq, and miRNA alignment and analysis. QSeq is fully integrated with ArrayStar, enabling you to take advantage of its powerful visualization and analytical tools, including using Gene Ontology (GO) annotations for ontology comparisons and gene characterization. Using QSeq, researchers can select gene sets and export associated reads through the rest of the DNASTAR software pipeline for sequence assembly, alignment, and detailed analysis.

GeneSifter http://www.geospiza.com/Products/default.shtml Analysis Kits offer a solution for scientists faced with large volumes of unprocessed data from AB SOLiD or Illumina Genome Analyzer DNA sequencing. Through Geospizas rapid analysis service, you can complete Next Generation Data Analysis as a service per file or per run. GeneSifter Analysis Edition now supports Whole Transcriptome Analysis with a more detailed view of each gene, including predicted splicing events and exon usage increasing your access to the depth of next generation sequencing. This new analysis capability also includes an integrated gene viewer to see your data as it relates to the genome and then tie it to publicly available information.

Geomatix Genome Analyzer http://www.genomatix.de/en/produkte /genomatix-genome-analyzer.html The Genomatix Genome Analyzer is our integrated solution for comprehensive second-level analysis of Next Generation Sequencing (NGS) data from ChIP-Seq, RNA-Seq or genotyping experiments. Each analyzer is brimming with state-of-the-art technology that sheds light on biological context essential to help you understand the big picture.

GenomeQuest RNA Seq http://www.genomequest.com /science/workflows/rna-seq/ RNA-Seq stands to replace existing transcript profiling technologies as it measures all RNA in a sample, not just the RNA that can be probed for using traditional chips. GenomeQuest provides a powerful workflow that integrates best-of-breed open source technologies in a commercially supported environment to measure gene expression and to discover novel splice variants. Ingenuity IPA 9.0 http://www.ingenuity.com/products /pathways_analysis.html IPA will support RNA-Seq processed datasets containing Ensembl, RefSeq or UCSC IDs. Researchers will now be able to analyze and interpret their RNA-Seq data. IPA helps you understand biology at multiple levels by integrating data from a variety of experimental platforms and

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providing insight into the molecular and chemical interactions, cellular phenotypes, and disease processes of your system. Even if you dont have experimental data, you can use IPA to intelligently search the Ingenuity Knowledge Base for information on genes, proteins, chemicals, drugs, and molecular relationships to build biological models or get up to speed in a relevant area of research.

Partek Genomics Suite (Partek GS) http://www.partek.com /ngs#rnaseq Using a whole-transcriptome sequencing approach, sequence data are analyzed for differential expression and alternative splicing based on known mRNA annotation. Sequencing reads that are not mapped to any known mRNA annotation are used to uncover the novel transcriptional regions. Partek GS will identify and quantify sequence variants (coding SNPs) within RNA-seq samples. With a collection of SNPs identified, Partek GS will then find allele specific expression that drive phenotypic change within the transcriptome. Both paired end and junction reads are supported.

RNA Baser Sequence Assembler http://www.rnabaser.com/ RNA Baser Sequence Assembler represents an extension of DNA Baser Sequence Assembler, specialized in processing rRNA sequences. It is optimized for early integration of contextual metadata. This way, the metadata will stay attached to the primary sequence information throughout the complete workflow of sequence analysis, until final submission. In addition, RNA Baser Sequence Assembler is optimized for data exchange with standard non-commercial software used for rRNA sequence analysis and submission.

SeqSolve http://www.integromics.com/ngs for Next Generation Sequencing: a unique approach for the downstream analysis of RNA-seq data. characterization of the reads, distributions into annotated genic regions, coverage profile within genes, read density over genomic sites, statistical support for differential gene expression, antisense transcription analysis, quality controls of the samples with library complexity, strand specificity, coverage asymmetry and much more! Get the best from your RNA-seq data with SeqSolve.

Softgenetics NextGENe http://www.softgenetics.com/NextGENe_11.html NextGENe Software includes analysis tools designed for RNA-Seq analysis of data DNA sequencing data from Roche/454 GS FLX, FLX Titanium & Junior, Applied Biosystems SOLiD System and Illumina GA & Hi-Seq systems. Transcriptome sequencing data is aligned to the reference sequence to allow for detection of alternative splice sites, gene fusions, exon skipping and intron retention. NextGENe detects which exons are present in the sample data and chooses the appropriate reference transcripts. Accurate expression levels can be evaluated and mutations are reported. Alignment of RNA-Seq data with NextGENe uses a specialized algorithm to accurately align reads along exon boundaries. Incoming search terms: commercial software for RNA seq

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commercial software for RNAseq analysis list of de package for rna-seq data partek tutorial understanding reads rna seq partek rna sequence analysis tutorial using partek rna-seq yeast partek seqsolve which gene ontology is used by partek

Apr
21

RNA-Seq News
Filed Under News | Leave a Comment

New Software Estimation of alternative splicing isoform frequencies from RNA-Seq data This algorithm, referred to as IsoEM, is based on disambiguating of information provided by the distribution of insert sizes generated during sequencing library preparation, and takes advantage of base quality scores, strand and read pairing information when available. The open source Java implementation of IsoEM is freely available at http://dna.engr.uconn.edu/software/IsoEM/ New Species BMC Genomics reports the transcriptome sequencing of two new species: Alfalfa and Guppy Alfalfa, (Medicago sativa [L.] sativa), a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. [abstract] The guppy (Poecilia reticulata) transcriptome, assembled do novo using 454 sequence reads, is presented and the authors evaluate potential uses of this transcriptome, including detection of sex-specific transcripts and deployment as a reference for gene expression analysis in guppies and a related species. Guppies have been model organisms in ecology, evolutionary biology, and animal behaviour for over 100 years. An annotated transcriptome and other genomic tools will facilitate understanding the genetic and molecular bases of adaptation and variation in a vertebrate species with a uniquely well known natural history. [abstract] New Deals Integromics and Ingenuity expand their co-operation with the integration of a fourth Integromics product to Ingenuitys IPA

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The SeqSolve analysis software, exclusively designed for Next Generation Sequencing (NGS), performs tertiary level analysis of RNA-seq data at the gene and transcript level for biologically relevant results. Integromics has already integrated three solutions with Ingenuitys IPA: Integromics Biomarker Discovery(R) for microarray data analysis, RealTime StatMiner(R) for the analysis of qPCR data as well as OmicsHub(R) Proteomics for the analysis and storage of proteomics data.

Apr
19

Construction of Normalized RNA-Seq Libraries for Next-Generation Sequencing

Filed Under Information | Leave a Comment

RNA-seq is a method for studying the transcriptome of cells or tissues by massively parallel sequencing of tens of millions of short DNA fragments. However, the broad dynamic range of gene expression levels, which span more than five orders of magnitude, necessitates considerable over-sequencing to characterize low-abundance RNAs at sufficient depth. Here, the authors describe a method that enables efficient sequencing of low-abundance RNAs by normalizing or reducing the range spanned by the most abundant RNA species to the least abundant RNA species. This normalization is achieved using an approach that was developed for generating expressed sequence tag (EST) libraries that uses the crab duplex-specific nuclease and exploits the kinetics of DNA annealing. That is, double-stranded cDNA is denatured, then allowed to partially re-anneal, and the most abundant species, which re-anneal most rapidly, are digested with crab duplex-specific nuclease. This procedure substantially decreases the proportion of sequence reads from highly expressed RNAs, facilitating assessment of the full spectrum of the sequence and structure of transcriptomes. Christodoulou DC, Gorham JM, Herman DS, Seidman JG. (2011) Construction of Normalized RNA-seq Libraries for Next-Generation Sequencing Using the Crab Duplex-Specific Nuclease. Curr Protoc Mol Biol. Chapter 4:Unit4.12. [abstract] Incoming search terms: rnaseq normalization normalization rna-seq normalization rna seq data normalisation rna-seq RNA-seq normalization RNA Seq normalization rna-seq pyrosequencing normalized RNA-Seq normalized library
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normalization of RNA-seq

Mar
25

Uncovering the Complexity of Transcriptomes with RNA-Seq

Filed Under Publications | Leave a Comment RNA-Seq is perhaps the most complex NGS application In recent years, the introduction of massively parallel sequencing platforms for Next Generation Sequencing (NGS) protocols, able to simultaneously sequence hundred thousand DNA fragments, dramatically changed the landscape of the genetics studies. RNA-Seq for transcriptome studies, Chip-Seq for DNA-proteins interaction, CNV-Seq for large genome nucleotide variations are only some of the intriguing new applications supported by these innovative platforms. Among them RNA-Seq is perhaps the most complex NGS application. Expression levels of specific genes, differential splicing, allele-specific expression of transcripts can be accurately determined by RNA-Seq experiments to address many biological-related issues. All these attributes are not readily achievable from previously widespread hybridization-based or tag sequence-based approaches. However, the unprecedented level of sensitivity and the large amount of available data produced by NGS platforms provide clear advantages as well as new challenges and issues. This technology brings the great power to make several new biological observations and discoveries, it also requires a considerable effort in the development of new bioinformatics tools to deal with these massive data files. The paper aims to give a survey of the RNA-Seq methodology, particularly focusing on the challenges that this application presents both from a biological and a bioinformatics point of view. J Biomed Biotechnol 2010 Costa V, Angelini C, De Feis I, Ciccodicola A

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nudus mature

Oct
22

Database of microRNA isomiRs from next-gen sequencing experiments

Filed Under Databases | Leave a Comment New RNA-Seq methods give us the ability to comprehensively and accurately assess the levels of the entire repertoire of miRNAs (including all sequence variants) in a given sample. Researchers at the Institute for Systems Biology have conducted a comprehensive survey of microRNA isomiRs from human and mouse samples using next generation sequencing platforms and constructed a database that catalogs all microRNA isomiRs. Read more Incoming search terms: rna seq database next generation sequencing database data base for next generation sequencing Next generation sequencing miRNA miRNA-seq true seq miRNA rna seq analysis mirna primers database isomir database iso-mirs rna seq for micro Rnas Next Page

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