ABRF - San Diego, CA

25 - 28 March 2017

ABRF 2017, "A forum for advancing today’s core technologies to enable tomorrow’s innovations," will bring together international leaders in our core disciplines to provide a vision of what can be done, and insight toward making further progress as we work in the present. Visit us at our booth #601 and snag some great giveaways!


When: 26 March 2017 - 10:30AM

10:30AM

Platinum Talk. Adult and Fetal Globin Transcript Removal for mRNA Sequencing Projects.

Piotr Mieczkowski

University of North Carolina, Chapel Hill

Piotr Mieczkowski1,3, Stephanie Marie DeYoung3, Neeta Vora2

1. Department of Genetics, Lineberger comprehensive Cancer Center, SOM, University of North Carolina at Chapel Hill; 2. Department of Ob/Gyn, Division of Maternal Fetal Medicine, SOM, University of North Carolina at Chapel Hill; 3. High Throughput Sequencing Facility, University of North Carolina at Chapel Hill

Preterm birth (PTB) is delivery prior to 37 completed weeks of gestation, which occurs following spontaneous labor or is medically induced. Our ultimate goal is to perform mRNAseq on umbilical cord blood and placentas obtained from pregnant women who deliver preterm and from matched controls who deliver at term. However, cord blood total RNA extracted from the umbilical cord samples demands a globin depletion protocol that will be applied to the RNA prior to RNA sequencing. It is known that globin mRNA does not contribute high value RNA sequencing information and because as much as 70% of the mRNA in a blood total RNA sample can be globin mRNA.  Thus, it is necessary to remove this globin RNA. Removing globin mRNA and rRNA from a blood RNA sample enables deeper sequencing for discovery of rare transcripts and splice variants and reduces the number of expensive sequencing reads that are wasted because they do not lead to prevention or treatment of disease. In this presentation we demonstrate NuGen globin reduction optimized protocol for mRNA sequencing from adult and fetal samples. 

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Piotr Mieczkowski

University of North Carolina, Chapel Hill

Research Associate Professor

Director of the High Throughput Sequencing Facility

University Of North Carolina

5:30PM

Poster Session. #B262. Harnessing the Power of SPIA Amplification for Viral Detection From Limited and Degraded Samples. 

Ashesh Saraiya

NuGEN Technologies, Inc.

Ashesh A. Saraiya1, Maureen Peterson1, I-Ching Wang1, Stephanie Huelga1, Bin Li1, Kurt S Schwalm2, Darrell L Dinwiddie2,3, Doug Amorese1

1. NuGEN Technologies, 201 Industrial Road, San Carlos, CA 94070 USA. 2. Department of Pediatrics, University of New Mexico Health Sciences Center, Albuquerque, NM, USA. 3. Clinical Translational Sciences Center, University of New Mexico Health Sciences Center, Albuquerque, NM, USA.

Viral detection from biofluids (blood, nasal swabs, CSF) is difficult due to a number of reasons, including low RNA quantity, poor RNA quality, low viral titers, and high background from the host.  qPCR is a highly sensitive method for viral detection but constraints in RNA availability only allow for limited testing of expected viruses.  Additionally, non-standard viruses, novel viruses, or potential bacterial infections can be missed.  RNA-Seq is an alternative method that has previously been used to identify unknown infections in an unbiased manner.   This method, however, suffers from high RNA input and quality requirements, background from unwanted reads and significant sequencing costs.  To address these challenges, we developed a single workflow that combines SPIA amplification, highly efficient library generation, and AnyDeplete (InDA-C) targeted depletion.  This workflow was used to study the causative agent(s) and host response in nasal swab samples obtained from asthmatic and non-asthmatic children with presumptive respiratory infections.  Even with limited and degraded RNA samples, the SPIA amplification produced sufficient cDNA to generate 2 libraries in parallel from a single sample.  Library 1 was depleted of all human rRNA and mitochondrial genes.  Microbial 16S rRNA reads from this library were aligned to databases to gain an understanding of the microbiome within the individual.  Library 2 was depleted of both abundant host transcripts as well as microbial rRNA transcripts enabling efficient viral detection as well as detection of rare expressed human transcripts (to determine allergic vs immune host response) with <1 million reads.  Data illustrating the efficiency of the depletion method, the coverage of the viral genome and the differences in host transcripts between asthmatic and non-asthmatic individuals will be presented.

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Ashesh Saraiya

NuGEN Technologies, Inc.

5:30PM

Poster Session. #B233. Customized Depletion of Unwanted Transcripts from RNA-Seq Libraries.

Benjamin Schroeder

NuGEN Technologies, Inc.

Benjamin Schroeder1, Zulfiqar Gulzar1, I-Ching Wang1, Lin Pham1 and Douglas Amorese1

1. NuGEN Technologies, San Carlos California

RNA-Seq has become a standard NGS tool for many avenues of research. There are, however, many variations to the technique, such as total RNA vs poly-A selected RNA input, methods of ribosomal RNA depletion, and various methods of maintaining strand orientation. Here we describe a flexible, modular workflow for generating RNA-Seq libraries from any organism. Users can select an upfront poly-A isolation module, or use total RNA as input. A high complexity, stranded library is generated from the core module using a mixture of oligo dT and random priming, resulting in complete 5’ to 3’ coverage of transcripts. Libraries can be sequenced directly, or further processed with a transcript depletion module. Targeted depletion of highly expressed transcripts can decrease sequencing costs and enrich low expressing transcripts which may be more biologically informative. Ribosomal transcripts are commonly targeted for depletion; however our method is capable of depleting any cDNA fragment from the sequencing library without affecting the other remaining cDNA fragments, due to the coupling of a simple oligonucleotide probe with enzymatic targeting specificity. For example, we have successfully applied this workflow to deplete globin transcripts from poly-A selected mRNA derived human whole blood. In another example of the utility of this modular, customizable workflow we show successful depletion of the 4 most abundant External RNA Controls Consortium (ERCC) spike-in transcripts, which comprise 70% of the total ERCC reads. Depletion of these highly abundant ERCC transcripts resulted in a 2 fold enhancement of low expressing ERCC transcript representation, making them detectable with less than 4 million reads. Depletion of highly abundant, non-informative transcripts provides the investigator a cost-effective method to detect low expressing transcripts which are biologically relevant to the experimental hypothesis.

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Benjamin Schroeder

NuGEN Technologies, Inc.