Advances in Genome Biology and Technology (AGBT) - Hollywood Beach, FL

13 - 16 February 2017

Stop by the poster session on Tuesday to meet two of our NGS experts and learn about advances in NGS library prep. Look for Doug Amorese at Poster #401 and Zulfiqar Gulzar at Poster #117! See below for details on the two posters we will be presenting.

Location: Poster Session
When: 14 February 2017

1:00PM

Simple library method enables selection for microbial populations, viral analysis and low abundance host transcript detection from nasal samples

Doug Amorese

NuGEN Technologies, Inc.

Maureen Peterson1, Stephanie Huelga1, Bin Li1 and Doug Amorese1

Darrell Dinwiddie2

(1)NuGEN Technologies Inc., San Carlos, California, USA (2)Department of Pediatrics, Clinical Translational Science Center, University of New Mexico Health Sciences Center

Finding the cause of a respiratory infection, especially in children, can be challenging.  They may have contracted a virus not tested for clinically, a novel virus, or possibly a bacterial infection.  Several studies exist where a previously unidentified infection was identified by performing RNA-Seq on cell-free RNA (cfRNA) by discarding reads that map to the host, and aligning the remaining reads to a pathogen database.  There are challenges to this approach: only small amounts of total RNA can be recovered, it is partially degraded, and pathogen RNA is contaminated with host and non-pathogen RNA.  Several reports have described the use of NuGEN’s isothermal amplification technology (SPIA) for the detection of viruses in human biological fluids.  Similarly, NuGEN’s AnyDeplete, an enzymatic target depletion technology, has been demonstrated to be a highly specific, customizable, and efficient solution for transcript depletion from RNA-Seq libraries.  

Here we describe the combination of these technologies: SPIA amplification with Ovation RNA-Seq V2, library generation via enzymatic fragmentation, and AnyDeplete (AnyDeplete) targeted depletion to study the causative agent(s) and host response in nasal samples obtained from asthmatic and non-asthmatic children with presumptive respiratory infections.  SPIA amplification of cDNA enabled 2 libraries to be generated 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.  This enabled efficient viral detection and detection of lowly 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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Doug Amorese

NuGEN Technologies, Inc.

Doug serves as Chief Scientific Officer for NuGEN and brings a wealth of experience and knowledge to the role as an accomplished leader in the development and commercialization of complex systems for nucleic acid analysis. He has extensive experience with microarray and DNA sequencing technologies, and has successfully directed a robust expansion of NuGEN’s product portfolio, including the introduction of novel sample preparation technologies for next-gen sequencing applications.  Prior to NuGEN, Doug led the research and development organization for Agilent Genomics. He has also held positions with DuPont and Life Technologies. Doug earned a PhD in biochemistry at Colorado State University, and served post-doctoral fellowships at the Salk Institute and at the University of Texas Health Science Center.  He is an avid bicyclist, and has used his bicycle as his primary mode of commuting for over 30 years.  Doug also volunteers at the local high school and enjoys woodworking in his spare time.

1:00PM

Customized depletion of unwanted poly-A transcript from the enriched mRNA-seq libraries

Zulfiqar Gulzar

NuGEN Technologies, Inc.

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

(1)NuGEN Technologies Inc., San Carlos, California, USA

Oligo dT based RNA Preparation is a common method for enrichment of mRNA from total RNA for sequencing. While two rounds of selection can deplete most of the rRNA, there continue to be uninformative, highly abundant mRNA transcripts such as housekeeping genes and mitochondrial genes still present in the sequencing libraries. These highly expressed genes are rarely differentially expressed between samples and a method to deplete these transcripts from the library can not only decrease the sequencing cost but also enrich remaining low expressing transcripts which may be more biologically informative. We describe a method capable of a customized depletion of any unwanted transcript from the enriched mRNA in addition to rRNAs.   The technique employs a simple sequence specific oligonucleotide probe and therefore is capable of custom depleting any cDNA fragment from the sequencing libraries without affecting the other remaining cDNA fragments. An illustration of the utility of this method has been demonstrated using libraries generated from ERCC control samples. We have shown successful depletion of the 4 most abundant transcripts which capture 70% of the total ERCC reads. Depletion of these ERCC transcripts resulted in a 2 fold increase in the enrichment of low expressing ERCC transcripts making them detectable with less than 4 million reads. We have also successfully applied this workflow to deplete globin mRNA from human whole blood mRNA and actin and myocin from Cardiac Skeletal mRNA. Depletion of the globin reads in human whole blood have allowed an increase in total number of remaining genes with a higher expression starting at the same number of reads. This method has allowed us to detect low expressing mRNA transcript which are biologically relevant to the experimental hypothesis.

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Zulfiqar Gulzar

NuGEN Technologies, Inc.

Zul serves as one of NuGEN’s lead Product Development Scientists. With extensive experience in DNA and RNA sequencing technologies, as well as microarrays, Zul is the scientific spearhead of our upcoming mRNA-Seq product. Prior to NuGEN, Zul was a Research Scientist at the Department of Urology at Stanford University leading a project for optimization of a platform and a workflow to isolate Prostate specific Circulating Tumor Cells and its analysis. Zul earned a PhD in Cancer Biology from Imperial College London. As an avid European Soccer fan, he spends early Saturday mornings catching up on the matches. He also volunteers tutoring the kids at a local community center in Milpitas, California and enjoys spending quality time with his family.