Abstracts

Abstracts received are listed here, in alphabetical order according to the surname of the presenter.
Additional abstracts will be listed as they are received.

MiSeq™ Personal Sequencing System

Graeme Bethel, Illumina

The MiSeq™ represents the next step in the evolution of Next Generation Sequencing leveraging 5 years of Illumina's proven TruSeq™ sequencing chemistry and its unmatched genomic coverage and accuracy. With the simplest and most scaleable targeted resequencing solutions, from 10's to 100,000's of targets, the MiSeq platform is uniquely suited to a range of applications from sample to analyzed report in as little as 8 hours. All applications, protocols, developed or produced on any of Illumina's sequencing platforms are compatible with the MiSeq as they share the same proven TruSeq™ chemistry:
· Small genome de novo sequencing, targeted resequencing, hybrid selection assays and structural variant detection are enabled by the integrated Paired-End read capability for 2 x 150bp reads coupled with the onboard assembler and variant caller.
· Pathogen screening, vaccine QC, clone checking, library QC, tissue typing and other rapid turn-around applications are enabled by the unmatched speed and scalable output range of 120Mb to >1.5 Gb, with full read length flexibilty and automated workflows.
· Capture and sequencing solutions for multiplexed-targetted and amplicon applications will be enabled for up to 384 amplicons in 96 samples.

The resequencing coverage limbo – how low can you go?

Mike Black, Rebecca Laurie, Les McNoe, Tony Merriman Department of  Biochemistry, University of Otago  

With  the  rapidly declining cost of whole genome sequencing,  there is considerable interest in using next generation sequencing technologies to conduct resequencing of human populations.  For studies of human disease, the goal is the identification of novel genetic variants (both SNPs and structural changes such as copy number variants) that are associated with modifying disease risk in a   population.  A major question when planning such studies involves calculating the amount of sequencing that is required, based on  the degree of genome coverage needed.  In this talk we will examine the relationships between depth of coverage and number of variants   discovered, both from theoretical and applied points of view.  These concepts will be illustrated using data from the 1000 Genomes Project, and from human resequencing data generated at the University of Otago.

 

Transcriptomics and Small RNAs from Trichomonas and Giardia

Lesley J. Collins  Institute of Fundamental Sciences, Massey University, Palmerston North, NZ

The two parasitic protists Giardia lamblia and Trichomonas vaginalis contain RNA-based systems found throughout eukaryotes, despite being very distant evolutionary-wise. Previously we sequenced small RNAs from these species to characterize small regulatory ncRNAs (~22nt) and medium length processing ncRNAs (>100 nt).  Combined with new RNA-seq transcriptomic data, we can now use this information to investigate Giardia and Trichomonas RNA systems in more detail, especially the relationship between their inherent dsRNA viruses and host viral defence systems.  This talk will highlight some of our latest research into protist RNA biology using NGS technology.

 

Adopting Optimal Technologies for Nucleic Acid Sequencing Experimental Outcomes

John Davis LifeTech

Genomics researchers have recently embarked upon the collection of unprecedented amounts of nucleic acid information. Such endeavours have positively impacted on a broad spectrum of scientific investigations and knowledge in a diverse range of fields spanning such themes as human health, speciation, agricultural selection and the study of complex ecosystems. Intrinsic to the benefits that the generation of such data provides, is the selection of appropriate technologies to both generate and analyse nucleic acid information. Life Technologies supports multiple platforms that provide benefits to researchers across a variety of experimental scales from single clone assessment to large genome experiments. Two platforms of particular interest in such endeavours are represented by the Ion Torrent Personal Genome Machine and the 5500 Genetic Analyzer.

The Ion Torrent Personal Genome Machine (PGM™) is simpler and quicker than any other sequencing technology. The PGM™ sequencer is a bench top system utilizing ground breaking and disruptive semiconductor technology that enables rapid and scalable sequencing experiments. Ion Torrent technology uses a massively parallel array of proprietary semi-conductor sensors to perform direct real time measurement of the hydrogen ions produced during DNA replication. A high-density array of wells on the Ion semiconductor chips provide millions of individual reactors while integrated fluidics allows reagents to flow over the sensor array. This unique combination of fluidics, micromachining, and semiconductor technology enable the direct translation of genetic information (DNA) to digital information (DNA sequence) rapidly generating large quantities of high quality data.

The 5500 Genetic Analyzer represents the newest enhancement to ligation based sequencing and caters to those researchers desiring very high sequencing accuracy and modular experiments through the potential utilization of a number of sample types during any sequencing run. Projects of these scales provide a number of challenges from a variety of perspectives. Aspects of the technical requirements and data resulting from Life Technologies will be presented.

 

Second generation sequencing and analysis of complex genomes

Kaitao Lai, Michal Lorenc, Paul Berkman, Kenneth Chan, Mike Imelfort, Sahana Manoli, Chris Duran, Hong Lee, Edmund Ling, Hana Šimková, Marie Kubaláková, Nela Rosic; Paulina Kaniewska, Emma Campbell, Pilar Hernandez, Jacqueline Batley, Jiri Stiller, Jaroslav Doležel and David Edwards

Second generation sequencing technologies and applied bioinformatics tools can provide an unprecedented insight into genome structure and variation. We have developed and applied novel bioinformatics tools and approaches for Illumina sequence data analysis with the aim of understanding complex genomes. These range from large genome polyploid crops such as wheat and Brassica species, to complex environmental metagenomic samples. Outcomes include more resilient crops and an understanding of how complex microbial populations interact with the environment.

 

Sequencing the NZ alpine cress Pachycladon: Lessons from EST library assembly and DEG analyses in an allopolyploid plant

Nicole Grünheit and Oliver Deusch, Institute of Molecular BioSciences, Massey University, Palmerston North

The New Zealand alpine cress Pachycladon originated between 1.6 and 0.8 million years ago by a hybridization event between two parental lineages that diverged 8 million years ago. As a consequence Pachycladon has two homeologous copies for most of its genes. Homeologous copies are on average 90 percent identical while homologous genes between P. cheesemanii and P. fastigiatum show 97 percent identity on the nucleotide level. The talk will focus on how the special characteristics described above affected the assembly of EST libraries from Illumina mRNA-Seq data as well as the identification of differentially expressed genes.

 

Best Practice approach to  NGS data processing: solutions and challenges

Chris Hartl Genome Sequencing and Analysis Group, Broad Institute, USA

One of the goals for Next Generation Sequence analysis is to map raw sequencer output to genetic variants, ideally variants with novel associations to one or more phenotypic traits. This talk walks through the Genome Sequencing and Analysis Group's best-practice approach to NGS data processing, detailing our solutions to many of the challenges posed by NGS data. In particular, this talk examines the pipeline we use to detect, genotype, and associate germline genetic mutations, the bulk metrics we use to assess the quality of our data at various stages, and the methods we employ to identify error and bias downstream of sequencing.


Whole Genome Sequencing to improve the NZ Dairy industry

Mike Keehan Livestock Improvement Corporation

LIC and ViaLactia BioSciences (VLB) in collaboration with the Primary Growth Partnership (PGP) fund have embarked on an ambitious whole genome sequencing programme. The goal of the program is the development of new capability and knowledge in the analysis and exploitation of the natural variation that exists in the genomic sequence. In this talk we present LIC as a biotechnology company with an extensive research and development investment and product line built on genomic technology. The whole genome sequencing will build on the previous investment and improve future applications of genomic selection in the LIC breeding scheme. Preliminary results, from the first sequenced genomes, will be presented on the validation of multiple common open source SNP calling pipelines based on a trio and a half sib family. The genomic distribution of variants within and between breeds will be discussed.  Conclusions for future sequencing strategies will be made. Practical experiences working with large NGS datasets will be shared.


Sequencing ancient Pacific voyagers using “home-made” in-solution hybridization capture

Michael Knapp1, 2, K. Ann Horsburgh1, 2, 3, Jo-Ann Stanton2, and Elizabeth A. Matisoo-Smith1, 2
1: Allan Wilson Centre for Molecular Ecology and Evolution
2: Department of Anatomy and Structural Biology, University of Otago
3: School of Geography, Archaeology and Environmental Studies, University of Witwatersrand

The Pacific is the last frontier of human settlement. The history of human settlement of the region only spans a few millennia. Nevertheless the origin of the first settlers is still controversial. The poor DNA preservation in the remains of early Pacific voyagers has long been an obstacle to genetic analyses of their origins. PCR enrichment and Sanger sequencing technologies are bias towards long molecules and have limited the success in sequencing ancient human remains. To overcome these limitations we have produced barcoded 454 sequencing libraries directly from DNA extracts of the remains of ancient Pacific voyagers using a protocol specifically designed for highly degraded DNA. Using “home-made” hybridization capture probes we were then able to enrich barcoded 454 libraries for human mitochondrial genome fragments of as little as 40 bp in length and sequence the complete mitochondrial genomes of pre-historic Polynesian settlers including that of one of the oldest burials from New Zealand, from the Wairau Bar site.

 

Identifying biological cause among millions of ‘bystander’ variations

Klaus Lehnert Vialactia 

A primary aim of the bovine genome sequencing programme recently commenced by ViaLactia Biosciences and LIC (funded by the Primary Growth Partnership) is the identification of sequence variations that are responsible for biological traits of commercial and scientific interest.

We hope to share our experiences with novel and established bioinformatics approaches, prediction of biological consequences, comparative genomics analysis, and classical genetics such as pedigree analysis. We aim to assess the utility of genome sequencing in principle, and individual techniques in particular, in the identification of a postulated genetic variation responsible for a dramatic change in milk composition. We also hope to provide insights into to efficient design of NGS studies aimed at the identification of causal variations.

 

New Zealand Genomics Limited: Off the drawing board and out to the scientists

Tony Lough New Zealand Genomics Limited, University of Otago

New Zealand Genomics Limited – NZGL – is a collaborative infrastructure providing New Zealand scientists with access to the significant equipment they need for large-scale genomics projects. Until recently, genomics based-projects in New Zealand could only be small in scale, limited by difficulties accessing analytical and bioinformatics expertise. The Ministry of Science and Innovation recognised these limitations and funded the establishment of NZGL. NZGL’s role is to provide genomics technology and bioinformatics services to New Zealand scientists – thereby underpinning research in a broad range of areas, including medicine, agriculture and the environment. NZGL also provides the framework for coordinating projects, analytical and bioinformatics support, data storage and sharing. The infrastructure has been a long time on the drawing board, but is now providing initial services to New Zealand scientists. In this presentation, NZGL Chief Executive will introduce the current services, outline what is scheduled for roll out in the medium-term and suggest future scope and possibilities.

 

SOLiD-SAGE analysis of the Deer Transcriptome

Paul Maclean, Rudiger Brauning, Nauman Maqbool, Jo Stanton and Colin Mackintosh University of Otago
 
Applied Biosystems SOLiD-SAGE technology was used to study differential gene expression in deer challenged with the causative agent of Johne’s disease. Susceptible and Resistant deer had their transcriptomes sampled over several time-points. This talk will detail the challenges of understanding a new technology and overcoming mapping difficulties due to the lack of reference deer gene set and incomplete genome assembly.


A tour through the tuatara transcriptome

Hilary Miller Allan Wilson Centre for Molecular Ecology and Evolution, School of Biological Sciences, PO Box 600, Wellington, New Zealand

The tuatara (Sphenodon punctatus) is a species of extraordinary zoological interest, being the only surviving member of an entire order of reptiles which diverged early in amniote evolution.  In addition to their unique phylogenetic placement, many aspects of tuatara biology, including temperature-dependent sex determination, cold adaptation and extreme longevity have the potential to inform studies of genome evolution and development. Despite increasing interest in the tuatara genome, genomic resources for the species are still very limited.  We aimed to address this by assembling a transcriptome for tuatara from an early-stage embryo, which will provide a resource for genome annotation, molecular marker development and studies of development and adaptation in tuatara.  RNA from the head, trunk and tail of the embryo was indexed and run in a single lane of an Illumina Genome Analyzer, producing a total of approximately 31 million paired-end 75 bp reads.  Using Velvet and Oases these reads were assembled into approximately 17,000 transcripts with an N50 of 724 and an average length of 522 bp.  Approximately 46% of these transcripts produce a significant match to mRNAs from chicken.  In this talk I will discuss the challenges in assembling and annotating a high-throughput sequencing dataset for an evolutionarily distinct organism. 

 

Next Generation Sequencing Simulations: Laying the Groundwork for Plant Genome Sequencing

Roger Moraga AgResearch Limited

Next Generation Sequencing (NGS) technologies have proven to be a boon for the study of non-model organisms, thanks to their affordability and wealth of data they provide. However, the rapidly evolving technology and humongous amounts of data present unique challenges that are not always obvious or easy to overcome. While the uncertainty of working with cutting-edge technologies cannot be completely eliminated, much of it can be minimized by spending some time developing a sequencing strategy tailored to the organism one wants to study. We present a brief overview of a range of simulation data from two plant genomes, Clover and Rice, and the kind of information which can be extracted from the in silico results, from the experiment design and the resource requirements point of view.

 

Parallel multi-amplicon sequencing using 454 GS FLX: a study of microsatellite evolution

Monika Zavodna1, Rudiger Brauning2, Andrew TM Bagshaw3 , Neil J Gemmell1
1Centre for Reproduction and Genomics, Department of Anatomy and Structural Biology, University of Otago, Dunedin
2AgResearch Limited, Invermay Agricultural Centre, Mosgiel
3Department of Pathology, University of Otago, Christchurch

Polymorphism in microsatellite DNA sequences derives mainly from variability in repeat number. Current models of microsatellite evolution regard replication slippage as the predominant mechanism through which variability in repeat number arises. Recombination, a common force in generating other forms of genomic variation may also contribute to microsatellite variability but if and to what extent recombination affects microsatellite mutational processes remains contentious.

To examine the role of recombination in microsatellite evolution, we located and designed primers for more than 250 microsatellite loci (in both, recombination hot spots and cold spots) in yeast (Saccharomyces cerevisiae) genome. All loci were PCR-amplified in eight separate replicate populations for both sexual and asexual, i.e. recombining and non-recombining, yeast strains that grew for 14 cycles of growth and sporulation in continuous culture (~700 generations). We used 454 GS FLX standard chemistry for parallel multi-amplicon sequencing and obtained in total over 350,000 reads for all amplicons. These reads were mapped to reference sequences and indels were detected using the CLC genomics workbench. Over 90% of obtained reads have been successfully mapped to references and about 95% of loci have been used for indel detection. In our presentation, we will report on the frequency of indels in microsatellite loci arising across the time, i.e. from generation zero to 700th within both sexual and asexual strains, and thus the frequency and nature of the mutations in relation to the presence or absence of genetic recombination.