Posts tagged Sequencing

Thule, Greenland Year Two

Sequence data from the previous year allowed us to determine the overall microbial population in each site and this year we decided to focus on the Rich Lake site which seem to have representation of nearly all microbes found in the other sites. So lucky for us we only had to work on one site this year rather than six. This in itself had me excited to go back to Thule. After a five-hour flight on a military plane from BWI I finally arrived to Thule Greenland where we were greeted by the Colonel as well as other high ranking military officials at the hanger. Once I cleared the customs processing area, I arrived to the dorm where the other scientists were living. It was a little different from last year’s accommodations but nevertheless the luxuries of WI-FI, Internet and cable TV were all available. As I am anxious to get to the field and see the changes in the Rich Lake site, we were given some interesting news. That day was not a good day to travel to the site because a mother polar bear and her two cubs were spotted nearby not too long ago by military police. However, we managed to get other work done by preparing the schedule for the sampling, cultivation and other labwork.

 

The next few days consisted of preparing culture media, cultivation traps and diffusion chambers, and going out into the field (polar bear spray in hand; yes it’s a real thing!). We were extra careful in the field since there was quite a bit of fog in the area that did not seem to go anywhere and fog happens to be the same color as polar bears. The fog did however make it a bit easier to sleep since most of the sunlight was covered and when there’s 24 hours of daylight from mid-April until September, a little fog can still serve a purpose.

Rich Lake Site

Rich Lake Site

Greenland

Greenland

JCVI Hosts South African Scientists to Share Microbiome Research Techniques

Two scientists from the University of Cape Town, South Africa have joined Dr. Bill Nierman’s lab for the next month as part of NIH’s Human Heredity and Health in Africa (H3Africa) Initiative, a training program designed to build out technical biological skills in the African research community. This training relates specifically to developing techniques around the area of microbiome analysis, a relatively new field in the biological sciences.

Microbiome analysis for the collaborative study is looking at entire community of microorganisms in the respiratory tract of South African infants to better understand how the microbiome is associated with infant pneumonia and wheezing episodes. The expectation is that the organisms that reside in the infant respiratory tract will provide protection from or a predisposition to the pneumonia or wheezing episodes.

 

The Nierman Group

The Nierman group left to right Sarah Lucas, Bill Nierman, Shantelle Claassen, Mamadou Kaba and Stephanie Mounaud (unpictured Jyoti Shanker and Lilliana Losada) welcomes visiting scientists Ms. Classeen and Dr. Kaba from University of Cape Town for a month long training in microbiome sequencing and analysis.

Mamado Kaba, MD, PhD and colleague Shantelle Claassen from the University of Cape Town will be working closely under the guidance of JCVI’s Stephanie Mounaud who is functioning as the project manager and coordinating the laboratory components of a similar project at JCVI studying the microbiomes of inafnts in the Philippines and also in South Africa. These studies are sponsored by the Bill and Melinda Gates Foundation. The training will focus initially on preparing samples for DNA sequencing on a modern DNA sequencing platform, the Illumina MiSeq instrument. Once the sequence reads are off the sequencer, the instructional focus will shift to analysis of the reads by means of an informatics pipeline that develop phylogenies, or family trees, of the microbes that are obtained from the infant respiratory tract so that the abundance and relatedness of the microbes can be established. The bioinformatics training will be provided by Jyoti Shankar, the statistical analyst working on the Gates Foundation Project.

Mamadou Kaba is a Wellcome Trust Fellow working in the Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town. Mamadou’s research interests include the molecular epidemiology of infectious diseases and the study of human microbiome in healthy and disease conditions. He has contributed in establishing a new research group conducting studies on how the composition of the upper respiratory tract, gastrointestinal, and the house dust microbial communities influences the development of respiratory diseases.

Prior to joining the University of Cape Town, Mamadou worked as Research Associate at the Laboratory of Medical Microbiology, Timone University Hospital, Marseille, France, where he studied the epidemiological characteristics of infection with hepatitis E virus in South-eastern France.

Shantelle Claassen is pursuing a Masters degree in the Division of Medical Microbiology at the University of Cape Town. She has completed a BSc (Med) Honours degree in Infectious Diseases and Immunology at the University of Cape Town, during which she examined the relative efficacy of extracting bacterial genomic DNA from human faecal samples using five commercial DNA extraction kits. The DNA extraction kits were evaluated based on their ability to efficiently lyse bacterial cells, cause minimal DNA shearing, produce reproducible results and ensure broad-range representation of bacterial diversity.

Mamadou and Shantelle are currently involved in an additional prospective, longitudinal study of which the primary objective is to investigate the association between fecal bacterial communities and recurrent wheezing during the first two years of life.

JCVI Viral Finishing Pipeline: a Winning Combination of Advanced Sequencing Technologies, Software Development and Automated Data Processing

JCVI viral projects are supported by the NIAID Genomic Sequencing Center for Infectious Disease (GSCID). The viral sequencing and finishing pipeline at JCVI combines next generation sequencing technologies with automated data processing. This allowed us to complete over 1,800 viral genomes in the last 12 months, and almost 8,800 genomes since 2005.

Viral Projects at JCVI

JIRA Viral Sample Tracking Workflow

Our NextGen pipeline, which utilizes SISPA-generated libraries with Roche/454 and Illumina sequencing, enables us to complete a wide variety of viral genomes including challenging samples. Automated assembly pipeline employs CLCbio command-line tools and JCVI cas2consed, a cas to ace assembly format conversion tool. Our complimentary Sanger pipeline software is currently being integrated with the NextGen pipeline. This will improve our data processing and will allow us to use validation software (autoTasker) more efficiently.

Assembly of Repetitive Viral Genomes

Genome Organization of Varicella-Zoster

Assembly of Novel Viral Genomes

CLC Assembly Viewer Representation

Promoter of Bat Genome

Promoter of Bat Genome

During the past year we have found that novel viruses, repetitive genomes, and mixed infection samples could not be easily integrated with our high-throughput assembly pipeline. We have developed an assembly and finishing process that utilizes components of the high-throughput pipeline and combines them with manual reference selection and editing. Using this approach we completed novel adenovirus genomes and mixed-infection avian influenza genomes, and improved assemblies of previously unknown arbovirus genomes. We are currently working on optimizing and automating this new pipeline.

Assembly of Mixed Viral Genomes

Consed Representation of Mixed Viral Sample

Consed Representation of Mixed Viral Sample

Repetitive genomes have long been known to present great challenges during assembly and finishing. We are presenting a new approach to assembly and finishing of repetitive varicella genome that is based on separating it into overlapping PCR amplicons followed by merging sequenced amplicons during assembly.

To streamline our viral pipelines, we have fully integrated them with JCVI’s LIMS and JIRA Workflow Management to create a semi-automated tracking interface that follows the progress of viral samples from acquisition through to NCBI submission. This allows us to process a large volume of samples with limited manual interaction and, at the same time, gives us flexibility to work on challenging and novel genomes.

Acknowledgements

The JCVI Viral Genomics Group is supported by federal funds from the National Institute of Allergy and Infectious Disease, the National Institutes of Health, and the Department of Health and Human Services under contracts no. HHSN272200900007C.

Bat coronavirus project is collaboration with Kathryn Holmes and Sam Dominguez, University of Colorado Medical Center.

The authors would like to thank members of the Viral Genomics and Informatics group at JCVI.

References

Viral genome sequencing by random priming methods. Djikeng A, Halpin R, Kuzmickas R, Depasse J, Feldblyum J, Sengamalay N, Afonso C, Zhang X, Anderson NG, Ghedin E, Spiro DJ. BMC Genomics. 2008 Jan 7;9:5A virus discovery method incorporating DNase treatment and its application to the identification of two bovine parvovirus species.  Allander T, Emerson SU, Engle RE, Purcell RH, Bukh J.

Note

This post is based on a poster by Nadia Fedorova, Danny Katzel, Tim Stockwell, Peter Edworthy, Rebecca Halpin, and David E. Wentworth.

Entamoeba histolytica research presented at the Molecular Parasitology Meeting

Entamoeba histolytica causes invasive intestinal and extraintestinal infections, known as amoebiasis, in about 50 million people and still remains a significant cause of human death in developing countries. However, for unknown reasons, fewer than 10% of E. histolytica infections are symptomatic (causing symptoms such as diarrhea, dysentery or liver abscess). The J. Craig Venter Institute is among the institutions awarded the NIAID Genome Sequencing Centers for Infectious Diseases (GSCID) contracts to provide high-quality genome sequencing and high-throughput genotyping of NIAID Category A-C priority pathogens.

Photo of Entamoeba histolytica

Entamoeba histolytica in the trophozoite stage.

A GSCID project led at JCVI by Dr. Elisabet Caler includes performing whole-genome sequencing of Entamoeba phenotypic variants from symptomatic, asymptomatic and liver abscess-causing strains chosen to include a range of clinical manifestations and taken from human cases, as well as strains grown under different conditions. Our objective is to develop a genome-wide landscape of Entamoeba diversity to understand how sequence variations in the parasite relate to pathogenicity (ability to cause disease) and clinical outcome.

The Molecular Parasitology Meeting held at the Woods Hole Oceanographic Institution, Woods Hole, MA last week provided a window into the exciting science of Parasitology.  The keynote speaker, Fotis Kafatos, spoke on “Major Challenges to Global Health in the Tropics and Beyond–Insect Vectors of Malaria and Other Parasitic or Viral Diseases.”  Dr. Kafatos stressed that a multi-pronged approach to the control of malaria is necessary to prevent the devastating loss of life that malaria causes.

Woods Hole Oceanographic Institution

A view of Woods Hole Oceanographic Institution.

The many excellent papers and posters provided an overview of the field, including   Plasmodium falciparum, Toxoplasma gondii, the trypanosomes, Giardia lamblia, Trichomonas vaginalis, Entamoeba histolytica, Schistosoma species, Babesia bovis, and associated vectors.  Topics spanned basic biology, drug design, sequencing and host-pathogen interactions.

I presented an overview of the Entamoeba sequencing project at the meeting.   Discussions as a result of the presentation included questions about the details of sequencing and handling the next-generation sequencing data.   We had animated discussions about methods for assembly of the DNA sequences, including reference-guided vs de novo assembly.   Many attendees were impressed with JCVI’s open-source METAREP metagenomic tool (J. Goll, et al., Bioinformatics 2010).  Determination of the best methods for the analysis of differences in the clinical isolates generated much discussion.  Entamoeba researchers see the sequences as a great resource and are looking forward to being able to mine the data.  One, from India, was very excited that he was going to have about 15 times the resources he has had in the past, since he has had only had one genome to mine up until now.

The Molecular Parasitology Meeting was an excellent venue for scientific exchange.  The Entamoeba histolytica GSCID project will help us understand the pathogenicity of Entamoeba histolytica, and has the potential to save lives in developing countries.

HMP Consortium – St. Louis Missouri

Human Microbiome Project Consortium – September 2010 – St Louis, Missouri

We received warm welcome messages from Dr George Weinstock and Dr Jane Petersen as well as a humorous welcome from Dr Larry Shapiro, Dean of Washington University Medical School. 

It was wonderful to see so many scientists come together to share the progress on their individual HMP related demonstration projects.  Our own demonstration project with Dr Zhiheng Pei, involving the esophagus microbiome and how that relates to esophageal adenocarcinoma (EA), was quite unique compared to the other projects as we were the only group to focus on the correlation between bacterial population and a form of cancer. 

With over 400 participants and 59 speakers, the conference was quite successful and very interesting.  JCVI Director Dr Karen Nelson did a wonderful job moderating one of the segments.  Dr Roger Lasken also gave a thorough presentation on his lab’s single cell approaches to genomic sequencing of uncultureable bacteria.  Johannes Goll gave a great presentation on his recent work with an open source tool called METAREP (recently published in Bioinformatics 8/26/2010), which is designed to help scientists with analyzing annotated metagenomic data.  And Dan Haft presented his interesting work with algorithmically tuning protein families from reference genomes for systems discovery. 

Overall the conference was quite interesting and informative.  I continue to wish all of the participating sequencing centers, PIs, and others involved with the HMP much success with their projects. 

Hope to see everyone in Vancouver!!!

Looking for a Few Good Genomes (to sequence)!

The JCVI is one of three centers funded by the National Institutes of Allergy and Infectious Disease (NIAID) to provide sequencing and genotyping services to the infectious disease community.  We are continually looking for researchers who would like to have organisms of research interest to them sequenced and become a resource for the community. The costs are covered under the NIAID contract to the JCVI Genome Sequencing Center for Infectious Diseases (GSC) and therefore of no cost to the investigators.

The JCVI GSC provides the infectious disease research community with rapid and cost-effective high-quality sequencing services for pathogenic microorganisms including viruses, bacteria, fungi, protozoa, and invertebrate vectors of disease. The center is focused on NIAID Category A-C priority pathogens, related organisms, clinical isolates, closely related species, and microorganisms responsible for emerging and re-emerging infectious diseases and their hosts. Genotyping services are offered by the Center in order to study the variation in host response. The center also offers expertise in pathogen biology with the ultimate goal to use the sequencing and genotyping data to develop new diagnostics, vaccines, and drugs.  Data generated from the sequencing and genotyping projects will be released to the scientific community in accordance with the NIAID Data and Reagent Sharing and Release Guidelines.

JCVI has completed many projects to date and over 15 are ongoing or about to start. See here for information about these projects and the collaborators.  We are currently in year 2 of our second five year contact and over 70 known publications have resulted from these collaborative efforts.

We particularly encourage multi-collaborator projects that will provide the most impact for the scientific community.  The sequencing and genotyping projects to be conducted by center are selected from white paper proposals that can be submitted by investigators worldwide, including academia, not-for-profit organizations, industry, and government.  Information about the application process is available here.

Please contact us with questions and for advice about developing a white paper proposal.  A JCVI project lead will be assigned to each person/group who submits a white paper to help them with the process and ensure the best chance for success.  We look forward to hearing from you!