Posts in category Sequencing

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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!!!

DNA microarrays vs RNAseq — The winner and new heavyweight champion is?… It’s a draw.

In the past year or so there have been several articles stating that the death of microarray technology is growing near. These proclamations are due to the more recently introduced methodology referred to as RNAseq. At first glance I wrote these claims off as being silly and premature. Over time though I am starting to appreciate that while the claim is still clearly wrong, the issue isn’t about technology displacement at all. My group works on a wide variety of gene expression problems ranging from the simple in vitro microbial gene expression studies to problems involving metagenomic samples of enormous complexity (http://pfgrc.jcvi.org). In my experience, the decision of whether to use DNA microarrays or RNAseq seems straight-forward and unambiguous. In reality the two technologies couldn’t be more complementary. Given the simple in vitro gene expression study as an example, the low cost, short turn-around time, exceptional quantitative accuracy and ease of data generation all make the glass slide microarray the clear choice.

About three years ago our laboratory began thinking about how to examine gene expression of pathogenic bacteria in the context of host infection. The challenge here is related to assay sensitivity since any RNA preparation derived from such an infection will yield host RNAs in an abundance 100 to 1000 times greater than that obtained from the infectious agent. Labeled RNAs from such an experiment would yield little useful information about the bacterial gene expression using standard DNA microarray procedures. This represents a clear case for RNAseq. The bewildering number of sequence reads we have come to enjoy from NextGen sequencing platforms is only going to get better. The extra bonus of applying RNAseq is that both the host and infectious agent can be profiled at the same time. There are still many technical problems to work out for routine use of RNAseq, such as effective rRNA removal and the development of appropriate data analysis tools, but the effort required seems quite justifiable.

I can think of only one application that is beginning to take on momentum where an investigator may truly ponder which strategy makes the most sense to apply. The approach is one that mimics EST sequencing as a means of defining genes and gene limits. Our ability to properly identify coding DNA sequences (CDS) in genomes ranges from, very good to relatively poor, depending on the genome in question. Members of the parasite research community, to name one, have struggled with this problem often. Generally speaking, substantial over-calling of genes occurs making it difficult for scientists to begin down the path of functional characterization of their favorite genome. We have worked with such groups recently to provide an independent means of substantiating gene calls via evidence of RNA expression. The design of such studies involves generating RNAs from a wide variety of experimental conditions to enhance the frequency for evidence based gene calls. DNA microarrays designed as a low or high density tiling array can be acquired at a reasonable cost and with good experimental outcomes. The case for applying RNAseq rests on the increased ability to detect transcripts that are expressed at low levels that defy routine detection using DNA microarrays.

In summary, I find very few instances where one might reasonably stop to wonder which technology are best suited for the biological/technical problem at hand. When sensitivity isn’t limiting, use DNA microarrays. When sensitivity is everything, look toward the short read sequencing technologies. In the end it turns out that it wasn’t really a contest at all. We should all feel fortunate that each strategy has its appropriate time and place for use. Those researchers, like myself, that have invested much time and effort working with DNA microarrays have nothing to fear, we just have more options now. This is a good thing to say the least. Most of our gene expression work is supported through a contract from NIAID to the PFGRC under contract N01-A115447.

Scott Peterson http://www.jcvi.org/cms/about/bios/speterson/

Professor, JCVI

Scientific Director, PFGRC at JCVI