February 15, 2010

A combined approach to genome sequencing

The aim of this research project is to sequence the genomes of four Pseudomonas fluorescens isolates from two different cave environments. The resulting genome sequences will allow us to estimate of the selective pressure on the same species in two caves and then understand the overall selection pressures in nutrient starved caves. The additional P. fluorescens genomes already available for Pf-5, SBW25 and Pf0-1 will also allow comparative genomics for completely different environments and further analysis of the P. fluorescens pan-genome. I’ll outline our sequencing strategy below.

De Novo genome sequencing

If there are genomes already available for P. fluorescens strains then the obvious choice should be comparative based sequencing and assembly. However comparative analysis of three P. fluorescens species shows the genomes of P. fluorescens strains are much more dissimilar than might be expected for strains of the same species. Therefore it is likely the genomes our cave strains will be similarly unrelated to existing sequences.

Based on this we’re going to use 454 sequencing and perform de novo assembly of two P. fluorescens isolates; we’ll sequence a single isolate from two different cave sites. We would have preferred to have sequenced four genomes but this was ruled out by the low coverage per genome (~9X) and additional sample preparation costs which I’ve discussed previously. So instead we’ll sequence two genomes on a 454 plate split in half using the standard rubber gasket. Each sample will be prepared as a combination of paired-end and shotgun reads. This will provide uniform 35-40X coverage for each genome with the paired reads to improve assembly.

Comparative genome assembly

We ruled out comparative assembly using short but high read sequencing, such as Illumina or AB SOLiD because of the anticipated low sequence identity between P. fluorescens strains. Nevertheless once we have two de novo assembled cave genomes hopefully we can do comparative assembly of other cave isolates using these as scaffolds. Previous P. fluorescens genomes show a low sequence identity but we hope that isolates of the same species from the same site will have enough genome similarity to allow one genome to act as a scaffold for the comparative assembly of a second.

Therefore we will take a two further isolates of the same species from each site and send them for AB SOLiD sequencing. This will provide 90X coverage for each genome at a much cheaper price compared with 454 sequencing. Hopefully the combination of 454 and AB SOLiD will produce large amounts of data to compare variability between strains and sites.

February 11, 2010

Adapting to generating my own data

I’ve spent the last five years as a computational scientist and my research begins with pulling data out of files. I’m far removed from the laboratories that generated the data in the first place. This past month however I’ve had to learn and decide about producing enough data to generate a complete genome. Prior to starting this post doc in November 2009 I assumed that second generation sequencing easily allowed small labs like us to obtain complete genome sequences. The reality however incurs problems I would have never considered.

For example: paired-end sequencing is useful for assembling sequence reads into a de novo scaffold because the distance between each pair of reads is known. However the extra effort required to prepare a paired-end sample results in an extra cost of a couple of thousand dollars. The expenses involved in research and how this affects the project outline are not something I have had to consider before because all I usually need is a computer and a desk.

Apart from just cost we also have to decide how many genomes we want to sequence and how to do this on a single 454 plate. One approach is to use a rubber gasket to divide the plate into 2, 4, 8, or 16 sections and allocate a single sample to each section. The downside of this approach is that the gasket covers the sequencing wells on the plate and therefore the more plate is divided the less the available sequencing capacity. The alternative to the rubber gasket is to label each sample with molecular barcodes however this will incur more costs because of the additional sample preparation.

When determining how many genomes to sequence we also had to consider the amount of sequence coverage for each genome. As we try to sequence more genomes there is less read depth for each individual genome and therefore each genome is harder to assemble. This is a constraint on our research aim of sequencing four Pseudomonas fluorescens isolates. This means our choice of research question is a balance of what we can theoretically achieve given the costs and amount of sequencing coverage available on a 454 plate.

Choices

I’m writing this as a from the the point of view of my initial surprise about the difficulties of planning sequencing project rather than to complain. The people who will do the sequencing for us been very helpful. Also it’s cheaper second generation sequencing that has made this research project possible.

January 5, 2010

Genomics in a small microbiology lab

My post-doc is doing genomics of micro-organisms from starved cave environments. Several universities in the Kentucky area have banded together to get a sequencer which allows a small microbiology lab like ourselves to do sequencing for a few thousand dollars. The biology department here doesn’t have the dedicated computing cluster required for genomic assembly and analysis however the availability of on demand computing resources means this isn’t a problem as we can rent a virtual machine with 64GB of RAM by the hour. The only bottleneck in my project will therefore be my ability to formulate a research question and properly analyse The genomic data.

The availability of cheaper sequencing and by-the-hour computer time means that smaller research laboratories are no longer restricted in their ability to do genomics. It’s not hard to imagine a few years ago that sequencing costs put novel genomics out of reach for most labs, while only labs at large institutions had access to dedicated computing facilities. From my experience of moving from a large to small university it seems the financial and infrastructure barriers for doing genomics are now much lower. Genomics, in microbes at least, can now be carried out by hundreds of smaller labs instead of clustered at a few large sequencing centres and universities.

I remember when I started doing my masters five years ago that most papers began by discussing the "explosion of sequence data", but I think the availability of cheaper sequencing means that the explosion is just beginning. Now is a great time to be a bioinformatician – sequencing and computational power are now much easier to access and the problem will be finding people that can manage and process the data.

December 22, 2009

Deciding what genomes to sequence



We’re aiming to sequence three genomes from three different cave environments, where each cave differs in the degree of nutrient starvation. We will sequence P. fluorescens isolates from each cave and examine how the genomes have adapted to the starved cave environments compared with the available genomes of P. fluorescens from soil or plants.

We’ll be using Roche/454 sequencing which provide ~800,000 reads of genomic DNA where each read is approximately 500 nucleotides in length. In total a single 454 plate should provide 400Mbp of sequence data. Previous sequencing of P. fluorescens has the shown the genome size is just under 7Mbp and therefore if we sequenced a single P. fluorescens genome this would generate 400Mbp / 7 Mbp = 57X coverage. We would however like to sequence multiple genomes and there are two options for this.

Rubber grid

A rubber grid can be placed over the sequencing plate to divide it into individual segments, where each segment can be used to sequence a genome. The rubber template however covers approximately one third of the sequencing plate and will reduce the amount of reads from 400Mbp to 266Mbp. Therefore if we sequenced four P. fluorescens genomes using the rubber template to divide the plate this would theoretically provide 10X coverage for each genome.

Sequence tags

The second approach to sequencing multiple genomes involves using sequencing tags. Each DNA fragment has a small oligonucleotide tag attached, where each tag is unique to one of the P. fluorescens isolates. As the fragment is sequenced the tag is also sequenced, and this allows the sequenced DNA to be attributed to a source genome based on the attached tag. There is therefore no need to use a rubber gasket and the full 400Mbp of sequence data can be produced from the plate. This could theoretically provide 14X coverage for 4 genomes, or 11X coverage for 5 genomes. This therefore may seem like the obvious choice, but the sequencing facility tried using sequencing tags before and therefore there is a risk in trying something for the first time.

Making a decision

Over the next few days we have to decide the aim of this project, especially since my funding is only for one year. One option is to sequence one genome from each cave and then compare the cave genomes with existing P. fluorescens genomes. This would give some indication of how the genomes differ between caves and how they are adapted to caves.

A second approach could be to sequence multiple genomes from two caves. This would allow not only examination of how each cave has shaped the genome but also how the variable the genome is between the same species in the same environment.

December 15, 2009

Starting a new post doc at NKU

Two weeks ago I started my new job as a post-doctoral researcher at Northern Kentucky University. I’ll be working in the Barton geomicrobiology lab doing bioinformatics. I’m looking forward to being a bioinformatician in a microbiology laboratory and I think the combination of computation and wet skills in the lab complement each other.

I’ll be working on doing comparative genomics of Pseudomonas species isolated from cave environments. Microbes growing in caves often have limited rRNA similarity with existing characterised microbes and a low rRNA similarity is indicative of a correspondingly low genome similarity. Several Pseudomonas soil and human pathogen species have already been sequenced which means the genomes of cave Pseudomonas can be compared with the genomes of sequenced Pseudomonas species.

My PhD at Manchester was in the area of systems biology and molecular evolution so the position here will be a chance for me to learn new skills in genome assembly and annotation. As my first post doc I think it’s also important for me to publish and establish my career as a scientist. Furthermore I will have to think about what direction I want to take my career and what I want to spend the next several years doing.

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