by: Mark Blaxter, Sujai Kumar, Gaganjot Kaur, Georgios Koutsouvoulos, Ben Elsworth
Parasite Immunol. 2011 Nov 2. doi: 10.1111/j.1365-3024.2011.01342.x.
PMID: 22044053
by: Paul Hunt, Axel Martinelli, Katarzyna Modrzynska, Sofia Borges, Alison Creasey, Louise Rodrigues, Dario Beraldi, Laurence Loewe, Richard Fawcett, Sujai Kumar, Marian Thomson, Urmi Trivedi, Thomas Otto, Arnab Pain, Mark Blaxter, Pedro Cravo
BMC Genomics, Vol. 11, No. 1. (2010), 499. doi:10.1186/1471-2164-11-499 Key: citeulike:8014259
http://dx.doi.org/10.1186/1471-2164-11-499
Abstract
BACKGROUND:Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.RESULTS:A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina(R) Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.CONCLUSIONS:This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.
]]>by: Sujai Kumar, Mark Blaxter
BMC Genomics, Vol. 11, No. 1. (2010), 571. doi:10.1186/1471-2164-11-571 Key: citeulike:8031121
http://dx.doi.org/10.1186/1471-2164-11-571
Abstract
BACKGROUND:Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base) reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC) to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode Litomosoides sigmodontis.RESULTS:Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects), which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs.CONCLUSIONS:Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies from different programs however gave a more credible final product, and this strategy is recommended.
]]>by: Laura Ferguson, Siu F. Lee, Nicola Chamberlain, Nicola Nadeau, Mathieu Joron, Simon Baxter, Paul Wilkinson, Alexie Papanicolaou, Sujai Kumar, thuan-Jin Kee, Richard Clark, Claire Davidson, Rebecca Glithero, Helen Beasley, Heiko Vogel, Richard Ffrench-Constant, Chris Jiggins
Molecular Ecology, Vol. 19, No. s1. (March 2010), pp. 240-254
http://dx.doi.org/10.1111/j.1365-294X.2009.04475.x
Abstract
The mimetic wing patterns of Heliconius butterflies are an excellent example of both adaptive radiation and convergent evolution. Alleles at the HmYb and HmSb loci control the presence/absence of hindwing bar and hindwing margin phenotypes respectively between divergent races of Heliconius melpomene, and also between sister species. Here, we used fine-scale linkage mapping to identify and sequence a BAC tilepath across the HmYb/Sb loci. We also generated transcriptome sequence data for two wing pattern forms of H. melpomene that differed in HmYb/Sb alleles using 454 sequencing technology. Custom scripts were used to process the sequence traces and generate transcriptome assemblies. Genomic sequence for the HmYb/Sb candidate region was annotated both using the MAKER pipeline and manually using transcriptome sequence reads. In total, 28 genes were identified in the HmYb/Sb candidate region, six of which have alternative splice forms. None of these are orthologues of genes previously identified as being expressed in butterfly wing pattern development, implying previously undescribed molecular mechanisms of pattern determination on Heliconius wings. The use of next-generation sequencing has therefore facilitated DNA annotation of a poorly characterized genome, and generated hypotheses regarding the identity of wing pattern at the HmYb/Sb loci.
]]>http://dx.doi.org/10.1101/gr.091231.109
Abstract
We inferred the rate and properties of new spontaneous mutations in Drosophila melanogaster by carrying out whole-genome shotgun sequencing-by-synthesis of three mutation accumulation (MA) lines that had been maintained by close inbreeding for an average of 262 generations. We tested for the presence of new mutations by generating alignments of each MA line to the D. melanogaster reference genome sequence and then compared these alignments base by base. We determined empirically that at least five reads at a site within each line are required for accurate single nucleotide mutation calling. We mapped a total of 174 single-nucleotide mutations, giving a single nucleotide mutation rate of 3.5 × 10−9 per site per generation. There were no false positives in a random sample of 40 of these mutations checked by Sanger sequencing. Variation in the numbers of mutations among the MA lines was small and nonsignificant. Numbers of transition and transversion mutations were 86 and 88, respectively, implying that transition mutation rate is close to 2× the transversion rate. We observed 1.5× as many G or C → A or T as A or T → G or C mutations, implying that the G or C → A or T mutation rate is close to 2× the A or T → G or C mutation rate. The base composition of the genome is therefore not at an equilibrium determined solely by mutation. The predicted G + C content at mutational equilibrium (33%) is similar to that observed in transposable element remnants. Nearest-neighbor mutational context dependencies are nonsignificant, suggesting that this is a weak phenomenon in Drosophila. We also saw nonsignificant differences in the mutation rate between transcribed and untranscribed regions, implying that any transcription-coupled repair process is weak. Of seven short indel mutations confirmed, six were deletions, consistent with the deletion bias that is thought to exist in Drosophila.
]]>I am doing a PhD in the evolutionary genomics of nematodes at the Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh
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Title: Evolutionary dynamics of conserved non-coding DNA elements: Big bang or gradual accretion?
Abstract:
Background Previous studies have found that DNA elements are highly conserved
in species from the same lineage, even though they do not code for proteins or RNA.
One proposed function of such conserved non-coding elements (CNEs) is that they
are cis-regulatory sequences for developmental genes which act as an abstraction of
genetic regulatory networks, thus allowing new animal body plans to be specified in
a modular way. This thesis tests the specific proposal by a previous study that CNEs
arose in a big bang in the Precambrian, approximately 600 million years ago.
Results The evolutionary dynamics of CNEs were studied by first identifying the
elements, and then examining their levels of identity over time. Pairwise comparative
sequence analysis of five contemporary nematode species provided a window into
the past because these species diverged at different points of time over the last ap-
proximately 700 million years. The number of CNEs and their basic properties for the
three most recently diverged species match the results obtained by other researchers,
although no clear trend is visible in the change in identity of CNEs with respect to
time since divergence. On adding two more species to the analysis, it was found that
no such elements could be identified for species pairs with deep divergences.
Conclusions The absence of CNEs for pairwise comparisons of species that diverged
earliest indicates that CNEs did not arise in a big bang. CNEs that were found for the
three Caenorhabditis species that diverged relatively recently (approximately 100 mil-
lion years ago) seem to be specific only to that clade. However, the big bang hypothe-
sis cannot be conclusively discarded because it is possible that the elements exist, but
are short, or have multiple components spread across the genome, and are therefore
difficult to detect. Missing CNEs could therefore be a limitation of computational ap-
proaches to discovering CNEs, and this study also suggests some ways to overcome
those limitations.
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