Next-gen. Haemonchus contortus genomics. CARS at ICOPA XII, August 2010. The life cycle of H. contortus. Refs.: Gasser, R.H., unpub. review; Nikolaou and Gasser (2006), Int. J. Parasitol. 36 , 859-868; Prichard and Geary (2008), Nature 452 , 157-158. H. contortus ' evolutionary context.
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Next-gen. Haemonchus contortus genomics CARS at ICOPA XII, August 2010
The life cycle of H. contortus Refs.: Gasser, R.H., unpub. review; Nikolaou and Gasser (2006), Int. J. Parasitol. 36, 859-868; Prichard and Geary (2008), Nature 452, 157-158.
H. contortus' evolutionary context C. briggsae C. remanei Elegans C. brenneri C. elegans Caenorhabditis C. japonica C. sp. 3 PS1010 Droso-philae C. drosophilae C. sp. 1 SB341 Heterorhabditis bacteriophora Haemonchus contortus Pristionchus pacificus Meloidogyne hapla Meloidogyne incognita Brugia malayi
Protein and DNA functions are conserved Ce-ant-1.1 * * * Hc-ant-1.1 *MonepantelR mutation Refs.: Rufener et al. (2009), PLoS Pathog. 5, e1000380 ; Hu et al. (2010), Biotechnol. Adv. 28, 49-60.
Next-generation sequencing in a nutshell Ref.: Miller et al. (2010), Genomics 95, 315-327.
Assembly strategy used for C. sp 3 PS1010 Assemble DNA reads Velvet Filter out E. coli contigs Map RNA reads onto assembly Repeat once ERANGE (Bowtie + BLAT) Scaffold contigs with RNA RNAPATH Final assembly
RNA scaffolding Velvet genomic supercontigs RNA-seq exons Within-supercontig RNA-seq reads Cross-supercontig RNA-seq reads RNA-mediated scaffolding of Velvet genomic supercontigs (RNAPATH) Velvet+RNAPATH supercontigs
PS1010 assembly statistics Genome size: 100 Mb [?]. Est. genomic coverage: ~170x. An additional 4.6 Mb of Velvet supercontigs matched E. coli.
H. contortus (McMaster) assembly statistics Genome size: ~290 to 340 Mb. Est. genomic coverage: ~35x.
Only 40% of reads map to the assembly Of reads which did map, 50% went to ≥1.8 kb contigs. Reads mapped with bowtie by Titus Brown.
What is to be done? 1. Brute force: push coverage up to at least 50x. Latest round of sequencing just raised it to ~47x. 2. Larger insert sizes: current libraries are ≤325 nt. ~500 nt goal; also, try jumping libraries (after PS1010 test). 3. Work smarter: remove erroneous or highly repetitive reads. New method for removing low-freq. 32-mers from Brown et al.: http://ivory.idyll.org/blog/jul-10/kmer-filtering.html http://ivory.idyll.org/blog/jul-10/illumina-read-phenomenology.html
Thanks: Robin Gasser Isolated genomic DNA Bronwyn Campbell Isolated stage- and sex-specific RNA Neil Young Aided DNA and RNA work Ali Mortazavi Devised RNAPATH; earlier H. contortus assembly Brian Williams cDNA library construction Lorian Schaeffer Illumina sequencing Igor Antoshechkin Optimized sequencing protocols Titus Brown k-mer filtering Jason Pell, Adina Chuang Jacobs Genome Center Infrastructure and funding ARC, NIH, and HHMI