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Additional file 1: Read length distribution and assembly coverage. (A) Distribution of read lengths as generated by the 454 Genome Sequencer FLX/Titanium. The read length in base pairs (bp) is on the horizontal axis and the read count is on the vertical axis. The plot displays a bimodal curve with one peak at 250 bp and another one at 500 bp, typical for each one of the two platform types used for sequencing. (B) Shows the number of assembly positions on the vertical axis with a certain level of coverage on the horizontal axis. The plot shows that the average coverage is around 47 times. (PDF 55 KB)

Authors’ original file for figure 6

Additional file 5: Evolutionary analyses of a recently acquired Giardia gene. Evolutionary analyses of a recently acquired Giardia gene. (A) A maximum likelihood tree of an acetyl transferase gene (GLP15_874). Only representatives of the homologs showing the highest similarity to the Giardia sequences are included. The tree is based on 139 unambiguously aligned amino acid positions. (B) Alignment of conserved hypothetical proteins from G. intestinalis GS (GL50581_4454) and WB (GL50803_10192) to all detected bacterial homologs. A putative amino acid sequence from a G. intestinalis P15 pseudogene is included and the positions of frameshift and in-frame termination codons are indicated. (C) A maximum likelihood tree based on 137 unambiguously aligned amino acid positions in the alignment shown in (B). Accession numbers are shown in parentheses. Only bootstrap values >50% are shown in the trees. Unambiguously aligned regions were identified manually and removed. Representative homologs with high sequence similarity to these genes were selected using the BLAST-EXPLORER software. (PDF 337 KB)

Additional file 7: VSP extended polyadenylation signal and VSP phylogenetic tree. (A) Comparative analysis of the extended polyadenylation sequence of VSP genes. The conservation of the VSP polyadenylation motif was investigated using the MEME software. Downstream regions (200 bp) of VSP genes were extracted and queried for the presence of conserved motifs. The VSP genes of all three Giardia isolates carries a very similar conserved motif that extends over at least 16 bp and is strictly positioned 0-15 bp from the translational stop codon. The P15 motif is more strictly conserved and carries a few more semi-conserved positions for a longer motif than the corresponding motifs in WB and GS. (B) A phylogenetic tree of VSP genes from P15 and WB. The VSP phylogenetic tree is based on a clustalw2 protein alignment of 156 VSP genes from WB and P15. VSP genes were selected for alignment by all-against-all BLASTP, and only VSP genes that showed at least 65% protein identity to the best-matching non-identical hit were used for the alignment (n = 156). In order to improve the alignment, sequences were trimmed (190 aa were removed from the 5' part of all sequences). Subsequent clustering was performed using maximum likelihood as implemented in the software RAxML version 7.0.4 using with the rapid bootstrap setting (-f a). The tree was rendered using the ape package for R. Black dots represent nodes with > = 50% bootstrap support. The gene id (genbank locus tag) is indicated at the tip of the branch. Blue labels represent genes from WB while black labels represent genes from P15. The tree revealed two primary groups (A and B), which are distinguished based on their distance in the tree. (PDF 172 KB)

Additional file 8: Synteny breaks and PCR primers. Experimental verification of 10 examples of structural variation discovered in the P15 genome. (PDF 785 KB)

Additional file of Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate

Additional file 6: Non-coding RNA conservation. Conservation of non-coding RNAs across the three Giardia genomes. Non-coding RNAs are generally well conserved, with P15-WB pairs showing the greatest similarity. Examples of ultraconserved non-coding RNAs across all genomes include GLsR2 and GLsR8. (PDF 93 KB)

Background Genome-wide association studies have so far identified 56 loci associated with risk of coronary artery disease (CAD). Many CAD loci show pleiotropy; that is, they are also associated with other diseases or traits. Objectives This study sought to systematically test if genetic variants identified for non-CAD diseases/traits also associate with CAD and to undertake a comprehensive analysis of the extent of pleiotropy of all CAD loci. Methods In discovery analyses involving 42,335 CAD cases and 78,240 control subjects we tested the association of 29,383 common (minor allele frequency >5%) single nucleotide polymorphisms available on the exome array, which included a substantial proportion of known or suspected single nucleotide polymorphisms associated with common diseases or traits as of 2011. Suggestive association signals were replicated in an additional 30,533 cases and 42,530 control subjects. To evaluate pleiotropy, we tested CAD loci for association with cardiovascular risk factors (lipid traits, blood pressure phenotypes, body mass index, diabetes, and smoking behavior), as well as with other diseases/traits through interrogation of currently available genome-wide association study catalogs. Results We identified 6 new loci associated with CAD at genome-wide significance: on 2q37 (KCNJ13-GIGYF2), 6p21 (C2), 11p15 (MRVI1-CTR9), 12q13 (LRP1), 12q24 (SCARB1), and 16q13 (CETP). Risk allele frequencies ranged from 0.15 to 0.86, and odds ratio per copy of the risk allele ranged from 1.04 to 1.09. Of 62 new and known CAD loci, 24 (38.7%) showed statistical association with a traditional cardiovascular risk factor, with some showing multiple associations, and 29 (47%) showed associations at p < 1 × 10−4 with a range of other diseases/traits. Conclusions We identified 6 loci associated with CAD at genome-wide significance. Several CAD loci show substantial pleiotropy, which may help us understand the mechanisms by which these loci affect CAD risk.

Biological characterization of the new assemblage AII isolates. Trophozoite growth rate in medium supplemented with either bovine or human serum. (DOCX 54 kb)

Distribution of read length and assembly positions. (DOCX 220 kb)

HCMP variability. Variability of HCMP genes in the different assemblage A isolates, WB, AS98 and AS175. (DOCX 686 kb)

Strcutural modellling of two Giardia BPI-like proteins. (DOCX 486 kb)

Localization of BPI-like proteins in Giardia using epitope tagging. (DOCX 244 kb)

Primers and cloning of BPI-like genes. (DOCX 126 kb)

HCMP variability. Variability of HCMP genes in the different assemblage A isolates, WB, AS98 and AS175. (DOCX 686 kb)

Localization of BPI-like proteins in Giardia using epitope tagging. (DOCX 244 kb)

Distribution of read length and assembly positions. (DOCX 220 kb)

Trypanosoma cruzi and Giardia intestinalis are two human pathogens and protozoan parasites responsible for the diseases Chagas disease and giardiasis, respectively. Both diseases cause suffering and illness in several million individuals. The former disease occurs primarily in South America and Central America, and the latter disease occurs worldwide. Current therapeutics are toxic and lack efficacy, and potential vaccines are far from the market. Increased knowledge about the biology of these parasites is essential for drug and vaccine development, and new diagnostic tests. In this thesis, high-throughput sequencing was applied together with extensive bioinformatic analyses to yield insights into the biology and evolution of Trypanosoma cruzi and Giardia intestinalis. Bioinformatics analysis of DNA and RNA sequences was performed to identify features that may be of importance for parasite biology and functional characterization. This thesis is based on five papers (i-v). Paper i and ii describe comparative genome studies of three distinct genotypes of Giardia intestinalis (A, B and E). Paper iii describes a genome comparison of the human infecting Trypanosoma cruzi with the bat-restricted subspecies Trypanosoma cruzi marinkellei. Paper iv describes the repertoire of small non-coding RNAs in Trypanosoma cruzi epimastigotes. Paper v describes transcriptome analysis using paired-end RNA-Seq of three distinct genotypes of Giardia intestinalis (A, B and E)., Comment: PhD thesis, Karolinska Institutet, November 2012

Carotid artery intima media thickness (cIMT) and carotid plaque are measures of subclinical atherosclerosis associated with ischemic stroke and coronary heart disease (CHD). Here, we undertake meta-analyses of genome-wide association studies (GWAS) in 71,128 individuals for cIMT, and 48,434 individuals for carotid plaque traits. We identify eight novel susceptibility loci for cIMT, one independent association at the previously-identified PINX1 locus, and one novel locus for carotid plaque. Colocalization analysis with nearby vascular expression quantitative loci (cis-eQTLs) derived from arterial wall and metabolic tissues obtained from patients with CHD identifies candidate genes at two potentially additional loci, ADAMTS9 and LOXL4. LD score regression reveals significant genetic correlations between cIMT and plaque traits, and both cIMT and plaque with CHD, any stroke subtype and ischemic stroke. Our study provides insights into genes and tissue-specific regulatory mechanisms linking atherosclerosis both to its functional genomic origins and its clinical consequences in humans.