9% saline was examined microscopically for the presence of erythrocytes, leukocytes, and E. histolytica trophozoites. The DNA was extracted using a slightly modified QIAamp DNA Stool Mini Kit protocol (Qiagen Inc., Valencia, CA) as described previously for specimens from ICDDR,B [54]. Stool samples are also listed in Additional file 1: Table S4. E. histolytica DNA derived from Amebic Liver Abscess (ALA) aspirates Aspirates from patients with amebic liver abscesses were obtained only from adults because ALA is an extremely rare complication
in children [55]. A presumptive diagnosis of ALA was based on clinical picture, ultrasound Emricasan price examination and positive serology using an E. histolytica antigen based ELISA (TechLab E. histolytica II) LY2090314 ic50 [6]. Abscess fluid was obtained under ultrasound guidance from patients with ALA and was purified using the modified QIAamp DNA Stool Mini Kit protocol described above (samples are listed in Additional file 1: Table S4) [6]. Primer design Primers for these experiments were designed using the
publically available Primer3 program and checked for specificity using the NCBI Primer-BLAST tool [56] (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). All primers used in this study are listed in either Additional file 1: Table S2 or Table S4. Whole genome Dolichyl-phosphate-mannose-protein mannosyltransferase sequencing of axenic cultured E. histolytica strains Whole genome sequencing of five of the E. histolytica strains used in this study was carried out
at the J. Craig Venter Institute. These sequence traces are deposited athttp:// http://www.ncbi.nlm.nih.gov/bioproject/9532dbSNPs Genbank(http://www.ncbi.nlm.nih.gov/projects/SNP/) and AmoebaDB (http://amoebadb.org/amoeba/)[57, 58]. This project is also fully described at the NCBI Bio Project page (Accession: PRJNA9532). Whole genome re-sequencing was performed at the Institute of Integrative Biology, (Centre for Genomic Research) University of Liverpool and results deposited at AmoebaDB [35, 57]. For a complete list of E. histolytica genomes, sequencing technology and Sequencing Center see Table 1 and Additional file 1: Table S1. SNP detection and selection of candidate informative SNPs For genome-wide SNP detection at JCVI the sequenced strains were analyzed using the CLC Genomics Workbench 4.0.2 SNP detection component as described below (see SNP detection and Tubastatin A molecular weight validation of amplicon sequences). In genomes sequenced at the Centre for Genomic Research, SNPs were identified according to the methods described Weedall et al. [35]. For a list of the SNP detection method used in each genome see Additional file 1: Table S1. SNPs are listed in Additional file 1: Table S5.