Data used to generate filters and reference sequences was obtained from the ImmunoPolymorphism Database and a set of fully-sequenced KIR haplotypes [ 61- 63].
From all the fecal samples, a dataset consisting of 409,812 high-quality 16S rRNA gene sequences was obtained (Table 1).
Alignment of the amino acid sequences was obtained using CLUSTALW with BOXSHADE (http://bioweb.pasteur.fr), in which gaps are marked as dashes and the conserved residues are highlighted in red, identical residues in yellow to orange and similar residues in green.
A repertoire of 408 distinct amoebic DNA sequences was obtained, with 256 of them corresponding to unique genes.
As Control datasets to this study, HIV-1 group M (all subtypes) reference sequence alignment (94 sequences) was obtained from the Los Alamos HIV database (http://www.hiv.lanl.gov/).hiv.lanl.gov/
In addition, a set of 3224 sequences was obtained from NCBI's reference sequence division, thus producing a final data set of 116638 ESTs.
A profile of overrepresented regulatory sequences was obtained for each of the seven signatures using the profiling tool GATHER [95] (See supporting information Table S5).
If the case arose where the second round varied significantly from the first, a third set of sequences was obtained and to determine if the results were reliable.
The underlying parsimonious phylogenetic tree of mtDNA coding sequences was obtained from Mitomap web page (www.mitomap.org) as of March, 2007 (Figure S1, File S1). Figure S1 shows 14 misannotations of substitutions found in the Mitomap tree.
No improvement of the forward sequences was obtained when the annealing temperature of the sequencing reaction was increased to 54°C, or when the JB2 primer was used to sequence the PCR product.
The MP tree of amino acid sequences was obtained using the Close-Neighbor-Interchange algorithm with search level 3 in which the initial trees were obtained with the random addition of sequences (10 replicates).
