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Figure S5. Methodology for T. cruzi CCNV estimations. (A) The CCNV estimations were performed using the median coverage of all T. cruzi genes, excluding those belonging to the largest multigene families in each one of the CL Brener 41 putative chromosomes as an estimate of its chromosome copy number. In brief, the median RDC of the selected genes in each of the 41 CL Brener chromosomes were generated by PERL scripts and normalized by the genome coverage. The genome coverage was estimated as the mean RDC of all single-copy genes in all chromosomes for each strain. (B) Heterozygous SNPs between the CL Brener chromosome and the mapped reads for the T. cruzi stains were obtained from the filtered SAMtools mpileup results. To be considered as a reliable SNP, the position RDC must be at least 10, with 5 reads supporting each variant. For each chromosome, the proportion of the alleles in each predicted heterozygous site was obtained and rounded to the second place. Base frequencies were rounded in ten categories, ranging from 0.01 to 1.00, and an approximate distribution of base frequencies for each chromosome was plotted in R. Disomic chromosomes have a peak in 0.50, while trisomic chromosomes have peaks in 0.33 and 0.66. Tetrasomic chromosomes have combination of peaks of 0.20, 0.80 and 0.50. (DOCX 120 kb)

Figure S2. Boxplot of the predicted ploidy of T. cruzi TcII field isolates. The predicted ploidy of each chromosome from the T. cruzi field isolates S11, S15, S154a, S162a, S23b, S44a and S92a using as a reference the 41 CL Brener chromosome sequences, was estimated based on the median coverage of all T. cruzi genes, excluding those belonging to the largest multigene families, and represented in boxplots. In this image, the predicted ploidy of each of the 41 chromosomes is represented by the median, first and third quartile, as well as maximum and minimum values. (A) Representation by strain. In this image, each quadrant corresponds to a TcII strain, containing the predicted ploidy of all 41 chromosomes. (B) Representation by chromosome. In this image, each quadrant corresponds to a chromosome, comprising the predicted ploidy of this chromosome in all seven TcII evaluated strains. (PPTX 4680 kb)

Figure S3. Read Depth Coverage of the chromosome 11 in the Y strain and clones. In this picture, the blue lines correspond to the normalized RDC of each position of the chromosome 11, estimated by the ratio between the RDC and the genome coverage. The red line corresponds to the 248 kb position in the chromosome. Below, the protein-coding genes are depicted as rectangles drawn as proportional to their length, and their coding strand is indicated by their position above (top strand) or below (bottom strand) the central line. Cyan and black rectangles represent multigene families and hypothetical/housekeeping genes, respectively. The initial 248-kb in this chromosome had a smaller RDC when compared to remaining sequence in the Y strain as well as in all three Y clones evaluated. (DOCX 226 kb)

Table S5. T. cruzi read libraries description. (DOCX 14 kb)

Table S6. Links to download T. cruzi reference genomes. (DOCX 12 kb)