Clone CL Brener (CLB) (T. cruzi Discrete Typing Unit (DTU) VI- lineage TcVI) (Zingales et al., 1997; Zingales et al., 2009; Zingales et al., 2012) and the G strain (T. cruzi DTU I - lineageTcI) (Yoshida, 1983; Briones et al., 1999; Zingales et al., 2009; Zingales et al., 2012) in axenic cultures at 28°C in liver-infusion tryptose medium (LIT) containing 10% fetal calf serum. Clone D11, which also belongs to the DTU I - lineageTcI (Lima et al., 2013), was isolated from the G strain by Santori (1991) using the protocol described by Lima and Villalta (1989). Log-phase epimastigotes were washed in phosphate buffered saline (PBS) and collected by centrifugation. Genomic DNA extraction was performed with the DNeasy Blood & Tissue (Qiagen) kit using 5x10⁷ cells/mL according to the manufacturer’s instructions.

Approximately 1 x 10⁷ epimastigotes in exponential phase were washed with PBS and collected by centrifugation; an equal volume of cell suspension and 2% low-melting point agarose were mixed as previously described (Souza et al., 2011). The chromosomes were separated by pulsed-field gel electrophoresis (PFGE) and hybridized with the indicated markers labelled with (Zingales et al., 1997)P as previously described (Cano et al., 1995; Souza et al., 2011). The genes used as probes are indicated in the figure legends.

The 8x60K array was designed and produced by Agilent (Agilent Technologies, Santa Clara, CA,US) based on the T. cruzi clone CLB genome, release 6, available in TriTrypDB (http://tritrypdb.org/tritrypdb/; see Supplementary Table S1). CLB genomic contigs were assembled into 41 in silico chromosome (TcChr) pairs varying in size from 78 kb to 2.3 Mb (Weatherly et al., 2009). Because of the hybrid origin of CLB, the genome of this strain is composed of two parental haplotypes, designated Esmeraldo-like (S) and non-Esmeraldo-like (P) (El-Sayed et al., 2005). Two groups of probes totaling 48,787 probes covering all the in silico chromosomes were selected. The first group of probes covered all the chromosomes, including coding and non-coding regions, with an average of one probe every 1.2 kb to give a total of 44,860 probes. To design the second group, we selected 422 specific chromosome markers (i.e., single copy genes present only in the homologous chromosomes). In order to obtain a higher coverage with these markers, probe density was increased to one probe every 120 bp.

Raw data was first normalized using Feature Extraction software (Agilent technologies). Agilent Genomic Workbench Standard Edition 6.5 was then used to perform CNV interval detection. The QC metrics motif of Workbench 6.5 ensured adequate quality control of the hybridization data. In our study, an array signal which met the requirements if intensity value >50 and signal-to-noise ratio >25 was included in the analysis. The Aberration Detection Method 2 algorithm was used with a threshold of 6 and bin size of 10 to identify genomic variation. The aCGH scanning step for data acquisition and extraction was carried out by the Agilent Feature Extraction Software using the default of 0.25 log2 ratio (log2 ratio > 0.25 for gains and log2 ratio < −0.25 for losses). Additionally, we applied a relatively stringent post-analysis filter to ignore small, spurious or low-quality alterations (Wang et al., 2015). We used as defining criteria of copy number alterations the presence of at least four consecutive probes altered in the region, minimum average absolute log2 ratio 0.5, Log2 ratios > 0.5 and < –0.5 as the threshold for gain and loss, respectively.

The raw aCGH data and sequence of probes involved in the aCGH design for Trypanosoma cruzi have been deposited into the GenBank GEO database (GSE197870). The GenBank GEO accession number (GSE197870) (http://www.ncbi.nlm.nih.gov/geo/).

qPCR was used to validate the aCGH data. Primer amplification efficiency for each gene was determined. Specific sequences of the reference and target genes were cloned into pGEM-T easy vector (Invitrogen). Known amounts of genomic DNA (from clones D11 and CLB and G strain) and recombinant plasmids containing the sequences of interest were incubated with 10 µL SYBR Green-Based Detection (Applied Biosystems), sense and antisense primers and water to a total reaction volume of 20 µL. The reaction mixture was distributed into 0.2 mL tubes and subjected to 40 cycles of amplification in the Rotor-Gene^® Q PCR cycler (Qiagen) according to the manufacturer’s instructions. The qPCR program was set as follows: initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 60 s. The results were analyzed with Rotor-Gene 6000 v1.7 software (Qiagen). A standard curve was constructed for each target gene. Data obtained by amplification with genomic DNA samples could be compared as the amount of genomic DNA was the same for the three T. cruzi isolates. To estimate the copy numbers of each target gene in the three isolates, data were normalized separately using the genome size of each T. cruzi isolate (Souza et al., 2011). All experiments were performed in triplicate.

To estimate gene content in amplified and deleted regions in clone D11 in relation to that of Esmeraldo-like and Non-Esmeraldo-like haplotypes, the coordinates of the regions that varied in copy numbers were crossed with the gene coordinates in CLB General Feature Format (GFF) files, version 9, downloaded from TriTrypdb (http://tritrypdb.org/tritrypdb/) using BEDTools intersect v2.23 ( Quinlan and Hall, 2010). Initially, all genes whose coordinates were inside the deleted or amplified regions were counted and classified as follows: 1- Multigene Families (MGFs.) if they were annotated as Trans-sialidase (TS), Mucins (TcMUC), Mucin Associated Surface Proteins (MASP), Retrotransposon Hotspot Protein (RHS), surface glycoprotein gp63 (GP63) or Dispersed Gene Family 1 (DGF-1); 2- Hypothetical if they were annotated as Hypothetical Proteins; and 3- Others if they did not meet any of the above criteria. To compare the MGF content of the amplified or deleted regions, the proportion of each multigene family was determined.

Visualization of the D11 amplified or deleted regions with the Esmeraldo-like or Non-Esmeraldo-like in silico chromosomal sequences was performed using Perl scripts and the R libraries grid (https://stat.ethz.ch/R-manual/R-devel/library/grid/html/00Index.html), ade4 (https://cran.r-project.org/web/packages/ade4/index.html) and genoPlotR (http://genoplotr.r-forge.r-project.org/). The length of the chromosomes was drawn according to the coordinates in the Esmeraldo-like and Non-Esmeraldo-like genome FASTA files, version 9, downloaded from TriTrypdb (http://tritrypdb.org/tritrypdb). Each gene was drawn in its position and strand based on the GFF file, where grey boxes correspond to housekeeping and hypothetical genes and cyan boxes denote MGF genes. The green and red boxes correspond to deleted and duplicated regions, respectively.

The whole genome sequence (WGS) of T. cruzi isolate Dm28c-version 2018 (lineage TcI) was downloaded from http://tritrypdb.org/tritrypdb. Whole genome alignments between CLB chromosomes (TcChr S - Esmeraldo-like haplotype and TcChr P - non-Esmeraldo-like haplotype) and Dm28c contigs were performed using the blastn algorithm and implemented with big_blast.pl script from the Sanger Institute. The annotation and graphical output of chromosome-specific markers were obtained using the Artemis Comparison Tool (ACT) (Carver et al., 2005) (http://www.sanger.ac.uk/resources/software/act).

Clone D11 and its parental G strain belong to the DTU I – lineage TcI of T. cruzi (Briones et al., 1999; Zingales et al., 2009; Zingales et al., 2012; Lima et al., 2013). The genome of clone CL Brener (CLB) of lineage TcVI (Zingales et al., 1997; El-Sayed et al., 2005; Zingales et al., 2009; Zingales et al., 2012) was used as the DNA reference in the microarray design because it was the only T. cruzi genome assembled into chromosomes (Weatherly et al., 2009). Genomic sequences of clone CLB, the reference strain of the T. cruzi genome, have been previously assembled into chromosome-sized scaffolds that allowed the array to be designed. CLB genomic contigs were assembled into 41 in silico chromosome (TcChr) pairs that varied in size from 78 kb to 2.3 Mb (El-Sayed et al., 2005). Because of the hybrid nature of CLB, the genome of this strain is composed of two parental haplotypes designated Esmeraldo-like (S) and non-Esmeraldo-like (P). Nearly 50% of the T. cruzi genome is comprised of repetitive sequences, such as multigene families (MGFs), retrotransposons and micro- and mini-satellites (El-Sayed et al., 2005). The final assembly of chromosomes was hampered by its hybrid nature and the many repetitive sequences, which means that a considerable number of unassigned contigs are found in the T. cruzi database. Although the G strain (TcI) and clone CLB (TcVI) belong to two distantly related lineages, there is evidence that large syntenic regions are conserved among the different T. cruzi lineages (Souza et al., 2011). This has recently been confirmed by Next Generation Sequencing (NGS) procedures (Berná et al., 2018; Callejas-Hernández et al., 2018). To compare sequence divergence between the genomes of isolates from TcVI and TcI lineages, we evaluated the homology of individual chromosomes of CLB (TcVI) with their counterparts in Dm28c (TcI) whose assembly in large contigs was recently published (Berná et al., 2018). We confirmed gene sequence identity between CLB and Dm28c, and the conservation of gene order (synteny) in the same relative positions in the chromosomes of these isolates (Supplementary Figure S1). The differences found between CLB and Dm28c are usually due to variation in the number of copies of MGFs and genes encoding small RNAs (snRNA, sonRNA, tRNA). These results support our aCGH analysis comparing the genomes of the G strain and clone D11 with that of CLB.

The DNA content of the G strain [genome size=89.8 Mb (Lima et al., 2013)] and clone D11 were assumed to be the reference and test DNAs, respectively. This assumption has taken into account that clone D11 was isolated from strain G. Therefore, alterations related to DNA gains in the G strain that did not occur in clone D11 were assumed to be DNA losses in clone D11; conversely, DNA losses in the G strain were assumed to represent DNA gains in clone D11. We assumed that each clone D11 chromosome is homologous to a CLB chromosome (TcChr). For example, TcChr12 chromosome markers identified DNA gain and loss in the homologous of clone D11, which will be referred to here as a D11 chromosome homologous to TcChr12 of CLB.

aCGH can be used to identify losses and gains, including imbalances associated with apparently balanced translocation, i.e., duplication followed by translocation. However, it is unable to detect balanced chromosomal rearrangements, e.g., inversions and balanced reciprocal translocations. The bulk of the detailed information on chromosomal alterations found in clone D11 is presented in Supplementary Table S2, which includes the in silico CLB chromosomes to which they were mapped, the genomic location (the beginning and end of the alteration), the size and type of alteration (DNA loss or gain) and the number of probes for each alteration. Of the 418 chromosomal imbalances identified in clone D11, 144 (34.45%) were classified as DNA gain/amplification, and 274 (65.55%) as DNA loss/deletion. It is interesting to note that the parameters used in our analysis to identify DNA alterations were not equally stringent for defining gain and loss. A more restrictive threshold was used to define DNA gain than DNA loss. To increase the stringency and selection of non-random alterations, we have used at least 4 consecutive altered probes. However, we can not rule out that the high proportion of DNA loss over DNA gain in clone D11 may also be due to a more stringent threshold for defining DNA gain. The average size of chromosomal alterations was 23 kb, the smallest being 1.5 kb, and the largest 405 kb (Supplementary Figure S2). Most chromosomal alterations (89.71%) were smaller than 50 kb, and only six were >200 kb.

Chromosomal alterations identified in clone D11 were mapped on both haplotypes (S, Esmeraldo-like and P, Non-Esmeraldo-like) of clone CLB. The proportion of chromosome alterations (loss or gain) in each chromosome was estimated based on the length of CLB chromosomes (Weatherly et al., 2009) (Supplementary Figure S3). CNVs were distributed in a heterogeneous manner throughout the D11 genome and their proportion to a given chromosome varied from 1.1% to 89.3%. Deletion and duplication proportions appear to vary among D11 chromosomes; notably, losses (65.55%) prevailed over gains (34.45%). The majority of D11 chromosomes exhibited some alteration, except the chromosome that shares homology with TcChr4 of CLB. Eighteen chromosomes had only DNA losses whereas in four chromosomes only DNA gain was observed. There was no apparent correlation between chromosomal length and number of chromosomal alterations. The proportion of chromosomal alterations in D11 was very similar to that in the haplotypes of CLB.

The distribution of CNVs in the chromosomes is shown in Figure 1. The bars are scaled according to chromosome size and show the predominance of DNA loss over DNA gain across the chromosomes. We selected several examples of chromosomes with different CNV profiles for a more comprehensive analysis (Supplementary Figure S4). For instance, a profile with a few loss/gain events was detected in the D11 chromosomes homologous to TcChr37, TcChr39 and TcChr8 of CLB.

Chromosomes TcChr4 and TcChr37 are part of a single chromosome and syntenic group that is highly conserved among different T. cruzi lineages (Souza et al., 2011). Another syntenic group was identified in TcChr39 (Souza et al., 2011). The syntenic groups (TcChr4+TcChr37) and TcChr39 are much larger than would be expected if rearrangements occurred randomly, suggesting that they have undergone positive selection (Souza et al., 2011). The aCGH analysis agreed with this hypothesis since chromosomes TcChr39 and (TcChr4+TcChr37) exhibited very few CNVs. In a number of cases, a large part of the chromosome had changed. The D11 chromosome homologous to TcChr6 lost a large region comprising 89% of the entire chromosome whereas the D11 chromosome homologous to TcChr31 showed duplication of a segment covering 41% of the chromosome. A mixed CNV pattern was found in the D11 chromosome that shares homology with TcChr12. This chromosome has a large segment with loss and gain regions that correspond to 80% of the chromosome. Several D11 chromosomes showed different hybridization patterns to those of the haplotypes of CLB. For example, the D11 chromosome homologous to TcChr22-P had a large DNA loss region (32% of the length of the chromosome) that was absent in the TcChr22-S haplotype. Another D11 chromosome had a deletion that corresponded to 36% and 57% of the Tchr24-S and Tchr24-P haplotypes, respectively. Notably, some D11 chromosomes are highly conserved with changes in less than 2% of their lengths, e.g., the D11 chromosomes homologous to TcChr39 and TcChr37. Alterations in TcChr39 correspond to only 0.76% of the whole chromosome (approximately 1.85 Mb). These results agree with previous works that showed a remarkable conservation of TcChr39 and TcChr37 in different T. cruzi lineages (Santos et al., 1999; Souza et al., 2011; Lima et al., 2013).

To determine the gene content of the chromosomal alterations, we analyzed 418 alterations in clone D11 using gene annotation (Supplementary Table S3). We identified 2,199 genes involved in DNA losses: 1,032 genes (of which 18.99% were pseudogenes) in Esmeraldo haplotypes and 1,167 genes (of which 24.67% were pseudogenes) in non-Esmeraldo haplotypes. A relatively small number of genes were identified in the amplified regions: 353 genes (of which 13.03% were pseudogenes) in Esmeraldo haplotytpes and 328 genes (of which 17.68% were pseudogenes) in non-Esmeraldo haplotypes ((Supplementary Table S3). The annotated genes were distributed into three groups: a 1^st group, consisting of MGFs encoding surface proteins, such as trans-sialidases, MASPs, TcMUC (mucins), GP63 and DGF1 (dispersed gene family 1), and nuclear proteins coded by the RHS gene family (retrotransposon hot spot protein); a 2^nd group, consisting of genes encoding hypothetical proteins without an assigned function; and a 3^rd group consisting of other genes that do not meet any of the above criteria, such as replication protein genes, kinases and phosphatases (Supplementary Figure S5). There was an increase in the proportion of MGFs in clone D11 in relation to the G strain. The gene content of MGFs in DNA gain regions clearly differs from those with DNA loss (Figure 2). We observed an increase in MASP, mucins (TcMucII) and Gp63 content in the amplification regions of clone D11 whereas in the deletion regions there was a decrease in the content of these genes and an increase in RHS and DGF1.

We speculated about a possible association between the presence of MGFs and chromosomal alterations and therefore mapped the alterations (deletions in green and amplifications in red) detected in the D11 clone with the gene annotation (MGFs in light blue and other genes in black) in the in silico S and P haplotypes of CLB chromosomes (Figure 3 and Supplementary Figure S6). However, we found that the CNVs were unevenly distributed along the chromosomes, and there seems to be no connection between the abundance or size of multigene family clusters (represented in light blue) and the number and size of chromosomal alterations found in clone D11. For instance, the presence of many members of MGFs throughout chromosomes TcChr18 and TcChr41 was in striking contrast to the small number of alterations in these chromosomes. On the other hand, chromosomes TcCrh6 and TcChr31 harbor a small number of genes belonging to MGFs, but many chromosomal alterations could be observed. In agreement with a previous report, the pattern of chromosomal alterations flanked by MGFs was observed (Minning et al., 2011). The authors reported that in several strains of T. cruzi, CNVs were particularly frequent in gene family-rich regions containing mucins and trans-sialidases.

The electrophoretic karyotypes of clone D11 and the G strain differ in number and size of chromosomes (Lima et al., 2013). To facilitate understanding and comparison of CNVs between the G strain and clone D11, the chromosomal bands of these isolates were separated by PFGE using the electrophoretic conditions established by Lima et al., 2013 (Lima et al., 2013) (Supplementary Figure S7). Chromosome size differences between clone D11 and G strain ranged from approximately 40 to 340 kb, suggesting small chromosome rearrangements in clone D11. In this study we carried out Southern blot hybridizations using chromosome-specific markers within or near the chromosomal regions showing alterations detected by aCGH. We also looked for possible balanced recombination events that could not be identified by aCGH such as translocations.

Representative examples of rearrangements in clone D11 are shown in Figure 4 (see raw images in Supplementary Figures S8A-D). The localization of CNVs in the in silico chromosomes and hybridization of chromosomal bands with chromosome-specific markers are shown at the top and bottom in each panel, respectively. Figure 4A shows the rearrangement identified with two specific markers from chromosome TcChr8 that hybridized with a single chromosomal band of 0.59 Mb in the G strain and two bands of 0.57 and 0.71 Mb in clone D11, indicating the presence of two homologous chromosomes of the same size (0.59 Mb) in the parental G strain and two different-sized homologous chromosomes in clone D11. Chromoblot hybridization results suggest that the 0.71 Mb chromosome of clone D11 was the result of an internal duplication of a 120 kb region in one of the homologous of the G strain. However, the duplication was not identified by aCGH in TcChr8 homologues (Figure 4A), suggesting that it may have occurred in a region that was not represented in the DNA microarray. For instance, some unresolved gaps located at the subtelomeric and non-syntenic regions of T. cruzi genome (El-Sayed et al., 2005; Berná et al., 2018). Only two short chromosomal alterations, a 33.8 kb deletion and a 11.3 kb duplication, were detected by aCGH in the D11 chromosome homologous to TcChr8 (Figure 4A).

Taken together, these results suggest the involvement of both homologous chromosomes of the G strain in the chromosomal rearrangement found in clone D11. We also suggest that the 0.71 Mb chromosome of clone D11 was the result of an internal segmental duplication in one of the homologous chromosomes of the G strain while the 0.57 Mb chromosome of D11 was generated by loss of a (20 kb) fragment in the other homologous chromosome. However, we cannot rule out the possibility of translocation of a 120 kb segment from a nonhomologous chromosome to the 0.59 Mb chromosome of the G strain giving rise to the 0.71 Mb chromosome of D11.

Hybridization of the TcChr12-specific markers identified a single chromosomal band in both isolates, one band of 0.59 Mb in the G strain and another of 0.64 Mb in clone D11 (Figures 4B and Supplementary Figure S8B). However, the aCGH analysis showed two chromosomal alterations adjacent to each other, a duplicated and a deleted region of approximately 139 kb and 278 kb, respectively. Gain and loss regions are flanked at both sides by members of MGFs, including pseudogenes of RHS, trans-sialidases and DGF, allowing misalignment between sister or homologous chromatids. The rearrangement may be explained by unequal sister chromatid exchange that resulted in deletion and duplication of chromosome segments in the same chromatid. As shown in Figure 5, an unequal sister chromatid crossing over resulted in two different-sized sister chromatids (Figure 5A). The smaller sister chromatid lost an ~250 kb segment (in green) while it gained a segment of ~140 kb, resulting in a duplication of ~280 kb (in red). In the meantime, the larger sister chromatid lost the 140 kb segment and gained 250 kb (Figure 5A). At the end of mitotic division there were two sets of homologous chromosomes in the daughter cells (Figure 5B). In clone D11, one homologous chromosome was represented by the smaller sister chromatid whereas the other homologous chromosome was similar to the parental chromatid. The use of aCGH allowed the identification of cryptic imbalances, which had not been detected by chromoblot hybridization.

Some considerations need to be made when comparing the size of an in silico chromosome with that of the chromosomal band separated by PFGE. The current sequenced genomes of T. cruzi are more complete and precise. However, a very few chromosomes have been finished end to end (telomere to telomere), and some unresolved gaps persist. In this way, in silico chromosomes can be smaller than the chromosomal bands in which they were assigned. The models proposed to explain the chromosomal rearrangements are based on the size of in silico chromosomes that makes difficult to correlate accurately the size of aCGH alterations with the size of chromosomal band. Regarding the chromosome TcChr6, the deletion identified by aCGH in one of the chromatids could have been balanced by DNA gain of the same size that occurred in the gaps or subtelomeric regions that are not represented in the DNA array. The presence of a single chromosomal band in clone D11 instead of two bands, as expected by the proposed model (Figure 5), could be explained by DNA gain occurring in the gaps and/or subtelomeric regions. In a previous work, Lima et al., 2013 (Lima et al., 2013) demonstrated that telomeric regions were involved in the sized-chromosome polymorphism in clone D11. These regions are very polymorphic and for this reason they were not included in the DNA array.

Figure 4C shows the rearrangement found in the D11 chromosome homologous to TcChr6. A specific-chromosome marker of TcChr6 hybridized with two chromosomal bands of 0.73 and 0.68 Mb in the G strain and with a single smaller-sized band of 0.64 Mb in clone D11, indicating the presence of two different-sized homologous chromosomes in the parental strain. Regarding the clone D11, the size of one of the homologous chromosomes can be estimated at 0.64 Mb while the other homologous chromosome would have a smaller size. Chromoblot hybridization corroborated the aCGH finding of a large deletion in TcChr6 (-1 log2 ratio). This rearrangement may be explained by unequal crossing over between homologous chromatids with deletion of an ~280 kb segment resulting in two different-sized DNA molecules (Supplementary Figure S9A). After chromosome segregation, clone D11 receives one recombinant homologous chromosome represented by the smaller chromatid, and the other homologous chromosome is similar to the parental chromatid (Supplementary Figure S9B).

TcChr22-chromosome markers hybridized with two chromosomal bands of the parental G strain (1.29 Mb and 0.96 Mb) but with only one band in clone D11 (1.17 Mb) (Figure 4D). The assignment of TcChr22 markers into two bands in the G strain indicated the presence of a pair of different-sized homologous chromosomes. The chromosome size differences between clone D11 and the parental strain were 0.12 Mb and 0.21 Mb, suggesting small chromosomal rearrangements. After analyzing the aCGH results, we were able to detect a 148 kb deletion in one of the haplotypes of clone D11. The hybridization of the marker TcCLB.509499.20 to the 1.17 Mb chromosomal band supports that the 148 kb segment identified by aCGH is present in one homologous chromosome of clone D11 (Figure 4D). One of the homologous chromosomes of clone D11 may have resulted from a deletion in the 1.29 Mb chromosome of the G strain while the other may have arisen from a segmental duplication in the 0.96 Mb chromosome of the G strain.

Although the chromosomal rearrangement in the D11 chromosome homologous to TcChr22 is quite complex, it allows a comprehensive analysis of the molecular mechanisms involved. The TcChr22-specific markers were mapped to two chromosomal bands of 1.29 and 0.96 Mb in the G strain and to a single band of 1.17 Mb in the clone D11. Lima et al., 2013 (Lima et al., 2013) suggested that fusion of the different-sized homologous chromosomes of 1.29 and 0.94 Mb of the G strain generating a dicentric chromosome was followed by fission, resulting in two similar-sized homologous chromosomes (1.17 Mb) in clone D11. However, the aCGH analysis showed a 148 kb deletion in one homologous chromosome of clone D11. Since the recombination model proposed by Lima et al., 2013 (Lima et al., 2013) did not foresee a DNA loss, we decided to propose a new model (Figure 6), according to which during the cloning process a double-strand break (DSB) occurred in the 1.29 Mb homologous chromosome of the G strain. The break occurred in a region that did not share any homology with the 0.94 Mb homologous chromosome of the G strain. The pairing of homologous chromosomal regions was followed by DNA repair through unequal homologous recombination (HR) via break-induced replication (BIR). First, the chromosomal extremity with the DSB recombination annealed with a homologous region of the DNA donor, initiating the replication repair. At the DSB end the strand resection occurred while the other strain invaded the homologous sequence in the intact donor DNA. The repair mechanism generated two homologous chromosomes measuring 1.17 Mb as a result of the addition of a 110 kb region to the donor chromosome.

Quantitative real time PCR (qPCR) was used to validate chromosomal alterations that represent different predicted copy number variations (loss, gain or null) in clone D11 (Table 1 and Supplementary Table S4). Triplicate qPCR assays were performed, and the results showed that 7 out of 9 aCGH results (77.7%) were confirmed (Table 1 and Supplementary Figure S10). Two alterations not validated by qPCR correspond to DNA loss and were covered by a large number of probes in the aCGH. These data lead us to suggest that the outcome of aCGH should be considered correct even when it cannot be validated by qPCR.

In our work, the CNV validation rate when we used qPCR was similar to that obtained in mammalian genomes (Li et al., 2012; Wang et al., 2013; Wang et al., 2015; Baldan et al., 2019) observed that the qPCR validation rate is directly related to the number of probes in the region where aCGH detected alterations. They suggested that validation is necessary when the alteration is detected by fewer than 10 probes. Here, two alterations detected by aCGH were not confirmed by qPCR (Table 1) but were confirmed by a large number of probes, 257 on chromosome TcChr 22 (TcCLB.509499.20) and 149 on chromosome TcChr 6 (TcCLB.507603.180) (Supplementary Table S2). Furthermore, some sequences are known not to amplify in the PCR reaction because Taq polymerase lacks 3’→5’ exonuclease activity (Tindall and Kunkel, 1988; Wu et al., 1989). The presence of a mismatch in the last nucleotide at the 3’ end of the primer is enough to block the extension of the oligonucleotide by Taq DNA polymerase as there is a lack of 3’→5’ exonuclease activity (Wu et al., 1989) It is possible that because of the large size of the probes in aCGH, the chances of finding SNP-type polymorphisms using this technique are higher. This would explain why aCGH is able to detect variants that cannot be detected (amplified) by PCR.