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Because this probe presented multiple hybridization signals on the chromosomes of D. willistoni, the inference of the main physical location was made by quantification.
Furthermore, the presence of repetitive sequences within the telomeric regions precludes the use of chromosome-specific telomeric probes for FISH analysis due to the potential for multiple hybridization signals.
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This is because hybridization signals from multiple probes are used to detect a CNV and so, within a complex CNV, only a fraction of probes are likely to detect changes in genome content.
For probe sets matching several Populus Hsp90 gene models, only these exhibiting consistently high hybridization signals across multiple samples were considered.
Expression levels were assessed using the Robust Multi-array Average algorithm (RMA; [ 25]), which summarizes hybridization signals from multiple probes per gene into a single expression value, and present/absent (P/A) calls were made as described in Methods.
A total of 95 BAC clones (14.2%) failed to produce distinct and reproducible FISH signals, even after multiple hybridization attempts.
To minimize redundant representation, we further reduced the list of 60 probes to those that have unique gene matches, and in cases of multi-probe representation, to those that exhibited the highest hybridization signals consistently across multiple samples.
The size of the eccDNA molecules ranged from over 8 kb down to 500 bp and in most cases their hybridization signals formed discrete spots corresponding to multiples of monomer or higher-order unit lengths (Table 1).
This fragment could produce strong hybridization signals even after short exposure times, indicating that multiple copies may be present in genomes of selected soybean cultivars or lines.
Second, a portion of the significant differences in hybridization signals may be due to the presence of multiple polymorphisms within the probe sequence between the reference and test sample genome.
Finally, array sequences representing duplicated genes and multiple-copy variants are expected to produce two-fold or higher hybridization signals than single-copy variants.
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