Abstract
The increasing interest in exploring the human genome and identifying genetic risk factors contributing to the susceptibility to and outcome of diseases has supported the rapid development of genome-wide techniques. However, the large amount of obtained data requires extensive bioinformatics analysis. In this work, we established an approach combining amplified fragment length polymorphism (AFLP), AFLP in silico and next generation sequencing (NGS) methods to map the malignant genome of patients with chronic myeloid leukemia. We compared the unique DNA fingerprints of patients generated by the AFLP technique approach with those of healthy donors to identify AFLP markers associated with the disease and/or the response to treatment with imatinib, a tyrosine kinase inhibitor. Among the statistically significant AFLP markers selected for NGS analysis and virtual fingerprinting, we identified the sequences of three fragments in the region of DNA repeat element OldhAT1, LINE L1M7, LTR MER90, and satellite ALR/Alpha among repetitive elements, which may indicate a role of these non-coding repetitive sequences in hematological malignancy. SNPs leading to the presence/absence of these fragments were confirmed by Sanger sequencing. When evaluating the results of AFLP analysis for some fragments, we faced the frequently discussed size homoplasy, resulting in co-migration of non-identical AFLP fragments that may originate from an insertion/deletion, SNP, somatic mutation anywhere in the genome, or combination thereof. The AFLP–AFLP in silico–NGS procedure represents a smart alternative to microarrays and relatively expensive and bioinformatically challenging whole-genome sequencing to detect the association of variable regions of the human genome with diseases.
Highlights
Efforts to explore the human genome through individual whole-genome analyses and identify genetic risk factors contributing to the susceptibility to and outcome of diseases are of vital importance
We described the amplified fragment length polymorphism (AFLP) fingerprinting of a cohort of patients and healthy donors, which enabled the identification of DNA regions associated with traits such as the disease itself and its response to the treatment, and we preselected loci in the human genome for further identification through next-generation sequencing
We analyzed the genome of 65 patients at the time of diagnosis in the chronic phase of chronic myeloid leukemia (CP-CML), who were treated at the Institute of Hematology and Blood Transfusion in Prague, as well as 30 healthy donors
Summary
Efforts to explore the human genome through individual whole-genome analyses and identify genetic risk factors contributing to the susceptibility to and outcome of diseases are of vital importance. The broad range of sequencing technologies incurs limitations due to the large volume of obtained data, requiring extensive bioinformatics analysis
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