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.

• MeSH
• analýza polymorfismu délky amplifikovaných restrikčních fragmentů metody   MeSH
• chronická myeloidní leukemie farmakoterapie   genetika   MeSH
• DNA fingerprinting metody   MeSH
• DNA nádorová genetika   MeSH
• dospělí MeSH
• genom lidský MeSH
• imatinib mesylát terapeutické užití   MeSH
• inhibitory proteinkinas terapeutické užití   MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• počítačová simulace MeSH
• protinádorové látky terapeutické užití   MeSH
• repetitivní sekvence nukleových kyselin * MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA metody   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA nádorová MeSH
• imatinib mesylát MeSH
• inhibitory proteinkinas MeSH
• protinádorové látky MeSH

Here, we present an improved amplified fragment length polymorphism (AFLP) protocol using restriction enzymes (AscI and SbfI) that recognize 8-base pair sequences to provide alternative optimization suitable for species with a genome size over 70 Gb. This cost-effective optimization massively reduces the number of amplified fragments using only +3 selective bases per primer during selective amplification. We demonstrate the effects of the number of fragments and genome size on the appearance of nonidentical comigrating fragments (size homoplasy), which has a negative impact on the informative value of AFLP genotypes. We also present various reaction conditions and their effects on reproducibility and the band intensity of the extremely large genome of Viscum album. The reproducibility of this octo-cutter protocol was calculated using several species with genome sizes ranging from 1 Gb (Carex panicea) to 76 Gb (V. album). The improved protocol also succeeded in detecting high intraspecific variability in species with large genomes (V. album, Galanthus nivalis and Pinus pumila).

• Klíčová slova
• amplified fragment length polymorphism, in silico AFLP, large genome, octo-cutter restriction enzyme, reproducibility, size homoplasy,
• MeSH
• analýza polymorfismu délky amplifikovaných restrikčních fragmentů metody   MeSH
• DNA rostlinná genetika   metabolismus   MeSH
• genom rostlinný * MeSH
• genotypizační techniky metody   MeSH
• reprodukovatelnost výsledků MeSH
• restrikční enzymy metabolismus   MeSH
• rostliny klasifikace   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Názvy látek
• DNA rostlinná MeSH
• restrikční enzymy MeSH

Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.

• MeSH
• analýza polymorfismu délky amplifikovaných restrikčních fragmentů metody   normy   MeSH
• DNA fungální chemie   genetika   MeSH
• houby chemie   genetika   izolace a purifikace   MeSH
• konformace nukleové kyseliny MeSH
• Phalaris mikrobiologie   MeSH
• polymorfismus délky restrikčních fragmentů * MeSH
• tranzitní teplota MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH