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SeqURE - a new copy-capture based method for sequencing of unknown Retroposition events
AY. Komkov, SZ. Urazbakhtin, MV. Saliutina, EA. Komech, YA. Shelygin, GA. Nugmanov, VP. Shubin, AO. Smirnova, MY. Bobrov, AS. Tsukanov, AV. Snezhkina, AV. Kudryavtseva, YB. Lebedev, IZ. Mamedov,
Jazyk angličtina Země Velká Británie
Typ dokumentu časopisecké články
Grantová podpora
18-14-00244
Russian Science Foundation
BioMedCentral Open Access od 2010
Directory of Open Access Journals od 2010
Free Medical Journals od 2010
PubMed Central od 2010
Europe PubMed Central od 2010 do 2020
ProQuest Central od 2015-01-01
Open Access Digital Library od 2010-01-01
Open Access Digital Library od 2010-01-01
Health & Medicine (ProQuest) od 2015-01-01
Springer Journals Complete - Open Access od 2010-12-01
Springer Nature OA/Free Journals od 2010-12-01
Odkazy
PubMed
33317630
DOI
10.1186/s13100-020-00228-6
Knihovny.cz E-zdroje
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated. RESULTS: Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions. CONCLUSIONS: This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.
Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow Russia
Shemyakin Ovchinnikov Institute of Bioorganic Chemistry Moscow Russia
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- $a Komkov, Alexander Y $u Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia. alexandrkomkov@yandex.ru. Dmitry Rogachev National Medical and Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia. alexandrkomkov@yandex.ru.
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- $a BACKGROUND: Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated. RESULTS: Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions. CONCLUSIONS: This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.
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- $a Urazbakhtin, Shamil Z $u Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
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- $a Saliutina, Maria V $u Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
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- $a Komech, Ekaterina A $u Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
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- $a Shelygin, Yuri A $u Ryzhikh National Medical Research Centre for Coloproctology of the Ministry of Health of Russia, Moscow, Russia.
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- $a Nugmanov, Gaiaz A $u Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
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- $a Smirnova, Anastasia O $u Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
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- $a Bobrov, Mikhail Y $u V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Moscow, Russia.
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- $a Tsukanov, Alexey S $u Ryzhikh National Medical Research Centre for Coloproctology of the Ministry of Health of Russia, Moscow, Russia.
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- $a Snezhkina, Anastasia V $u Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
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- $a Kudryavtseva, Anna V $u Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
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- $a Lebedev, Yuri B $u Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
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- $a Mamedov, Ilgar Z $u Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia. imamedov@mx.ibch.ru. Dmitry Rogachev National Medical and Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia. imamedov@mx.ibch.ru. V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Moscow, Russia. imamedov@mx.ibch.ru. Central European Institute of Technology, Masaryk University, Brno, Czech Republic. imamedov@mx.ibch.ru.
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