A Pipeline for the Error-Free Identification of Somatic Alu Insertions in High-Throughput Sequencing Data

Nugmanov, G; Komkov, Alexander; Saliutina, M. V.; Minervina, Anastasia A.; Lebedev, Yuri B.; Mamedov, Ilgar Z.

doi:10.1134/s0026893319010114

Cited by 3 publications

(8 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We used a custom computational pipeline [27] to map all the sequenced insertions' flanks to the human reference genome. The method specificity that was calculated as the percentage of target RE flanks among all sequences was 0.87-0.96 for AluYa5, 0.86-0.92 for AluYb8, and 0.83-0.98 for L1HS.…”

Section: Validation Of Candidate Tumor-specific Insertionsmentioning

confidence: 99%

The Absence of Retroelement Activity Is Characteristic for Childhood Acute Leukemias and Adult Acute Lymphoblastic Leukemia

Urazbakhtin

Smirnova

Volakhava

et al. 2022

IJMS

View full text Add to dashboard Cite

Retroelements (RE) have been proposed as important players in cancerogenesis. Different cancer types are characterized by a different level of tumor-specific RE insertions. In previous studies, small cohorts of hematological malignancies, such as acute myeloid leukemia, multiple myeloma, and chronic lymphocytic leukemia have been characterized by a low level of RE insertional activity. Acute lymphoblastic leukemia (ALL) in adults and childhood acute leukemias have not been studied in this context. We performed a search for new RE insertions (Alu and L1) in 44 childhood ALL, 14 childhood acute myeloid leukemia, and 14 adult ALL samples using a highly sensitive NGS-based approach. First, we evaluated the method sensitivity revealing the 1% detection threshold for the proportion of cells with specific RE insertion. Following this result, we did not identify new tumor-specific RE insertions in the tested cohort of acute leukemia samples at the established level of sensitivity. Additionally, we analyzed the transcription levels of active L1 copies and found them increased. Thus, the increased transcription of active L1 copies is not sufficient for overt elevation of L1 retrotranspositional activity in leukemia.

show abstract

Section: Validation Of Candidate Tumor-specific Insertionsmentioning

confidence: 99%

The Absence of Retroelement Activity Is Characteristic for Childhood Acute Leukemias and Adult Acute Lymphoblastic Leukemia

Urazbakhtin

Smirnova

Volakhava

et al. 2022

IJMS

View full text Add to dashboard Cite

show abstract

“…After sequencing, we use a custom computational pipeline [ 24 ] to map all sequenced insertions flanks to the human genome. Coordinates of insertions in compared samples (e.g.…”

Section: Resultsmentioning

confidence: 99%

“…After sequencing (see Table 2 for sequencing read numbers) and data processing (as described previously in [ 24 ]) we searched for 5′ flanks of Alu insertions characteristic for genomes of individuals D2-D4 (from Table 1 ) in the corresponding datasets. As a result, we were able to identify 44 out of 44 insertions present in 1% (individual D2) of cells, 42–43 out of 45 insertions present in 0.3% (individual D3) of cells and 34–39 of 50 insertions present in 0.1% (individual D4) of cells (See Table 2 ).…”

Section: Resultsmentioning

confidence: 99%

“…Raw sequencing reads were processed as described previously [ 24 ]. As a result, we obtained a metatable containing a list of identified AluYa5 insertions in all four individuals with their genomic positions and read counts for each of the four individuals.…”

Section: Methodsmentioning

confidence: 99%

“…These methods are more sensitive in respect of the catching somatic insertion events and obviously are more cost-effective compared to WGS. However, all the existing enrichment techniques can generate various artificial sequences (chimeras) that mimic true RE insertions and lead to many false-positive results [ 23 , 24 ]. Thus, independent confirmation of insertions by locus-specific PCR starting from initial genomic DNA samples with subsequent Sanger sequencing and identification of Target Site Duplication (TSD – the sign of true retrotransposition event) became a gold standard in somatic RE studies [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

SeqURE – a new copy-capture based method for sequencing of unknown Retroposition events

et al. 2020

View full text Add to dashboard Cite

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.

show abstract

Mobile retroelements induced by hypomethylating agents are restricted to transpose in myeloid malignancies

Plevova,

Pavlova,

Krzyzankova

et al. 2023

Preprint

View full text Add to dashboard Cite

Retroelements (RE) present in the human genome are silenced via multiple mechanisms, including DNA methylation, to prevent their potentially mutagenic effect. RE activity, demonstrated by their expression and somatic retrotransposition events, is deregulated in multiple tumor types but not in leukemia. We hypothesized that treatment with hypomethylating agents (HMA), commonly used in myelodysplastic syndromes and acute myeloid leukemia, could lead to increased RE activity and somatic retrotranspositions, and contribute to disease progression. We induced expression of ORF1p protein encoded by long interspersed nuclear element-1 (L1) after 72h treatment with HMA in DAMI and HL-60 cell lines. ORF1p was predominantly localized in the cytoplasm, as evidenced by fluorescent microscopy of the DAMI cell line. To study whether long-term HMA therapy may induce somatic retrotranspositions, we (i) treated both cell lines for four weeks, (ii) analyzed a cohort of 17 MDS patients before and on treatment with HMA. Using a previously established sensitive NGS-based method, no RE events were identified. To conclude, we show that although HMA induces the expression of L1-encoded proteins in tumor myeloid cell lines, de novo somatic retrotransposition events do not arise during the long-term treatment of MDS patients and myeloid cell lines with these agents.

show abstract

A Pipeline for the Error-Free Identification of Somatic Alu Insertions in High-Throughput Sequencing Data

Cited by 3 publications

References 26 publications

The Absence of Retroelement Activity Is Characteristic for Childhood Acute Leukemias and Adult Acute Lymphoblastic Leukemia

The Absence of Retroelement Activity Is Characteristic for Childhood Acute Leukemias and Adult Acute Lymphoblastic Leukemia

SeqURE – a new copy-capture based method for sequencing of unknown Retroposition events

Mobile retroelements induced by hypomethylating agents are restricted to transpose in myeloid malignancies

Contact Info

Product

Resources

About