An Improved Sequencing-Based Bioinformatics Pipeline to Track the Distribution and Clonal Architecture of Proviral Integration Sites

Diagnosis and Early Prediction of Lymphoma Using High-Throughput Clonality Analysis of Bovine Leukemia Virus-Infected Cells

“…For BLV clonality analysis, several molecular methods have been developed to analyze proviral integration sites of BLV-infected cells, including ligation-mediated PCR ( 5 ), LAM-PCR ( 6 ), target capture sequencing ( 7 ), and inverse PCR ( 8 , 12 ). Most of these methods requires HTS analysis for the detection of integration sites ( 5 – 7 , 12 ), but the high cost of the analysis makes it unsuitable for clinical diagnosis with large numbers of samples.…”

“…For BLV clonality analysis, several molecular methods have been developed to analyze proviral integration sites of BLV-infected cells, including ligation-mediated PCR ( 5 ), LAM-PCR ( 6 ), target capture sequencing ( 7 ), and inverse PCR ( 8 , 12 ). Most of these methods requires HTS analysis for the detection of integration sites ( 5 – 7 , 12 ), but the high cost of the analysis makes it unsuitable for clinical diagnosis with large numbers of samples. Additionally, these current methods utilize restriction enzymes or ultrasound sonication for DNA fragmentation, which raises concerns about the bias of amplification or detection, causing their poor sensitivity and reproducibility.…”

“…Therefore, the clonality of proviral integration sites in BLV-infected cells has been considered a useful marker to diagnose the onset of lymphoma ( 5 – 8 ). Several methods have been recently developed to analyze transgene integration sites using high-throughput sequencing (HTS), such as ligation-mediated PCR ( 5 , 9 ), target capture sequencing ( 7 , 10 , 11 ), inverse PCR ( 12 ), linear amplification-mediated PCR (LAM-PCR) ( 6 ), and nonrestrictive LAM-PCR ( 13 ). However, it would be difficult to analyze multiple specimens sensitively, rapidly, and inexpensively by these current methods.…”

Meeting FDA Guidance recommendations for replication-competent virus and insertional oncogenesis testing

“…Indexing strategies using oligos containing random sequences (unique molecular identifiers or UMIs) can also be leveraged to aid the quantitation of integration-site frequency in a population of cells. 50 , 51 , 54 , 55 Over time, Illumina platforms have become the NGS technology employed by most ISA protocols, triggering the need for novel and more sophisticated bioinformatic pipelines capable of managing short paired-end sequencing readouts made of tens of millions of reads. The operations performed by most bioinformatic pipelines can be divided into three main tasks: preprocessing of reads, mapping to the reference genome, and identification of integration site coordinate and clone abundance estimation.…”

CELLULAR AND EXTRACELLULAR LEVELS OF RETROVIRUS—HOST INTERACTIONS ON THE EXAMPLE OF THE BOVINE LEUKOSE VI-RUS. 2. CRITICAL STAGES — MULTIPLICITY AND VERSATILITY (review)

“…Например, интеграция провируса BLV в районе генов хозяина, вовлекаемых в процесс онкогенеза, влияет на их экспрессию (52)(53)(54)(55). В ряде работ отмечается, что достаточно часто интеграция провируса BLV выявляется в районах локализации ретровирусов (56)(57)(58)(59)(60).…”