PCIP-seq: simultaneous sequencing of integrated viral genomes and their insertion sites with long reads

Artesi, Maria; Hahaut, Vincent; Cole, Basiel; Lambrechts, Laurens; Ashrafi, Fereshteh; Marçais, Ambroise; Hermine, Olivier; Griebel, Philip; Arsic, Natasa; Meer, Frank van der; Burny, Arsène; Bron, Dominique; Bianchi, Elettra; Delvenne, Philippe; Charlier, Carole; Georges, Michel; Vandekerckhove, Linos; Broeke, Anne Van den; Durkin, Keith

doi:10.1186/s13059-021-02307-0

Cited by 27 publications

(35 citation statements)

References 74 publications

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“…Further expansion of this analysis for a larger number of samples and a functional assessment of the cis -perturbed transcripts associated with the viral integration sites will shed light on the diverse mutations in subclonal architecture among patients with different symptoms. Improvements in sensitivity (e.g., targeted amplification 17 , 18 ) and read length (e.g., long-read technology 50 ), as well as utilization of future applications enabling simultaneous DNA-seq and RNA-seq in depth at a single-cell resolution, should lead to the development of approaches for the early diagnosis and pre-emptive therapeutic strategy for this disease, which currently has a poor prognosis. Early detection and characterization of a minimal premalignant clone, which can develop into a fully malignant clone, may be achieved through the integration of biological and reliable genetic data to provide the shortest path for personalized care.…”

Section: Discussionmentioning

confidence: 99%

Chronological genome and single-cell transcriptome integration characterizes the evolutionary process of adult T cell leukemia-lymphoma

et al. 2021

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Subclonal genetic heterogeneity and their diverse gene expression impose serious problems in understanding the behavior of cancers and contemplating therapeutic strategies. Here we develop and utilize a capture-based sequencing panel, which covers host hotspot genes and the full-length genome of human T-cell leukemia virus type-1 (HTLV-1), to investigate the clonal architecture of adult T-cell leukemia-lymphoma (ATL). For chronologically collected specimens from patients with ATL or pre-onset individuals, we integrate deep DNA sequencing and single-cell RNA sequencing to detect the somatic mutations and virus directly and characterize the transcriptional readouts in respective subclones. Characteristic genomic and transcriptomic patterns are associated with subclonal expansion and switches during the clinical timeline. Multistep mutations in the T-cell receptor (TCR), STAT3, and NOTCH pathways establish clone-specific transcriptomic abnormalities and further accelerate their proliferative potential to develop highly malignant clones, leading to disease onset and progression. Early detection and characterization of newly expanded subclones through the integrative analytical platform will be valuable for the development of an in-depth understanding of this disease.

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Section: Discussionmentioning

confidence: 99%

Chronological genome and single-cell transcriptome integration characterizes the evolutionary process of adult T cell leukemia-lymphoma

et al. 2021

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“…Integration site analysis (ISA) pinpoints the chromosomal location of proviruses and is frequently used as a marker to study clonal expansion of infected cells 16 , 17 . More recently, NFL provirus sequencing and ISA were combined into a single assay, allowing the study of the relationship between proviral IS and genome structure 26 – 28 . However, because these assays are usually performed on bulk CD4 T cell DNA, they mainly identify defective proviruses, as it has been estimated that only 2–5% of the total proviruses are genome-intact 22 , 29 , 30 .…”

Section: Introductionmentioning

confidence: 99%

In-depth single-cell analysis of translation-competent HIV-1 reservoirs identifies cellular sources of plasma viremia

et al. 2021

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Clonal expansion of HIV-infected cells contributes to the long-term persistence of the HIV reservoir in ART-suppressed individuals. However, the contribution from cell clones that harbor inducible proviruses to plasma viremia is poorly understood. Here, we describe a single-cell approach to simultaneously sequence the TCR, integration sites and proviral genomes from translation-competent reservoir cells, called STIP-Seq. By applying this approach to blood samples from eight participants, we show that the translation-competent reservoir mainly consists of proviruses with short deletions at the 5’-end of the genome, often involving the major splice donor site. TCR and integration site sequencing reveal that cell clones with predicted pathogen-specificity can harbor inducible proviruses integrated into cancer-related genes. Furthermore, we find several matches between proviruses retrieved with STIP-Seq and plasma viruses obtained during ART and upon treatment interruption, suggesting that STIP-Seq can capture clones that are responsible for low-level viremia or viral rebound.

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“…(Patro et al, 2019) and Artesi et al . (Artesi et al, 2021), respectively called Matched Integration site and Proviral sequencing ( MIP-Seq ), Multiple Displacement Amplification Single-Genome Sequencing ( MDA-SGS ) and Pooled CRISPR Inverse PCR sequencing ( PCIP-seq ). These assays combine the qualitative strength of NFL HIV-1 sequencing with IS analysis, shedding light on the integration profile of intact versus defective proviruses.…”

Section: Introductionmentioning

confidence: 99%

Extensive characterization of HIV-1 reservoirs reveals links to plasma viremia before and during analytical treatment interruption

Cole

Lambrechts

Boyer

et al. 2021

Preprint

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The HIV-1 reservoir is composed of cells harboring latent proviruses that are capable of refuelling viremia upon antiretroviral treatment interruption. This reservoir is in part maintained by clonal expansion of infected cells. However, the contribution of large, infected cell clones to rebound remains underexplored. Here, we performed an in-depth study on four chronically treated HIV-1 infected individuals that underwent an analytical treatment interruption (ATI). A combination of single-genome sequencing, integration site analysis, near-full length proviral sequencing and multiple displacement amplification was used to identify infected cell clones and link these to plasma viruses before and during an ATI. A total of six proviruses could be linked to plasma sequences recovered during an ATI. Interestingly, only two of six proviruses were genome intact, one of which is integrated in the ZNF141 gene. To our knowledge, this is the first instance of an intact provirus with its matched IS being matched to plasma virus during an ATI. These findings demonstrate that with in-depth reservoir characterization, clones of infected cells harboring genome-intact proviruses can be linked to rebound viremia, confirming the previously proposed notion that infected clonal cell populations play an important role in long-term HIV-1 reservoir maintenance.

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PCIP-seq: simultaneous sequencing of integrated viral genomes and their insertion sites with long reads

Cited by 27 publications

References 74 publications

Chronological genome and single-cell transcriptome integration characterizes the evolutionary process of adult T cell leukemia-lymphoma

Chronological genome and single-cell transcriptome integration characterizes the evolutionary process of adult T cell leukemia-lymphoma

In-depth single-cell analysis of translation-competent HIV-1 reservoirs identifies cellular sources of plasma viremia

Extensive characterization of HIV-1 reservoirs reveals links to plasma viremia before and during analytical treatment interruption

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