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. 2018 Jul 20;9(7):365. doi: 10.3390/genes9070365

Table 1.

Summary of Hepatitis B Virus (HBV) DNA integration site detection methods.

Technique Biases Drawbacks Advantages Suitable Uses Ref.
Southern blot
  • Dependent on restriction enzyme sites to resolve different integration events

  • Time consuming

  • Technically demanding

  • Low sensitivity (>103 copies)

  • No sequence information

  • Low cost

  • Classical robust technique

  • Absolute quantification possible (low precision)

Detecting presence of integrated HBV DNA in highly clonal samples [39,40,42,43,44,45,46]
Direct cloning and Sanger sequencing
  • Dependent on restriction enzyme sites for cloning

  • Technically demanding

  • Low-throughput

  • Complete integrated genome sequenced

Determining structure of integrated HBV genome in highly clonal samples [45,47]
Alu PCR
  • Dependent on Alu sequences

  • Biased towards larger clones

  • Multiple copies required for detection

  • Alu-Alu products in low clonal samples

  • No absolute quantification

  • Inexpensive

  • Relatively simple

Detecting and sequencing integrated HBV DNA in clonal samples [48,49,50]
invPCR
  • Dependent on restriction enzyme sites for detection

  • Biased towards larger clones (as based on limiting dilution)

  • Time-consuming

  • Technically demanding

  • Only finds DNA sequence immediately adjacent to junctions

  • Absolute quantification

  • High sensitivity (single copy)

  • High specificity (detection of 1 in 106 cells)

  • Biases can be controlled for by in silico models

Detecting and quantifying rare HBV DNA integrations [16,26,28,29,31,32,33]
WGS
  • Biased away from poorly mappable (e.g., transposon sequences) regions

  • Low-depth

  • Cost

  • No absolute quantification

  • Full genome coverage

Integration site detection in highly clonal samples [18,19,20,21,22,23]
WES
  • Dependent on being in (or close to) coding regions

  • Coverage only of coding regions

  • No absolute quantification

  • Greater depth than WGS

Integration site detection in coding regions [51,52]
RNA-Seq
  • Biased towards more highly expressed genes

  • Coverage of expressed coding regions only

  • No absolute quantification

  • Greater depth than WGS

  • Data on transcriptional activity

Virus-fusion transcripts [19,22,53,54,55]

invPCR, inverse-nested PCR; WGS, Whole Genome Sequencing; WES, Whole Exome Sequencing; RNA-Seq, RNA Sequencing.