Evaluating a Ligation-Mediated PCR and Pyrosequencing Method for the Detection of Clonal Contribution in Polyclonal Retrovirally Transduced Samples

Abstract

Retroviral gene transfer has proven therapeutic potential in clinical gene therapy trials but may also cause abnormal cell growth via perturbation of gene expression in the locus surrounding the insertion site. By establishing clonal marks, retroviral insertions are also used to describe the regenerative potential of individual cells. Deep sequencing approaches have become the method of choice to study insertion profiles in preclinical models and clinical trials. We used a protocol combining ligation-mediated polymerase chain reaction (LM-PCR) and pyrosequencing for insertion profiling and quantification in cells of various tissues transduced with various retroviral vectors. The presented method allows simultaneous analysis of a multitude of DNA-barcoded samples per pyrosequencing run, thereby allowing cost-effective insertion screening in studies with multiple samples. In addition, we investigated whether the number of pyrosequencing reads can be used to quantify clonal abundance. By comparing pyrosequencing reads against site-specific quantitative PCR and by performing spike-in experiments, we show that considerable variation exists in the quantification of insertion sites even when present in the same clone. Our results suggest that the protocol used here and similar approaches might misinterpret abundance clones defined by insertion sites, unless careful calibration measures are taken. The crucial variables causing this variation need to be defined and methodological improvements are required to establish pyrosequencing reads as a quantification measure in polyclonal situations.

Efficiency and Safety of O6-Methylguanine DNA Methyltransferase (MGMTP140K)-MediatedIn VivoSelection in a Humanized Mouse Model

“…This was especially true for the PB samples and could not be overcome by reanalysis of the same sample. This technical limitation reflects the current concerns associated with clonality assessment based on sequential PCR-based techniques (Giordano et al, 2011;Brugman et al, 2013;Rittelmeyer et al, 2013).…”

Clonal dynamics of native haematopoiesis

“…The LM-PCR method currently applied is not quantitative, and is likely to underestimate the full clonal repertoire 22 (Extended Data Fig. 4g, Supplementary Information).…”

Quantitative Shearing Linear Amplification Polymerase Chain Reaction: An Improved Method for Quantifying Lentiviral Vector Insertion Sites in Transplanted Hematopoietic Cell Systems

“…Without controls containing a multiplex of known integration sites at defined frequencies, it can be difficult to assess data arising from complex samples for the failure to detect clones that are present (false negatives) and/or the presence of amplicons not based on true integration events within the genome (false positives). 17 In our experience, this is more common than generally realized. In addition, many current methods are reliant upon pyrosequencing, relatively expensive, and not generally available to gene therapy investigators outside of collaboration with centers of expertise.…”

“…4,[32][33][34] However, these methods sometimes lack quantitative standards and may not accurately measure clone size in all instances, particularly when restriction enzyme digestion is used to facilitate adapter ligation. 13,17,35 Recently, random DNA shearing by sonication and count of shear site was introduced and appear to have better accuracy and sensitivity. 20,21,23,28,36,37 We further improved this method by minimizing the number of exponential PCR cycles used for template generation and by including an internal control sample with a relatively high number of quantitatively defined VIS.…”

“…[13][14][15] When these assays are based on early stage restriction enzyme digestion, such as in the linear amplification mediated PCR (LAM PCR) assay, 16 they can give nonquantitative results based on variability in the location of the genomic restriction enzyme site and resultant differences in PCR amplification efficiency. 13,[17][18][19] Newer methods that eliminate the requirement for restriction enzyme digestion can circumvent this particular limitation, [20][21][22][23][24][25] but are often accompanied by reduced overall sensitivity and lack of rigorous quantitative controls. Without controls containing a multiplex of known integration sites at defined frequencies, it can be difficult to assess data arising from complex samples for the failure to detect clones that are present (false negatives) and/or the presence of amplicons not based on true integration events within the genome (false positives).…”