Estimating abundances of retroviral insertion sites from DNA fragment length data

Abstract

A likelihood function is proposed for the discrete lengths observed in each of a collection of insertion sites and is maximized with a hybrid expectation-maximization algorithm. Patient data illustrate the method and simulations show that relative abundance can be estimated with little bias, but that variation in highly abundant sites can be large. In replicated patient samples, variation exceeds what the model implies-requiring adjustment as in Efron (2004) or using jackknife standard errors. Consequently, it is advantageous to collect replicate samples to strengthen inferences about relative abundance.

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

“…The clonal repertoire reflected at this sequencing depth, however, is not exhaustive, partly because of the limitations imposed by the use of a given specific restriction enzyme (Harkey et al, 2007;Gabriel et al, 2009;Wu et al, 2013) and partly because of sampling issues. Moreover, the LM-PCR method can be skewed, as can any method that employs PCR amplification of intermediate products, toward amplification of smaller PCR products, among other limitations (Berry et al, 2012;Bystrykh et al, 2012), although we could not show that small amplicons had an effect on the extent to which the pyrosequencing reads correlate with the qPCR data ( Supplementary Fig. S3).…”

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

“…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. The internal control allowed us to define the sensitivity, accuracy, and overall quality of each MiSeq run, and provides a standard for measuring clonal abundance in complex samples.…”

“…While our data support the use of shear site counts in place of read counts, shear site counts could potentially underestimate the absolute abundance of a relatively large dominant clone because the number of different shear sites for a given VIS could be limited by the length of sheared DNA fragments. 21 Therefore, the gold standard for measuring the frequency of dominant clones should remain being VIS-specific qPCR using primers specific for a given insertion site. 18,38 Alternatively, random barcoding could be built into the adaptor to enhance the quantitativeness.…”

“…[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).…”

The MET Gene Is a Common Integration Target in Avian Leukosis Virus Subgroup J-Induced Chicken Hemangiomas

“…In order to measure the relative abundance of each integration, we quantified the number of sonication breakpoints for each site. Previous studies have shown that sonication breakpoints can be used as a measure of clonal expansion; if an integration has more than one breakpoint, the cell carrying that integration has undergone clonal expansion (43).…”