A quantitative model of error accumulation during PCR amplification

Pienaar, Elsje; Theron, M.; Nelson, Michael; Viljoen, Hendrik J.

doi:10.1016/j.compbiolchem.2005.11.002

Cited by 45 publications

(32 citation statements)

References 41 publications

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“…Other sources of error in dPCR that may also contribute to the variability in the measurement include chamber volume variation and sample distribution within a panel [38], degradation of the template due to prolonged periods of heating [39], [40], PCR inhibitors that affect one assay more than the other [3] as well as possible manual shearing of the DNA during microfluidic loading of the array. Overall, our findings suggest that care must be taken when linearised plasmid DNA is used to assess or control for more complex templates like genomic DNA.…”

Section: Discussionmentioning

confidence: 99%

Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

Whale¹,

Cowen²,

Foy³

et al. 2013

PLoS ONE

138

111

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Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA.

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Section: Discussionmentioning

confidence: 99%

Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

Whale¹,

Cowen²,

Foy³

et al. 2013

PLoS ONE

138

111

View full text Add to dashboard Cite

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“…The standard immunosequencing ( Rep-seq ) protocols include an amplification step that introduces amplification errors that can be propagated by consecutive amplification cycles, thus generating pseudo-diversity of an antibody repertoire (17, 18). These errors are further compounded by sequencing errors in reads (estimated at 0.5% per base on average in Illumina reads), leading to error-prone Rep-seq datasets, which trigger errors in the constructed repertoires and complicate downstream analysis of antibodies.…”

Section: Introductionmentioning

confidence: 99%

Reconstructing Antibody Repertoires from Error-Prone Immunosequencing Reads

Shlemov

Bankevich

Bzikadze

et al. 2017

The Journal of Immunology

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Transforming error-prone immunosequencing datasets into antibody repertoires is a fundamental problem in immunogenomics, and a prerequisite for studies of immune responses. Although various repertoire reconstruction algorithms were released in the last three years, it remains unclear how to benchmark them and how to assess the accuracy of the reconstructed repertoires. We describe an accurate IgReC algorithm for constructing antibody repertoires from high-throughput immunosequencing datasets and a new framework for assessing the quality of reconstructed repertoires. Surprisingly, antibody repertoires constructed by IgReC from barcoded immunosequencing datasets in the blind mode (without using information about unique molecular identifiers) improved upon the repertoires constructed by the state-of-the-art tools that use barcoding. This finding suggests that IgReC may alleviate the need to generate repertoires using the barcoding technology (the workhorse of current immunogenomics efforts) because our computational approach to error correction of immunosequencing data is nearly as powerful as the experimental approach based on barcoding.

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“…When this phenomenon takes place, in vitro-generated recombinant molecules will be present in the amplified product, which will result in overestimation of the diversity in the starting material (27,28). Another potential complication is the introduction of point mutations during PCR amplification due to misincorporation by the DNA polymerase (16). In an effort to elucidate the origin of recombinants in the clinical material, we first performed experiments that showed the in vitro source of the recombinant sequences.…”

mentioning

confidence: 99%

Frequent In Vitro Recombination in Internal Transcribed Spacers 1 and 2 during Genotyping of Pneumocystis jirovecii

2007

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Pneumocystis jirovecii is the causative agent of Pneumocystis pneumonia (PCP) in immunocompromised persons. Knowledge of the transmission and epidemiology of PCP is still incipient, and investigations on these subjects are based exclusively on applications of molecular typing techniques. The polymorphic internal transcribed spacers ITS1 and ITS2 in the ribosomal DNA operon, which in the P. jirovecii genome exist as single-copy DNA, are commonly used as target loci for isolate typing. In the course of genotyping P. jirovecii in respiratory specimens from PCP patients by amplification and cloning of a large number of ITS sequences, we found mixed infections (two or more types) in 50% of the samples. In a majority of the specimens with mixed infections, we detected many ITS haplotypes (combinations of ITS1 and ITS2 types) that appeared to be products of recombination between globally common ITS haplotypes present in the same sample. Here we present results of a series of experiments showing that essentially all ITS recombinants are chimeras formed during the genotyping process. Under standard conditions, as many as 37% of the amplified sequences could be hybrid DNA artifacts. We show that by modifying PCR amplification conditions, ITS chimera formation could be largely abolished and the erroneous establishment of artifactual haplotypes avoided. The accurate assessment of genetic diversity is fundamental for a better understanding of the epidemiology and biology of P. jirovecii infections.The infectious fungus Pneumocystis jirovecii is the etiological agent of Pneumocystis pneumonia (PCP) in immunocompromised individuals. Since P. jirovecii cannot be cultivated in vitro, investigations of the transmission and epidemiology of this organism have been based on applications of molecular typing techniques (1). For P. jirovecii, sequence analysis of the internal transcribed spacer (ITS) regions in the nuclear ribosomal DNA (rDNA) gene complex, which in this fungus exists as a single-copy locus (2, 24), is the most informative epidemiological tool available at present. ITS1 is located between the coding regions of the 18S and 5.8S rRNA genes, and ITS2 is located between the 5.8S and 26S rRNA genes. The sequence diversity of ITS1 and ITS2 among different strains of P. jirovecii and the fact that these segments are removed during ribosomal biogenesis and should not be subjected to selective pressure make them suitable targets for genotyping (10). To date, more than 30 ITS1 genotypes and 40 ITS2 genotypes with more than 90 haplotypes (combinations of ITS1 and ITS2 types) have been described worldwide, based on two different but similar typing systems (8, 26). Some ITS haplotypes are globally more common, and coinfections with multiple types of P. jirovecii often occur (4,8,11,15,17,25,26). Although these typing systems have been useful, questions have been raised about the extensive polymorphism and stability of the ITS locus and its use for the genotyping of P. jirovecii.During the genotyping of specimens from patients with ...

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A quantitative model of error accumulation during PCR amplification

Cited by 45 publications

References 41 publications

Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

Reconstructing Antibody Repertoires from Error-Prone Immunosequencing Reads

Frequent In Vitro Recombination in Internal Transcribed Spacers 1 and 2 during Genotyping of Pneumocystis jirovecii

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