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

Abstract

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 ...

Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA

“…That is lower than the frequency we obtained. The reasons could be [16] the length of the fragment they used to detect crossovers of 14 signature nucleotides (RT amino acid positions 181, 184, 188, 190, 210, 215, and 219) is shorter than ours (positions 41, 65, 67, 70, 74, 100, 103, 181, 184, 188, 190, 215, 219) [29]. We have signature nucleotide from positions 41 to 103.…”

“…Mild et al [22] used 100,000 templates in PCR and observed 0.89% recombinants while we used 1,000,000 templates in our PCR and observed 11.65% recombinants. PCR mediated recombination results from incompletely extended primers annealing to heterologous templates and extending in the next round of elongation [14,16]. By modifying PCR conditions to reduce the probability of premature termination, we found that that PCR mediated recombination could be reduced by 27 fold.…”

“…[14]. Other studies have reported >20% [15], and even 37% [16] of recombinant products after PCR amplification. These high rates of in vitro recombination represent a substantial limitation for determining linkage and haplotype composition [17,18].…”

Nosocomial Pneumocystis jirovecii Pneumonia: Lessons From a Cluster in Kidney Transplant Recipients

“…Upper case italics as (␤-TUB), Using DNA extracts from 11 confirmed PJP cases, the ITS1/2, ␤-TUB, ␤-TUB, mtLSU, and DHPS loci were amplified using primers and amplification conditions as specified (see Table, SDC 3, http://links.lww.com/TP/A553) (32)(33)(34)(35). Amplicons were purified and then sequenced commercially (Macrogen Inc., Seoul, Korea).…”

Reducing cloning artifacts for recovery of allelic sequences by T7 endonuclease I cleavage and single re-extension of PCR products — A benchmark

“…Host cell DNA repair system can also produce variants. When the host cell assimilates a plasmid containing heteroduplexes, a mismatch repair system works randomly utilizing either strand as template and thus makes chimeric sequences (Speksnijder et al, 2001;Longeri et al, 2002;Kanagawa, 2003;Beser et al, 2007). These PCR or cloning errors give rise to risks on over-estimation of number of haplotypes or alleles.…”