A Survey of Virus Recombination Uncovers Canonical Features of Artificial Chimeras Generated During Deep Sequencing Library Preparation

Abstract

Chimeric reads can be generated by in vitro recombination during the preparation of high-throughput sequencing libraries. Our attempt to detect biological recombination between the genomes of dengue virus (DENV; +ssRNA genome) and its mosquito host using the Illumina Nextera sequencing library preparation kit revealed that most, if not all, detected host–virus chimeras were artificial. Indeed, these chimeras were not more frequent than with control RNA from another species (a pillbug), which was never in contact with DENV RNA prior to the library preparation. The proportion of chimera types merely reflected those of the three species among sequencing reads. Chimeras were frequently characterized by the presence of 1-20 bp microhomology between recombining fragments. Within-species chimeras mostly involved fragments in opposite orientations and located less than 100 bp from each other in the parental genome. We found similar features in published datasets using two other viruses: Ebola virus (EBOV; -ssRNA genome) and a herpesvirus (dsDNA genome), both produced with the Illumina Nextera protocol. These canonical features suggest that artificial chimeras are generated by intra-molecular template switching of the DNA polymerase during the PCR step of the Nextera protocol. Finally, a published Illumina dataset using the Flock House virus (FHV; +ssRNA genome) generated with a protocol preventing artificial recombination revealed the presence of 1-10 bp microhomology motifs in FHV–FHV chimeras, but very few recombining fragments were in opposite orientations. Our analysis uncovered sequence features characterizing recombination breakpoints in short-read sequencing datasets, which can be helpful to evaluate the presence and extent of artificial recombination.

The discovery, distribution and diversity of DNA viruses associated withDrosophila melanogasterin Europe

“…However, the vast majority of these (5,124 out of 5,169) aligned internally to TEs, more than 5 bp away from the start or end position of the TE, which is inconsistent with insertion (Gilbert et al 2016, Loiseau et al 2020. Instead, this pattern suggests PCR-mediated recombination, and assuming that all chimeras we found were artefactual, their proportion among all reads mapping to the Kallithea virus (0.01%) falls in the lower range of that found in other studies (Peccoud et al 2018). We therefore believe there is no evidence supporting bona fide transposition of D. melanogaster TEs into genomes of the Kallithea virus in these natural virus isolates.…”

“…We aligned these to 135 D. melanogaster TEs curated in the November 2016 version of Repbase (Bao et al 2015) using blastn (-task megablast). All reads for which one portion aligned to the virus (Genome reference KX130344.1) and another portion aligned to a D. melanogaster TE were identified as chimeric using the R script provided by (Peccoud et al 2018), and those for which the read-pair spanned TE ends were considered evidence of a TE insertion.…”

Recombination Signal in Mycobacterium tuberculosis Stems from Reference-guided Assemblies and Alignment Artefacts

“…For example, the contribution of intrasegmental recombination to rotavirus evolution has been rejected due to the observation that recombined segments occur only once and, thus, might have originated as errors during sequencing ( Woods 2015 ). Similarly, technical chimeras originating from in vitro recombination impede the detection of biological recombination between dengue virus and its host ( Peccoud 2018 ). Assembly errors in the analysis of mixed species samples can result in overestimating the abundance of LGT in the focal genome ( Koutsovoulos et al.…”

Characterization of a new case of XMLV (Bxv1) contamination in the human cell line Hep2 (clone 2B)

“…Characteristics of HeLa-HPV18 junctions supported by both DNA-and RNA-seq reads. a Several steps involved in the construction of an Illumina library (including cDNA library synthesis and illumina PCR) may generate artificial chimeras 46,47 . Thus, relying only on one read to identify a breakpoint is not good practice.…”