Nanopore sequencing enables multigenic family reconstruction despite highly frequent PCR-induced recombination

Abstract

This study developed a new bioinformatics pipeline to acquire all the different copies of multi-copy gene families based on Oxford Nanopore Technologies sequencing of PCR products. We used this pipeline to acquire the sequences of highly similar copies of the cidA and cidB genes present in the genomes of Wolbachia pipientis (wPip) bacteria infecting the cells of Culex pipiens mosquitoes. The approach is based on read mapping, SNP calling and haplotyping, using our already wide existing reference database for the cid genes obtained by cloning and Sanger sequencing. We addressed problems commonly faced when using mapping approaches for multi-copy gene families with highly similar variants (or haplotypes). In addition, we confirmed that PCR amplification causes frequent chimeras which have to be carefully considered when working on families of recombinant genes. We tested the robustness of the pipeline through a combination of analyses of simulated reads and of gene sequence acquisitions through cloning and Sanger sequencing. For genes of which the haplotype cannot be reconstructed from short reads sequencing, this pipeline confers a high throughput acquisition, gives reliable results as well as insights of the relative copy numbers of the different variants.

Intra-lineage microevolution ofWolbachialeads to the emergence of new cytoplasmic incompatibility patterns

“…To decipher the genomic changes that led to this recent CI shift, we sequenced the cidA and cidB repertoires present in Wolbachia infecting four individuals from the Ist isofemale line and eight females from the Slab isofemale line (four compatible and four incompatible) using Nanopore Technologies sequencing of cidA and cidB polymerase chain reaction (PCR) products [39]. While Wolbachia present in all Ist individuals had the same cid repertoires (S1 Table ), we found polymorphism in cid repertoires of Slab individuals: they all had the same cidB (i.e., toxin) diversity, composed of cidB-III-ae3 and cidB-III-ag1 variants, whereas cidA (i.e., antidotes) repertoires varied among individuals.…”

“…Previous investigations describing the cid repertoire of a given Culex line were performed by acquiring the sequences of cidA and cidB variants separately [22,27,[39][40][41]. Here, we also amplified and Nanopore sequenced a region with both cidA and cidB variable regions in a single amplicon (S5 Fig) . We could thus reconstruct the cidA-cidB tandems associated in the genome, resulting in the complete repertoires shown in Figure 3.…”

“…We acquired the cidA and cidB repertoires of eight individuals from the Slab isofemale line, four individuals from the Ist isofemale line, and two individuals from each of the newly established isofemale lines following the pipeline described in [39]. We also acquired cidAB combinations for all newly established isofemale lines.…”

“…In short, the target sequence was amplified using the adequate primer set (S5 Table, S6 Fig) . PCR products were then sequenced by Nanopore sequencing with the MGX Platform (Montpellier) using a MinION (Oxford Nanopore Technologies). Repertoires were then acquired following the pipeline described in [39]. For cidAB combinations, the pipeline was adapted: cidA and cidB repertoires were first obtained, and then the combinations between all cidA and cidB were artificially created and used as a reference base to run the pipeline.…”

“…We took advantage of a recent methodological development that enables the rapid and extensive acquisition of cidA and cidB repertoires using Nanopore Technologies sequencing [39] to address these three hypotheses. We were able to rule out the contamination hypothesis and show that no less than three distinct Wolbachia sublineages with different cidA repertoires now coexist in the Slab isofemale line (but do not appear to coinfect the same individuals).…”