Sequence and structural variation in the genome of the Biomphalaria glabrata embryonic (Bge) cell line

Abstract

Background: The aquatic pulmonate snail Biomphalaria glabrata is a significant vector and laboratory host for the parasitic flatworm Schistosoma mansoni, an etiological agent for the neglected tropical disease schistosomiasis. Much is known regarding the host-parasite interactions of these two organisms, and the B. glabrata embryonic (Bge) cell line has been an invaluable resource in these studies. The B. glabrata BB02 genome sequence was recently released, but nothing is known of the sequence variation between this reference and the Bge cell genome, which has likely accumulated substantial genetic variation in the ~50 years since its isolation.

Results: Here, we report the genome sequence of our laboratory subculture of the Bge cell line (designated Bge3), which we mapped to the B. glabrata BB02 reference genome. Single nucleotide variants (SNVs) were predicted and focus was given to those SNVs that are most likely to affect the structure or expression of protein-coding genes. Furthermore, we have highlighted and validated high-impact SNVs in genes that have often been studied using Bge cells as an in vitro model, and other genes that may have contributed to the immortalization of this cell line. We also resolved representative karyotypes for the Bge3 subculture, which revealed a mixed population exhibiting substantial aneuploidy, in line with previous reports from other Bge subcultures.

Conclusions: The Bge3 genome differs from the B. glabrata BB02 reference genome in both sequence and structure, and these are likely to have significant biological effects. The availability of the Bge3 genome sequence, and an awareness of genomic differences with B. glabrata, will inform the design of experiments to understand gene function in this unique in vitro snail cell model. Additionally, this resource will aid in the development of new technologies and molecular approaches that promise to reveal more about this schistosomiasis-transmitting snail vector.

Keywords: Bge; Biomphalaria glabrata; Genome sequence; Karyotype; Schistosoma mansoni; Variant calling.

Fig. 1

Average read depth coverage (RDC) for the Bge3 cell line genome mapped to the 18 largest B. glabrata linkage groups (LGs). Illumina reads from the Bge3 genome were aligned to the B. glabrata BB02 reference and mapped to LGs. Each point represents the average RDC for one scaffold, and only scaffolds > 10,000 base pairs are shown. Horizontal lines represent the mean RDC for the entire LG, which is displayed at the bottom of each panel

Fig. 2

Annotation of genome-wide single-nucleotide variants (SNVs) and analysis for Gene Ontology (GO) term enrichment. a SNVs were filtered for quality and biallelism, and the filtered SNVs were annotated by their predicted impact on protein-coding genes. The number of variants for each classification of predicted impact is shown. b High-impact SNVs (a, red) were analyzed for GO term enrichment using Fisher’s exact test. The ten GO terms with lowest p-value in each of the three main GO classes are shown

Fig. 3

Molecular karyotyping of Bge3 cell line with read depth coverage and single-nucleotide variant allele frequency. Short paired-end Illumina reads from Bge3 and B. glabrata were mapped to version 1 of the strain BB02 reference genome. a Total normalized coverage for Bge3 and B. glabrata reads were similar (left), while coverage distributions for 7 of the 18 largest linkage groups (LGs) were significantly different (right, P < 0.05, highlighted in red). The Kolmogorov-Smirnov test was used to test the null hypothesis that the two LG RDC samples are drawn from the same distribution. b SNVs were filtered for quality and biallelism and grouped by LG. Reads supporting the alternative allele for each SNV were counted and plotted as a frequency to total reads at that site. Given a population with stable chromosome copy number (CCN), alternative allele frequency from independently inherited LGs should correlate to CCN. Allele frequencies greater than 0.875 and less than 0.125 were trimmed to theoretically accommodate octosomies. Frequencies in our data do not coalesce around predictable ratios, suggesting that CCN as measured by allele frequency is masked by a mixed aneuploid population

Fig. 4

Karyotype of Bge3 cells reveals mixed aneuploidy and differences among other subcultures of Bge. a Chromosomes were tallied for 20 cells; each point represents a single cell. Modal counts from karyotypes of other Bge subcultures are included for comparison [20]. b Karyotype counts and box plots for 6 groups previously established based on chromosome size and centromere position, and one group of unassigned remainders. Group means from karyotypes of other Bge subcultures are included. c A representative karyogram of one cell from the Bge3 analysis. d An example of a putative tetraploid cell