DOI: 10.5061/dryad.37pvmcvpc
title: Population genetic structure of the gastropod species Bulinus truncatus

description: Background: Gastropod snails remain strongly understudied, despite their
 important role in transmitting parasitic diseases. Knowledge on their
 distribution and population dynamics increases our understanding of
 processes driving disease transmission. This is the first study using High
 Throughput Sequencing (HTS) to elucidate the population genetic structure
 of the hermaphroditic snail Bulinus truncatus (Gastropoda, Heterobranchia)
 on a regional (17 to 150 km) and an inter-regional (1,000 – 5,400 km)
 scale. This snail species acts as an intermediate host of Schistosoma
 haematobium and Schistosoma bovis, which cause human and animal
 schistosomiasis respectively. Methods: Bulinus truncatus snails were
 collected in Senegal, Cameroon, Egypt and France and identified through
 DNA barcoding. A single-end Genotyping by Sequencing (GBS) library,
 comprising of 87 snail specimens from the respective countries, was built
 and sequenced on an Illumina HiSeq 2000 platform. Reads were mapped
 against S. bovis and S. haematobium reference genomes to identify
 schistosome infections and Single Nucleotide Polymorphisms (SNPs) were
 scored using the Stacks pipeline. These SNPs were used to estimate genetic
 diversity, assess population structure and to construct phylogenetic trees
 of Bulinus truncatus. Results: A total of 10,750 SNPs were scored and used
 in downstream analyses. The phylogenetic analysis identified five clades,
 each consisting of snails from a single country but with two distinct
 clades within Senegal. Genetic diversity was low in all populations,
 reflecting high selfing rates, but varied between locations due to habitat
 variability. Significant genetic differentiation and isolation by distance
 patterns were observed at both spatial scales, indicating that gene flow
 is not strong enough to counteract the effects of population bottlenecks,
 high selfing rates and genetic drift. Remarkably, the population genetic
 differentiation on a regional scale (i.e. within Senegal) was as large as
 between populations on an inter-regional scale. The blind GBS technique
 was able to pick up parasite DNA in snail tissue, demonstrating the
 potential of HTS techniques to further elucidate the role of snail species
 in parasite transmission. Conclusions: HTS techniques offer a valuable
 toolbox to further investigate the population genetic patterns of
 schistosome intermediate host snails and the role of snail species in
 parasite transmission.
description: Samples of<em> B.
 truncatus </em>(numbers of specimens ranging from four to 16 per
 locality) were collected from 2011 to 2014 in Cameroon (one site,
 Cam_BAK), Senegal (four sites, Sen_DIAM, Sen_GUEC, Sen_MBO, Sen_NDO),
 Egypt (two sites, Egy_BEK, Egy_MAN) and France (one site,
 Cor_SUT). A single-end GBS reduced representation library
 (RRL) was built and sequenced on an Illumina HiSeq 2000 platform (Cornell
 University Biotechnology Resource Center). The library was prepared by
 digesting DNA with EcoT22I, ligating P1 and P2 Illumina sequencing primers
 (the former including molecular identifiers for sequence demultiplexing)
 and Illumina adapters, before proceeding with titration and library
 enrichment. The choice of the restriction enzyme followed preliminary
 comparisons between EcoT22I and PstI on representatives of <em>B.
 truncatus</em>. <em>HTS data
 processing</em> The HiSeq run produced
 257x10<sup>6</sup> single-end raw reads of 101 bp. FastQC
 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used for
 sequence quality check before trimming the last 7 bp of raw sequences as
 implemented in FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).
 We explored if, and to what extent, parasite infection could be detected
 using GBS data by adding two snail samples from an additional site of
 Senegal (Sen_PAK), which were known to be infected with <em>S.
 bovis </em>(i.e. cercariae collected after shedding). To remove
 possible contaminant sequences originating
 from <em>Schistosoma </em>spp<em>.</em> flukes,
 reads were mapped against the <em>S.
 bovis </em>and <em>S. haematobium </em>reference genomes
 (GenBank assembly GCA_003958945.1 and GCA_000699445.1,
 respectively)<em> </em>using DeconSeq 0.4.3 applying default
 settings. Decontaminated data were processed using the Stacks 1.21
 pipeline and implementing (1) process_radtags for demultiplexing and
 quality filtering and (2) denovo_map.pl to call SNPs. After some pilot
 runs with various parameter settings, denovo_map.pl considered a minimum
 threshold of 10 raw reads per stack (m), maximum two mismatches between
 loci from a single individual (M), maximum two mismatches when aligning
 secondary reads to primary stacks (N), and maximum two mismatches between
 loci when building the catalog (n). SNPs were scored when occurring in at
 least four samples. VCFtools v0.1.14 was used to filter out specimens with
 more than 60 % missing data, and SNPs with minimum allele frequencies
 &lt; 0.05. To eliminate putative paralogs we conducted a
 heterozygosity excess test for each SNP using VCFtools and applied a
 threshold level of significance of 0.01. The data were further filtered by
 selecting one single SNP per RAD tag. This generated a main dataset of
 10,750 polymorphic sites scored in 87 specimens.
description: Linux platform