- The capture reaction performed with these probes was highly efficient with 97% of the targets recovered on the focal species. - Conclusion: The target capture protocol used in this study was effective in capturing genome-wide variation in the focal species L. - 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - 1 Marine Biodiversity, Naturalis Biodiversity Center, Leiden, The Netherlands Full list of author information is available at the end of the article. - They constitute an important part of the global marine zooplankton assemblage e.g. - In spite of their vulnerability to ocean acidification and their im- portant trophic and biogeochemical roles in the global marine ecosystem, little is known about their resilience to- wards changing conditions [5].. - For Limacina helicina, the Arctic and Antarctic populations were discovered to be separate species through differences in the COI gene [16, 17].. - RADseq [23], which involves the enzymatic fragmentation of genomic DNA followed by the selective sequencing of the regions flanking the restric- tion sites of the used enzyme(s), is attractive for non- model organisms as no prior knowledge of the genome is required. - Prior knowledge of the genome is required for probe de- sign, however, this information is usually limited for non- model organisms. - In this study, we designed target capture probes for the shelled pteropod Limacina bulimoides based on the method developed in Choquet et al. - bulimoides to develop a set of target capture probes, and tested the success of these probes through the number of single nucleotide polymorphisms (SNPs) recovered in the focal species. - Limacinoid pteropods have a high abundance and biomass in the world’s oceans and have been the focus of most ocean acidification research to date e.g. - Draft genome assembly. - Scaffolding resulted in a slight improvement, with an in- crease in the N50 to 893 bp and a decrease in the L50 to 994,289 contigs. - Therefore, a predicted 60.4% of the complete genome was sequenced.. - Genome completeness based on the assembled draft genome was measured in BUSCO version and resulted in the detection of 60.2% of near universal orthologues that were either completely or partially present in the draft genome of L. - SRR to assess the completeness of the coding sequences and aid in the design of capture probes. - of the genome was assembled of the transcripts could be mapped onto it using the splice- aware mapper GMAP version . - About half of the transcripts (46,701 transcripts) had single mapping paths and the other half (46,605 transcripts) had multiple mapping paths. - Of the singly mapped transcripts, 8374 mapped to a scaffold that contained two or more distinct exons separated by introns. - 3) and 87% of the recovered targets (2446 of the 2822 targets) had a sequence depth of 15x or more across at least 90%. - Of the 2822 targets, 643 targets Table 1 Summary of draft genome statistics for Limacina. - Number of scaffolds. - Table 2 Summary of BUSCO analysis showing the number of metazoan near universal orthologues that could be detected in the draft genome of Limacina bulimoides. - accounted for 50% of the total aligned reads in L. - Of the 10 mitochondrial genes included in the capture, surprisingly, only the COI target was recovered.. - The hybridisation of the probes and targeted re- sequencing worked much less efficiently on the four related species. - trochiformis) targets were covered with a mini- mum of 15x depth across 90% of the bases (Additional file 1: Table S1). - The number of targets that accounted for 50% of the total aligned reads varied across species, with 4 of 620 targets for L. - The number of SNPs ranged between 1371 (H.. - Across the five species of Limacinoidea, we found an exponential decrease in the efficiency of the targeted re- sequencing congruent with the genetic distance from the focal species L. - Only 62 targets were found in common across all five species, comprising 14 conserved pteropod orthologues, 47 coding regions, and a 700 bp por- tion of the 28S nuclear gene. - Based on the differences in profiles of number of SNPs per target and total number of SNPs, the hybridisation worked differently between the focal and non-focal species. - bulimoides, the median number of SNPs per target was 45, whereas in the remaining four species, most of the targets had only one SNP and the median number of SNPs per target was much lower: 11 for L. - The number of SNPs per target varied between one and more than 200 across the targets (Fig. - The hard-filtered SNPs were subsequently filtered to keep those with a minimum site coverage of 5x and present in at least 80% of the individuals. - There was an initial 10-fold decrease in number of SNPs between L. - The subsequent decrease in number of SNPs was smaller in L.. - Number of targets. - Bars on the right of the dashed vertical line represent the number of targets where more than 90% of the bases in each target was sequenced with ≥ 15x depth. - 60% of the genome was sequenced. - In comparison, previously sequenced molluscan genomes have shown a wide variation in size across spe- cies, ranging from 412 Mbp in the giant owl limpet (Lot- tia gigantea) [47] to 2.7 Gbp in the Californian two-spot octopus (Octopus bimaculoides) [48]. - Fur- ther, when considering marine gastropod genome size estimates in the Animal Genome Size Database [50], genome sizes range from 430 Mbp to 5.88 Gbp with an average size of 1.86 Gbp. - Roughly 350 million paired-end (PE) reads were used for the de novo assembly, but 50% of the assembly is still largely unresolved with fragments smaller than 893 bp. - The absence of peaks in the k-mer distribution histogram and low mean coverage of the draft genome may indicate insufficient sequencing depth caused by a large total genome size, and/or high heterozygosity which complicates the assembly. - In the 1.6 Gbp genome of another gastropod, the big-ear radix, Radix auricu- laria, approximately 70% of the content consisted of repeats [51]. - Number of SNPs per target. - Numbers in brackets represent the total number of targets in that category on the set of target probes designed for Limacina bulimoides Species Biomin. - Our results show that generating a draft genome and tran- scriptome to serve as a reference in the design of target capture probes is a promising and cost-effective approach to allow population genomics studies in non-model spe- cies of small sizes. - Despite the relatively low N50 of the as- sembled genome, we were able to map 79.8% of the transcript sequences onto it. - The combined use of the transcriptome and fragmented genome allowed us to identify the expressed genomic regions reliably and in- clude intronic regions, which may have contributed to the probe hybridisation success [59]. - The target capture was highly successful in the focal spe- cies L. - The number of SNPs recovered (Table 4) and percentage of properly paired reads mapping uniquely to the tar- gets (Table 3) are comparable to the results from a similar protocol on copepods [26].. - Surprisingly, only the COI gene was recovered out of the 10 mitochondrial genes in- cluded in the set of probes. - Only 13 of the 41 non-coding targeted regions were recovered, which may indicate that these re- gions were also too divergent to be captured by the probes.. - The success of targeted re-sequencing of the four related pteropod species (L. - The number of SNPs recovered also decreased rapidly with genetic distance (Fig. - While direct com- parisons are not possible due to differences in the probe design protocol and measurements used, we also see a decreasing trend in success of target capture applied with increasing levels of genetic divergence in other studies e.g. - This probably indicates that high levels of genetic diversity and divergence from the focal species resulted in the targeted regions not being able to hybridise to the probes. - Indeed, from the four non-focal pteropod species, most of the recovered targets had low diversity, containing only a single SNP (Fig. - 3 Density of single nucleotide polymorphisms (SNPs, present in 80% of individuals) plotted against coverage for each of the five pteropod species (a: Limacina bulimoides , b: L. - levels of gene flow in the focal species. - The statistical strength from analysing many genomic loci overcomes the limitation of an incomplete sampling of the meta- population [74] and increases the capacity to detect even subtle patterns in population structure. - Back in the lab, 147.2 ng of genomic DNA was extracted from the whole specimen using the E.Z.N.A. - 4 Log-scaled number of SNPs against genetic divergence from the focal species Limacina bulimoides shows that there is a sharp reduction in the SNPs recovered with genetic distance. - The total genome size was roughly estimated using MaSuRCA (as a by-product of calculating optimal assembly parameters), based on the size of the hash table containing all error corrected reads. - A second estimate of the genome size was made by searching for k-mer peaks in sequencing reads using JELLYFISH version with various k- mer lengths between 15 and 101. - To assess the complete- ness of the generated draft genome, the in-built BUSCO metazoan dataset containing 978 near-universal ortholo- gues of 65 species was used to search for key orthologous genes with BUSCO version 3.0.1 [42]. - Target capture probes design. - We designed the target capture probe set by using the draft genome and transcriptome as a reference, following the workflow recommended by Choquet et al. - Firstly, we aimed to select only single-copy coding DNA sequences (CDS) in order to achieve a high specificity of the target capture probes and to reduce false-positive SNPs from multi-copy genes. - We then mapped these selected transcript se- quences (with splicing allowed) directly to the contigs of the genomic assembly to identify expressed regions and their respective exon-intron boundaries. - Additionally, 643 transcripts that mapped to unique contigs in the draft genome were selected from a set of conserved orthologues from a phylogenomic ana- lysis of pteropods [43] to give a set of 2812 single copy coding nuclear targets. - Of the 63 transcripts that showed homology to biomineralisation proteins [45, 46], we in- cluded 35 of these candidate biomineralisation genes in the final probe set as they could be mapped to contigs in the draft genome (Additional file 2).. - A frag- ment of the COI gene (NCBI: MK642914), obtained by. - bulimoides, to evaluate the efficiency of the target capture probes on species of vary- ing genetic relatedness. - inflatus) were collected across various sites during the AMT22 and AMT24 cruises in the Atlantic and from two sites in the Pacific Ocean (Table 6 and Additional file 1: Table S2). - The captured library of the species L. - The captured libraries of the other species were sequenced together on the same NextSeq500 mid-output v2 chip.. - The processed SNPs were further filtered using VCFtools version to keep those with a minimum coverage of 5x and rep- resented in at least 80% of the individuals.. - In order to investigate the relative effect of the differ- ent SNP filters, other less conservative VCFtools filtering settings such as a reduced genotyping rate of 50% or re- duced depth requirement of 2x were used, and the rela- tive increase in number of SNPs recovered for each species was recorded. - For each species, the resulting VCF files were then annotated with the names and coor- dinates of the original targets using retabvcf.pl [83]. - The targets represented in each species and the number of SNPs per target were then extracted from the annotated VCF files (Additional file 1: Appendix S4).. - bulimoides and each of the four other species was calculated from the branch lengths of a maximum likelihood (ML) phylogeny of pteropods based on tran- scriptome data [43]. - The number of SNPs recovered per species using the most conservative filtering settings (80% genotyping rate and 5x depth) was plotted against sequence divergence from L. - We thank Erica Goetze, Nina Bednar š ek, Alice Burridge, and Lisette Mekkes as well as the captains and crews of the research cruises for support and assistance with collecting zooplankton samples.. - Three individuals per site were included from localities in the Atlantic and Pacific Oceans.. - Latitude and longitude are presented in the decimal system, with positive values indicating North and East and negative values, South and West, respectively. - All authors provided feedback and approved of the final manuscript.. - SAMN11131519), and raw sequencing data of the target capture are available in NCBI Genbank, under BioProject PRJNA527191. - The global distribution of pteropods and their contribution to carbonate and carbon biomass in the modern ocean. - Impact of ocean acidification and elevated temperatures on early juveniles of the polar shelled pteropod Limacina helicina: mortality, shell degradation, and shell growth. - Assessing species boundaries in the open sea: an integrative taxonomic approach to the pteropod genus Diacavolinia . - a case study of the marine zooplankton Calanus finmarchicus . - Exposure to CO2 influences metabolism, calcification, and gene expression of the thecosome pteropod Limacina retroversa . - Near-future pH conditions severely impact calcification, metabolism and the nervous system in the pteropod Heliconoides inflatus. - The origin and diversification of pteropods predate past perturbations in the Earth ’ s carbon cycle. - Characterization of the pigmented shell-forming proteome of the common grove snail Cepaea nemoralis . - NurkowskiKA, Smets P, Rieseberg L, et al.. - Mitochondrial DNA hyperdiversity and its potential causes in the marine periwinkle Melarhaphe neritoides (Mollusca: Gastropoda)
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