The European Reference Genome Atlas: piloting a decentralised approach to equitable biodiversity genomics.

ABSTRACT: A global genome database of all of Earth’s species diversity could be a treasure trove of scientific discoveries. However, regardless of the major advances in genome sequencing technologies, only a tiny fraction of species have genomic information available. To contribute to a more complete planetary genomic database, scientists and institutions across the world have united under the Earth BioGenome Project (EBP), which plans to sequence and assemble high-quality reference genomes for all ∼1.5 million recognized eukaryotic species through a stepwise phased approach. As the initiative transitions into Phase II, where 150,000 species are to be sequenced in just four years, worldwide participation in the project will be fundamental to success. As the European node of the EBP, the European Reference Genome Atlas (ERGA) seeks to implement a new decentralised, accessible, equitable and inclusive model for producing high-quality reference genomes, which will inform EBP as it scales. To embark on this mission, ERGA launched a Pilot Project to establish a network across Europe to develop and test the first infrastructure of its kind for the coordinated and distributed reference genome production on 98 European eukaryotic species from sample providers across 33 European countries. Here we outline the process and challenges faced during the development of a pilot infrastructure for the production of reference genome resources, and explore the effectiveness of this approach in terms of high-quality reference genome production, considering also equity and inclusion. The outcomes and lessons learned during this pilot provide a solid foundation for ERGA while offering key learnings to other transnational and national genomic resource projects.info:eu-repo/semantics/publishedVersio

The Carniolan Honeybee from Slovenia-A Complete and Annotated Mitochondrial Genome with Comparisons to Closely Related Apis mellifera Subspecies

The complete mitochondrial genome of the Carniolan honeybee (Apis mellifera carnica) from Slovenia, a homeland of this subspecies, was acquired in two contigs from WGS data and annotated. The newly obtained mitochondrial genome is a circular closed loop of 16,447 bp. It comprises 37 genes (13 protein coding genes, 22 tRNA genes, and 2 rRNA genes) and an AT-rich control region. The order of the tRNA genes resembles the order characteristic of A. mellifera. The mitogenomic sequence of A. m. carnica from Slovenia contains 44 uniquely coded sites in comparison to the closely related subspecies A. m. ligustica and to A. m. carnica from Austria. Furthermore, 24 differences were recognised in comparison between A. m. carnica and A. m. ligustica subspecies. Among them, there are three SNPs that affect translation in the nd2, nd4, and cox2 genes, respectively. The phylogenetic placement of A. m. carnica from Slovenia within C lineage deviates from the expected position and changes the perspective on relationship between C and O lineages. The results of this study represent a valuable addition to the information available in the phylogenomic studies of A. mellifera-a pollinator species of worldwide importance. Such genomic information is essential for this local subspecies' conservation and preservation as well as its breeding and selection

Functional loss of two ceramide synthases elicits autophagy-dependent lifespan extension in <i>C. elegans</i>

Ceramide and its metabolites constitute a diverse group of lipids, which play important roles as structural entities of biological membranes as well as regulators of cellular growth, differentiation, and development. The C. elegans genome comprises three ceramide synthase genes; hyl-1, hyl-2, and lagr-1. HYL-1 function is required for synthesis of ceramides and sphingolipids containing very long acyl-chains (≥C24), while HYL-2 is required for synthesis of ceramides and sphingolipids containing shorter acyl-chains (≤C22). Here we show that functional loss of HYL-2 decreases lifespan, while loss of HYL-1 or LAGR-1 does not affect lifespan. We show that loss of HYL-1 and LAGR-1 functions extend lifespan in an autophagy-dependent manner, as knock down of the autophagy-associated gene ATG-12 abolishes hyl-1;lagr-1 longevity. The transcription factors PHA-4/FOXA, DAF-16/FOXO, and SKN-1 are also required for the observed lifespan extension, as well as the increased number of autophagosomes in hyl-1;lagr-1 animals. Both autophagic events and the transcription factors PHA-4/FOXA, DAF-16, and SKN-1 have previously been associated with dietary restriction-induced longevity. Accordingly, we find that hyl-1;lagr-1 animals display reduced feeding, increased resistance to heat, and reduced reproduction. Collectively, our data suggest that specific sphingolipids produced by different ceramide synthases have opposing roles in determination of C. elegans lifespan. We propose that loss of HYL-1 and LAGR-1 result in dietary restriction-induced autophagy and consequently prolonged longevity

A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds

Background The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map. Results Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor &gt;98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features. Conclusions The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.Andreas Wallberg and Ignas Bunikis contributed equally to this work.</p

Phenotypes associated with lifespan extension and stress resistance are observed in <i>hyl-1;lagr-1</i>.

<p>(A) Pumping rates of N2, <i>hyl-1;lagr-1,</i> and an <i>eat-2</i> mutant. The latter displays a pronounced reduction in pumping rate and is commonly used as a genetic model for dietary restricted animals. Bars represent the mean number of pumps per minute. Compared to N2 displaying a mean pumping rate of 201±2 pumps/min, <i>hyl-1;lagr-1</i> shows a 17.4% decrease with a mean pumping rate of 166±2, P<0.0001, while <i>eat-2</i> shows a 67.2% decrease with a mean pumping rate of 66±3, P<0.0001. The data represents an mean ± SEM of 15 measurements in 5 worms of each genotype. (B) Quantification of fluorescent beads in the pharynx and the anterior part of the intestine following a feeding period of 30 minutes. Compared to N2, <i>hyl-1;lagr-1</i> displays 59% less fluorescence, P = 0.0026. Mean ± SEM is shown, n indicates the number of worms. (C) Mean total brood size of N2 and <i>hyl-1;lagr-1</i>. Compared to N2 which displays a mean brood size of 304±9, <i>hyl-1;lagr-1</i> shows a 33% decrease with a mean brood size of 204±6, P<0.0001. Mean ± SEM is shown, n = number of worms examined. (D) Survival curves of N2 and <i>hyl-1;lagr-1</i> subjected to heat-shock at 37°C. Compared to N2, <i>hyl-1;lagr-1</i> shows increased resistance, P = 0.0016. A total of 60 worms of each strain were assayed. Mean ± SD of 3 experiments is shown. N2-worms (6) and <i>hyl-1;lagr-1</i> worms (22) were censored but are incorporated in the analysis until the time they were censored. (E) Bars represent mean median heat shock survival from the 3 experiments shown in D. Compared to N2 which has a median survival of 7 hours, <i>hyl-1;lagr-1</i> displays a 29% increase in heat shock resistance with a median survival of 9 hours. Error bars represent ± SD.</p

Knock-down of PHA-4, DAF-16, SKN-1, ATG-12, or SPHK-1 affect the extended longevity of <i>hyl-1;lagr-1.</i>

<p>Cumulative survival curves of N2 and <i>hyl-1;lagr-1</i> worms grown at 20°C subjected to either empty vector control bacteria (L4440) or the indicated RNAi from the early adult stage. (A) When subjected to <i>atg-12</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>atg-12(RNAi)</i> control animals, P = 0.3053. (B) When subjected to <i>pha-</i>4 RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>pha-4(RNAi)</i> control animals, P = 0.2369. (C) When subjected to <i>daf-16</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is decreased beyond the extent of <i>daf-16(RNAi)</i> control animals, P = 0.0002. (D) When subjected to <i>skn-1</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>skn-1(RNAi)</i> control animals, P = 0.5476. (E) When subjected to <i>daf-2</i> RNAi, <i>hyl-1;lagr-1</i> lifespan is further extended compared to both <i>hyl-1;lagr-1</i> control animals, P<0.0001, and <i>daf-2(RNAi)</i> control animals, P<0.0001. (F) When subjected to <i>eat-2</i> RNAi, <i>hyl-1;lagr-1</i> lifespan is decreased compared to <i>hyl-1;lagr-1</i> control animals, P = 0.0002, while the lifespan of <i>eat-2(RNAi)</i> animals is extended compared to wild-type control animals, P<0.0001. (G) When subjected to <i>aak-2</i> RNAi, <i>hyl-1;lagr-1</i> lifespan is decreased compared to <i>hyl-1;lagr-1</i> animals, P = 0.0009, while no lifespan effect is seen when comparing <i>aak-2(RNAi)</i> animals to wild-type control animals, P = 0.0975. (H) When subjected to <i>sphk-1</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>sphk-1(RNAi)</i> control animals, P = 0.8002. For additional details about these experiments, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070087#pone-0070087-t001" target="_blank">Table 1</a>.</p

Autophagy is increased in <i>hyl-1;lagr-1</i> and the response mechanism differs from that of wild type.

<p>LGG-1 is part of autophagosomal membranes and widely used as an indicator of autophagy in <i>C. elegans</i>. Bars represent the mean number of LGG-1::GFP-containing puncta per seem cell in non-starved wild type and <i>hyl-1;lagr-1</i> worms grown at 20°C subjected to either empty vector control bacteria (L4440) or the indicated RNAi. The number in each bar indicates the total number of seam cells observed. (A) Knock-down of <i>atg-12</i> lowers the level of autophagy in both wild type and <i>hyl-1;lagr-1</i>. (B) Knock-down of <i>pha-4</i> does not change the increased level of autophagy in <i>hyl-1;lagr-1</i> but increases autophagy in wild type. (C) Knock-down of <i>daf-16</i> lowers the increased level of autophagy in <i>hyl-1;lagr-1</i> but increases autophagy in wild type. (D) Knock-down of <i>skn-1</i> lowers the increased level of autophagy in <i>hyl-1;lagr-1</i> but increases autophagy in wild type. (E) Knock-down of <i>daf-2</i> increases autophagy to the same extent in wild type and <i>hyl-1;lagr-1</i>. (F) Knock-down of <i>sphk-1</i> increases the level of autophagy in <i>hyl-1;lagr-1</i> beyond wild type level. Statistical analyses were performed by unpaired two-tailed t-test (with Welch’s correction if variances were significantly different) using GraphPad Prism version 6.0 (GraphPad Software). The Bonferroni method was used to correct for multiple comparisons and P values below 0.0125 were considered statistically significant equivalent to a significance level of 0.05. (*) P≤0.0125, (**) P≤0.001, and (***) P≤0.0001. N used for analysis is the total number of worms observed for each treatment (23–45 worms, two trials). Mean ± SEM is shown.</p

Adult lifespan of <i>hyl-1;lagr-1</i> and N2 control worms subjected to empty vector control or the indicated RNAi at 20°C.

a<p>Median/mean RNAi lifespan of N2 and <i>hyl-1;lagr-1</i> fed the specified RNAi-bacteria.</p>b<p>Some animals were censored as they crawled of the plate, ruptured, or died as a “bag of worms”, however they are incorporated in the data set up until the day they were censored. The number of individual trials is in parentheses.</p>c<p>Median/mean control lifespan fed vector-only control bacteria.</p>d<p>P-values were determined using the Gehan-Breslow-Wilcoxon test using GraphPad Prism version 6.0 (GraphPad Software). The Bonferroni method was used to correct for multiple comparisons and P- values below 0.0125 are considered statistically significant equivalent to a significance level of 0.05 with four pair-wise comparisons. Cumulative statistics is shown in this table as experimental animals subjected to the same treatment behaved similarly between trials. Data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070087#pone-0070087-g001" target="_blank">Figure 1</a>.</p

Lipidomic analysis reveals a modified sphingolipid composition in <i>hyl-1;lagr-1</i>.

<p>Relative abundance of detected sphingolipid species showing significant changes in <i>hyl-1;lagr-1</i>. Sphingolipids containing C24-26 fatty acids and sphingomyelin species containing C16-18 fatty acids are lowered in <i>hyl-1;lagr-1</i>, while sphingolipids containing C21-22 fatty acids acids are more abundant. <i>C. elegans</i> sphingolipids predominately contain C17 long-chain bases, thus making the fatty acid chain length readily deducible. The number of carbon atoms indicated is without head groups. (A) Sphingomyelins containing C16-18 fatty acids are significantly reduced in <i>hyl-1;lagr-1</i>. (B) All significantly changed sphingolipid species containing C21 fatty acids are more abundant in <i>hyl-1;lagr-1</i>. (C) All significantly changed sphingolipid species containing C22 fatty acids are more abundant in <i>hyl-1;lagr-1</i>. (D) The only significantly changed sphingolipid species containing C23 fatty acids is more abundant in <i>hyl-1;lagr-1</i>. (E) All significantly changed sphingolipid species containing C24 fatty acids are less abundant in <i>hyl-1;lagr-1</i>. (F) All significantly changed sphingolipid species containing C25 fatty acids are less abundant in <i>hyl-1;lagr-1</i>. (G) All significantly changed sphingolipid species containing C26 fatty acids are less abundant in <i>hyl-1;lagr-1</i>. Statistical analyses were performed by one way analysis of variance followed by Dunnett’s multiple comparisons test using GraphPad Prism version 6.0 (GraphPad Software). Results from two biological experiments are shown for each strain. Mean ± SD is shown. All species shown are at least significant different at the P<0.05 level. The entirety of detected sphingolipid species can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070087#pone.0070087.s007" target="_blank">Figure S7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070087#pone.0070087.s008" target="_blank">Figure S8</a>.</p