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Follow-up meeting 7 GENBAS Location: RBINS May 18 Authored by: Sofie Derycke Follow-up meeting 7 GENBAS 18/05/2016 Present : Loic Kéver, Pascal Poncin, Koen Herten, Gregory Maes, Maarten Van Steenberge, Erik Verheyen, Sofie Derycke, Nicolas Lichilin Start meeting: 9.30h Gregory Maes replaces Jeroen Van houdt and manages now the GENBAS project for the Genomics Core. WP1: Beha vioura l and acoustic experiments Task 1.1: Installation of the tanks, experimental set up. Task 1.2: Recording and characterisation of the behaviour during con- and heterospecific encounters. Task 1.3: Recording and characterisation of acoustic communication during con- and heterospecific encounters. Task 1.4: Data analysis behaviour and acoustics. Overview: The first three tasks have been completed. Follow-up meeting 7 GENBAS | May 18 Ethograms have been constructed based on three reproductions for O. ventralis, four for O. nasuta and one for O. boops. During these matings, sound production was almost absent. The most prevalent difference between the three species was the ‘inviting’ behaviour of O. ventralis, which has not been observed in the two other species. A sound was recorded accompanying this inviting behaviour. The ethogram and sound data are currently being processed for a publication by the Ulg partners. 1 A mate choice experiment has been conducted in which female O. nasuta have been exposed to a male O. nasuta, O. ventralis, O. boops, a female O. nasuta and to nothing. Exposure lasted 45 minutes, during which the fish were recorded and after which the brain parts were dissected and fixed in RNAlater for gene expression analysis. Analysis of the movement of the two fish in each tank with ImageJ tracking software is ongoing, preliminary results do not show much difference before and after removal of the opaque wall. Comparison of behavioural aspects shows a clear difference in activity before and after removal of the opaque wall and between the control fish that are not exposed to another fish and the fish that have been exposed to another fish. Females appear to be much more aggressive when confronted with another female, while they fled more when confronted with a male. However, these differences are not statistically significant. The most important aspect here was to verify that the fish respond when they see another fish, and this is clearly the case! Next to the mate choice experiment, females were killed when they had deposited their first egg. This moment was considered to be the point of no return, the moment at which we can be absolutely sure that the female has chosen her mating partner. This experiment involved a conspecific setting of O. ventralis and O. nasuta and a heterospecific setting with an O. ventralis female and an O. nasuta male. We have 4, 4 and 3 replicates respectively. At this point, the contract of Loic Kéver (postdoc, Ulg) is finished. The juveniles will be kept in Brussels and used for an experiment to see whether the microbiome of Ophthalmotilapia differs between the species. The adult fish can stay in Liege. Loic remains interested in the project, and will be included in all our future correspondence. Actions to be taken: 1. Continue paper with ethograms (Loic) 2. Explore other statistical methods than Kruskal- Wallis (eg Permanova) for the behavioural aspects of the mate choice experiment (Loic) 3. Movement of fish in the mate choice experiment: instead of taking the average across 45 min, plot points for every 30s for each specimen and then compare the point clouds across specimens (Maarten) 4. Preparation of paper on Ophthalmotilapia with morphological data, mtDNA and GBS data (Maarten) WP2 : Specimen and tissue collection Task 2.1: Purchase, import and determination of live fishes. Task 2.1: DNA extraction samples genotyping by sequencing. Task 2.2: Brain dissection and RNA extraction samples. Overview: All brains have been dissected, and RNA extraction is completed for the mate choice experiment. RNA extraction for the female egg deposition experiment is halfway. Actions to be taken: 1. Finalise RNA extraction (18 samples remain) by end of May (Sofie) WP3: NGS Task 3.1: Genotyping by sequencing Task 3.2: RNA sequencing Overview: GBS data has been generated for 48 O. heterodonta, 88 O. nasuta, 38 O. ventralis, 6 cf. O. ventralis and 1 O. boops. Five samples have low read numbers, while six samples recieved a Follow-up meeting 7 GENBAS | May 18 The first two tasks are completed. 2 very high number of reads. The majority of samples show read numbers that differ at most two times. The number of raw reads between technical replicates can differ with a factor 2 (510 501 – 1 025 081 reads). This means that the differences in read numbers between samples can vary twofold with our adjusted GBS protocol just because of technical issues. Five tissue samples have been extracted with the Nucleospin protocol and again with the Paulino protocol. The latter protocol does not use a spin filter, and produces a clear intact band of high molecular weight DNA. The Nucleospin samples used for this test were partially degraded and did not have the intact DNA band. We expected to obtain more reads from the tissues extracted with the Paulino protocol. Three of the five samples yielded comparable read numbers between both extraction protocols. For the two other samples, the nucleospin protocol clearly outperformed the Paulino protocol in terms of read numbers. The GBS library protocol as tested now yields good data, the only point of adjustment is that the samples will be measured 2 times with the picogreen before going into the pool. RNA quantseq libraries have been prepared, containing all brainparts from the mate choice experiment (90 samples) and more than half of the samples from the female egg experiment. Actions to be taken: 1. GBS: library prep pool 2: 2 * 96 samples so that we can determine one or two lanes. Before pooling, measure samples 2 (or 3) times with picogreen. Pay attention to size selection (was probably not correctly performed for pool 1). Sequencing run scheduled for July the latest (Gregory) 2. RNA Quantseq remaining 18 samples (scheduled for June latest) (Gregory) Follow-up meeting 7 GENBAS | May 18 WP4: Genomics data analysis 3 Task 4.1: Preparatory data analysis for genotyping by sequencing. Task 4.2: Data analysis genotyping by sequencing. Task 4.3: Data analysis RNA sequences. Overview: Task 1 has been completed. Raw reads of the 192 specimens from pool 1 were mapped against O. niloticus and Metriaclima. The latter genome resulted in a higher number of reads mapped (mean of 65 vs 79.4%, respectively). SNP calling against O. niloticus yielded a total of 318 456 SNPs. After filtering for genotype quality, read depth, minor allele frequency, indels and missing data, 10 277 SNPs are retained for further analysis. The filtered SNPs were used to create a phylogenetic tree using SNPhylo. The resulting tree nicely shows the clustering of samples according to species, and within species, a clear geographic clustering can be observed. For each SNP, Fst was calculated between and within species. Manhattan plots of the resulting Fst values showed that SNPs were located throughout the genome, and that Fst values ranged from 0 to 1 in all genomic regions. No ‘islands of divergence’ could be observed. When plotting nucleotide diversity over 10 kb windows, 6 regions with high diversity were detected in all comparisons within and between species. Mapping of the RNA quantseq data was done against the genome of O. niloticus and the transcriptome of O. ventralis. In both cases, a substantial part of the reads are not mapped (45 and 42% respectively). Preliminary analysis of the female egg experiment shows a clear distinction in the 500 most expressed genes between O. nasuta females in conspecific setting, O. ventralis females in conspecific setting and O. ventralis in heterospecific setting for two brain parts. Analysis of the mate choice experiment is ungoing. RNA Quantseq 1. to increase the mapping results, Gregory proposed to create an O. ventralis brain transcriptome. The cost for this ranges between 4000 and 6000 euro. When preparing such a transcriptome, we have to be sure that all genes are expressed, otherwise this will not yield more reads to be mapped. This requires a set of different experimental conditions to increase the number of genes turned on. It may be a better strategy to focus on the O. ventralis genome instead, because this will contain all genes. The annotation file for the O. ventralis transcriptome and the status of the O. ventralis genome need to be known. Walter Salzburger will be contacted (Sofie) 2. Reads will be mapped against the Metraclinia genome (Koen) Follow-up meeting 7 GENBAS | May 18 Actions to be taken: GBS 1. Read mapping will be conducted based on de novo assembly. This may yield higher read mapping results than mapping against a reference genome (Koen) 2. Filter the SNP file obtained from mapping against O. niloticus for missing data after each step, so that we can see where we loose most of the data. Filter order: Total SNPs, missing data, indels, missing, 2 alleles, missing, homozyguous, missing, read depth 10 (instead of 20), missing, genotype quality 30, missing, maf, missing (Sofie) 3. Filter the SNP file obtained from mapping against Metriaclina (Sofie) 4. For the phylogenetic tree: reduce the number of missing data (use 75% instead of 50%), this may increase bootstrap support for O. ventralis clades. 5. For Manhattan plots: filter the SNP files per species, instead of on the whole dataset (Sofie) 6. For nucleotide diversity: check whether those regions have higer read depth than the others, as this may explain the observed pattern (Sofie) 4 3. DESeq analysis of the behavioural experiment is nearly finished. Results and a gene list will be transferred to Sofie beginning of June the latest (Koen) WP5: data integration Task 5.1: Integration of the behaviour and acoustics with the gene expression. Task 5.2: Integration of behaviour and acoustics with introgression patterns. Task 5.3: Integration of the gene expression analysis with the genotyping by sequencing. Overview: Nothing to report at this point WP6: Project coordination Task 6.1: Coordination of the project. Task 6.2: Reporting and administration. Task 6.3 Communication with the follow up committee. Task 6.4 Dissemination of the results. (All) From July 18-22, the FSBI Meeting is held in Bangor, UK. Sofie is going and gives an oral presentation. The next meeting is scheduled for Octobre. We intend to invite an external scientist that has expertise in integrating data from behavioural, genomic and transcriptomic data. Follow-up meeting 7 GENBAS | May 18 The meeting was ended at 14.30h. 5