Download Report_FollowUp7_18052016

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.
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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.
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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
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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)
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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.
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