Follow-up meeting 3 GENBAS
Location: Genomics Core (Leuven)
February 27
Authored by: Sofie Derycke
Follow-up meeting 3 GENBAS
27/02/2015
Present : Loic Kéver, Jeroen Van Houdt, Koen Herten, Jos Snoeks, Erik Verheyen, Sofie Derycke
Excused : Maarten Van Steenberge, Eric Parmentier, Pascal Poncin
Participants arrived at 9.30h.
Start meeting: 9.45h
Koen Herten (Genomics Core, KULeuven) has joined the GENBAS project. He is a
bioinformatician and is in charge of the data analyses for the GBS and RNAseq datasets.
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:
Seven aquaria are up and running at two locations (F=female; M=male):
Poncins lab:
- 6F + 3M of nasuta and 6F+2M of boops (tank 1, species separated by a net)
- 4F+1M of nasuta and 5F+1M of boops (tank 2, species separated by a net)
- 5F boobs (tank 3, controls)
Follow-up meeting 3 GENBAS | February 27
Ulg:
1
-
5F+1M ventralis (tank 4)
3F+1M ventralis (tank 5)
5F nasuta (tank 6, controls)
5F ventralis (tank 7, controls)
On February 26th, one female in tank 4 and one in tank 5 laid eggs!! This was captured on
video, and indicates that the fish have reached an equilibrium. A lot of fish have died
(especially O. ventralis). Over the last month, dead rate has substantially declined, and fish
start to show mating behavior. We expect the other species will also soon start to mate.
No progress on the acoustic part.
Control females (females that have not been exposed to males) will be left alone for one
month.
Actions to be taken:
1. If all proceeds as expected, the ethograms of O. ventralis should be ready soon. We
aim to have observations from at least three females (Loic).
2. Record acoustics during mating (Loic).
3. Post video of egg laying female on website (Loic and Sofie).
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:
The fish that have died have been sequenced to verify their origin. Haplotype of 1 O.
ventralis has been found around Ulwile island, which corresponds to the information from
the trader.
A database containing all Ophthalmotilapia specimens and tissues from the 92, 95, 2007,
2010 and 2014 campaigns has been compiled.
NJ trees and haplotype networks have been constructed and confirm the very close
relationships between the four Ophthalmotilapia species, and Cyathopharynx.
For GBS: all 442 specimens will be analysed. In this way, we have replication of the process
in different areas, and we will catch the maximum intraspecific genetic variability. Having a
full genome of 1 species may help the analyses substantially, but data analyses of the first
GBS test will indicate the necessity of this.
Actions to be taken:
1. sequences of ca 20 fish from the aquaria need to be compared with the database to
ensure the reliability of the trader (Maarten*).
2. morphological ID from specimens of the 2014 campaign and check morphology of
the few specimens that cluster as outlier in the TCS network (Maarten*)
3. Plot geographical distribution on haplotype networks (Maarten* and Sofie)
4. Perform RNA extraction on brain parts that have been in the freezer for three
months (Sofie)
5. Kill control females, dissect brains end of March/first week of April (Loic and Sofie)
6. Perform RNA extraction on all brain parts from all 15 females (Sofie)
*: Actions by Maarten are scheduled from June onwards
Follow-up meeting 3 GENBAS | February 27
Brain dissection and RNA extraction have yielded high quality RNA.
2
WP3: NGS
Task 3.1: Genotyping by sequencing
Task 3.2: RNA sequencing
Overview:
GBS libraries have been prepared with 9 test samples (2 from each species + 1
Cyathopharynx), for two restriction enzymes (ApeKI and PStI). Initial test indicate that PstI
yields many more fragments in the 200-300bp area than ApeKI. Size selection works good,
PCR with 15 cycles yields enough material to sequence. We want as many loci as possible
shared between the four species.
RNA sequencing will be done on the control females to perform power analysis => this will
give us an idea of the number of replicates we need in the real experiment and will ensure a
good experimental design.
Actions to be taken:
1. GBS: Sequence the two test libraries (planned for first week of March, Jeroen)
2. RNA seq on control females (planned for end of April/beginning May, Jeroen)
WP4: Genomics data analysis
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:
The GBS test data will be analysed using the GBS pipeline setup by Koen. After first
analyses, Sofie will join Koen to go through the pipeline.
Follow-up meeting 3 GENBAS | February 27
Actions to be taken:
1. Setup the GBS pipeline (Koen)
2. Check available genomes from closely related species (Sofie)
3
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)
The follow up committee has not been contacted yet because we do not have actual
project data yet. We will invite an expert in transcriptomics when we will have our first
RNA dataset.
From September 7-9, the Cichlid Science Meeting is held in Austria. Maarten is in the
organising committee, depending on the results we obtain from the experimental work,
GBS and RNAseq, we could all go to the meeting and try to organise an extra meeting
there with all experts that are present and relevant for our project. We should try to get
the list of participants as soon as possible so that we can contact those experts.
Follow-up meeting 3 GENBAS | February 27
The meeting was ended at 12.00h, and was followed by a visit to the Genomics Core facility
and by a lunch at the restaurant of AZ Gasthuisberg.
4