Notes on peak reading of Msats of Paramuricea clavata from Portugal
Joana Boavida 05-Nov-2013/12-Jun-2014. CCMAR, University of Algarve.
joanarboavida@gmail.com
Supervisors of genetic work: Prof. Ester Serrão University of Algarve, Dr. Sophie Arnaud-Haond
IFREMER, Dr. Didier Aurelle IMBE.
Genotyped in a ABI3130 at IMBE, France. Software STRand + MsatAllele R package.
ATTENTION: This is a work in progress and might have mistakes; it is incomplete and is not in
any way a formal document.
In the pictures below notice the differences in the shape of the peaks between different
sampling locations (a.k.a. populations).
Locus: PARCLA 09
Sample FAR182
Figure 1 - Run: 247. PCR type: simple. Genotype: 118120 HETEROZYGOTE
Figure 2 - Run: 378. PCR type: Genotype: 120120 HOMOZYGOTE
Figure 3 - Run 457. PCR type: multiplex. Genotype: 120120 HOMOZYGOTE
Consider only the last peak from all the stutters of this locus.
Consider heterozygotes only the ones with clearly separated peaks or groups of stutters
(Figure 4).
Previously i considered heterozygotes for this locus peaks as the one in Figure 1. After scoring
more alleles realized the mistake and corrected the readings.
Figure 4 – True heterozygote for Locus PARCLA 09.
Figure 5 – Sample FAR147 at Locus PARCLA 9 and Right: sample FAR153 heterozygote with
very different sizes of alleles (consistent in two different runs: simple PCR and multiplex PCR).
PROBLEMS:
For the problems below the only options are to 1) repeat the PCR and genotyping for such
samples; 2) discard the sample in question.
Figure 6 – Sample FAR147 at Locus PARCLA 9 with Left: a heterozygote genotype and Right: a
homozygote genotype.
Figure 7 – FAR154. Left homozygote, Right heterozygote.
Figure 8 – Same sample FAR157 genotyped in two different events: Two different clear
heterozygote genotypes: 113118 and 099120.
Figure 9 – FAR161 with two different genotypes for the same locus: 113118 and 113116.
Figure 10 – Sample TAV38. Left: an incorrect heterozygote assigning based on a slightly higher
peak at size 139, genotype 139151. Right: a correct assigning of homozygote for the same
sample genotyped in a different run, genotype 151151. Notice the less obvious shape of the
139 peak.
Sample from Lagos: Heterozygote due to equal size of max peaks.
Figure 11- cumulative plot of alleles scoring.
Alleles 163 and 164 in the plot are actually from the same sample from two different runs in
the sequencer (see R output below). One was simple PCR (run228) and the other was multiplex
(run350). How to choose?
DFrow
Sample Reading
Gel
1 124 TAV57 (E3) 162.53 run0350
2 1209 TAV57 (B3) 164.47 run0228
i chose the simple (non-multiplex) PCR output. Samples inconsistent across different
genotyping events were discarded.
From 2014:
Below is a picture of the same sample, same PCR product, it’s the same sequencer mix (PCR
product diluted 25x + Formamide + size standard). I genotyped it twice in different days. Gives
different genotypes for one locus: First is homozygote 128128 and the second run gives a
heterozygote 153155! How is this possible? How to chose - > new pcr dilution and new
genotyping.
Locus Parcla 10
Heterozygote, Tavira, scored in 2013.
Heterozygote, Cape Espichel deep, scored in 2013.
Heterozygote, Cape Espichel deep, scored in 2013.
Homozygote, Tavira scored in 2013.
Homozygote, Segredo cave in Sagres scored in 2014.
Homozygote, Cape Espichel scored in 2014.
Heterozygote, Cape Espichel scored in 2014.
Heterozygote, Sines scored in 2014. Very different genotype.
Heterozygote, Sines scored in 2014. Very different genotype.
Homozygote, Sines scored in 2014. Very different genotype.
Locus Parcla 12
Berlengas. Homozygote. Genotype 108108
Berlengas. Heterozygote. Genotype 106126. Not perfect peaks.
For some locations this marker is not very polymorphic and always retrieves the 104104
genotype. The tall peak at left is not an allele. Sagres.
Heterozygote from Catrapona, Cape Espichel, 2014.
Locus Parcla 14
Berlengas. Homozygote. Genotype 177177
Berlengas. Heterozygote. Genotype 185189
Sagres. Heterozygote. Genotype 165183
Sines. Heterozygote.
Heterozygote. Tavira.
Heterozygote. Tavira.
Homozygote. Tavira.
The same heterozygote of Parcla 14: Top image shows just the NED; Bottom image shows NED
and PET: the PET peak (=parcla 17) shows the effect on the NED caused by that large peak,
which creates the artificial parcla 14 peaks in the middle seen in the top image. Portimão.
Heterozygote. Tavira.
Heterozygote from Sines (SIN) with confusing right peak caused by Parcla 17.
Locus 17
Berlengas. Heterozygote. Genotype 170178
Tavira. Less optimal heterozygote.
Tavira. Homozygote. Genotype 174174.
Portimão. Heterozygote.
Homozygote of the same population. Portimão.
Homozygote same population. Portimão.
Sagres, Berlengas and Espichel.
Locus Par_d
Homozygote, Berlengas, 2013.
Homozygote, Tavira, 2013.
Heterozygote, Berlengas, 2013.
Heterozygote, Tavira, 2013.
Heterozygote, Tavira, 2013.
Heterozygote, Cape Espichel, 2014.
Heterozygote, Cape Espichel, 2014.
Homozygote, Tavira, 2013.
Homozygote, Cape Espichel, 2013.
Heterozygote, Segredo cave in Sagres, 2014. Top image: by mistake used the method
“FragmentsCourts” and the sequencer didn’t read until the end (~600bp) only up to ~300bp;
that gave an untrue genotype very different (much shorter) than the true genotype (image
below, with method “Fragments”).
Heterozygote, Lagos, 2014. Very different genotype.
Notice how the peak at the left is not an allele (Lagos 2014). Now look to the next pictures:
The top image (Lagos 2014) seems to be heterozygote but it’s not, it’s the same right peak; the
picture below is from Berlengas (2014) and is a true heterozygote showing the same right peak
that is not our allele.