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A new method for isolation of purified genomic DNA from haemosporidian parasites inhabiting nucleated red blood cells
Accepted Manuscript A new method for isolation of purified genomic DNA from haemosporidian parasites inhabiting nucleated red blood cells Vaidas Palinauskas, Asta Križanauskienė, Tatjana A. Iezhova, Casimir V. Bolshakov, Jane Jönsson, Staffan Bensch, Gediminas Valkiūnas PII: DOI: Reference:
S0014-4894(12)00374-8 http://dx.doi.org/10.1016/j.exppara.2012.12.003 YEXPR 6575
To appear in:
Experimental Parasitology
Received Date: Revised Date: Accepted Date:
24 October 2012 11 December 2012 13 December 2012
Please cite this article as: Palinauskas, V., Križanauskienė, A., Iezhova, T.A., Bolshakov, C.V., Jönsson, J., Bensch, S., Valkiūnas, G., A new method for isolation of purified genomic DNA from haemosporidian parasites inhabiting nucleated red blood cells, Experimental Parasitology (2012), doi: http://dx.doi.org/10.1016/j.exppara.2012.12.003
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A new method for isolation of purified genomic DNA from haemosporidian parasites
2
inhabiting nucleated red blood cells
3 4
Vaidas Palinauskasa, Asta Križanauskienėa, Tatjana A. Iezhovaa, Casimir V.
5
Bolshakovb, Jane Jönssonc, Staffan Benschc and Gediminas Valkiūnasa
6 7
a
8
[email protected], tel: +37069887279, fax: +37052729352.
9
b
Nature Research Centre, Akademijos 2, Vilnius, LT-08412, Lithuania. E-mail:
Biological Station Rybachy of the Zoological Institute, Russian Academy of
10
Sciences, Rybachy, 238535, Kaliningrad Region, Russia.
11
c
12
University, Ecology Building, S-223 62 Lund, Sweden.
Department of Biology, Molecular Ecology and Evolution Laboratory, Lund
13 14 15 16 17 18 19 20 21 22 23 24 25
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1
Abstract
2
During the last 10 years, whole genomes have been sequenced from an increasing
3
number of organisms. However, there is still no data on complete genomes of avian
4
and lizard Plasmodium spp. or other haemosporidian parasites. In contrast to
5
mammals, bird and reptile red blood cells have nuclei and thus blood of these
6
vertebrates contains high amount of host DNA; that complicates preparation of
7
purified template DNA from haemosporidian parasites, which has been the main
8
obstacle for genomic studies of these parasites. In the present study we describe a
9
method that generates large amount of purified avian haemosporidian DNA. The
10
method is based on a unique biological feature of haemosporidian parasites, namely
11
that mature gametocytes in blood can be induced to exflagellate in vitro. This results
12
in the development of numerous microgametes, which can be separated from host
13
blood cells by simple centrifugation.
14
Our results reveal that this straight forward method provides opportunities to collect
15
pure parasite DNA material, which can be used as a template for various genetic
16
analyses including whole genome sequencing of haemosporidians infecting birds and
17
lizards.
18 19
Key Words: Avian malaria, haemosporidians, Haemoproteus tartakovskyi,
20
hSISKIN1, template, DNA purification, next generation sequencing.
21 22
1. Introduction
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The research of avian malaria parasites has a long history, starting in the end of XIXth century (Garnham, 1966). For a long time, these widespread parasites were
2
1
used as models to assist human malaria research (Trager, 1950; Davey, 1951; Coatney
2
et al., 1953; Ball and Chao, 1961; Ball, 1964; McGhee et al., 1977). The theoretical
3
importance of avian Plasmodium and related haemosporidian parasites remains
4
because of their astonishing diversity and phylogenetic relationships with the most
5
dangerous human malaria parasite Plasmodium falciparum (Pick et al., 2011).
6
Additionally, avian haemosporidian parasites continue to be of high interest in
7
evolutionary, ecological as well as in host-parasite interaction studies (Bensch et al.,
8
2000, 2004, 2009; Ricklefs et al., 2004; Palinauskas et al., 2008; Perkins, 2008; Levin
9
et al., 2009; Knowles et al., 2010; Ricklefs and Outlaw, 2010; Marzal et al., 2011).
10
During the last 10 years, whole genomes have been sequenced from an
11
increasing number of organisms. This innovative technique has progressively become
12
more accessible and the first genomes of mammals, plants and important pathogens
13
such as human malaria parasites have been determined (Arabidopsis Genome
14
Initiative, 2000; Lander et al., 2001; Gardner et al., 2002; Carlton et al., 2005; Pain et
15
al., 2008; Warren et al., 2010). The first malaria parasites to be sequenced was the
16
human parasite species Plasmodium falciparum (Gardner et al., 2002). Since then the
17
genomes of other malaria parasites, P. chabaudi, P. yoelii and P. knowlesi have been
18
completed and published (Janssen et al., 2001; Carlton et al., 2002; Pain et al., 2008).
19
Surprisingly, even though the cost of whole genome sequencing has been reduced to a
20
fraction of the costs of the first attempts to sequence genomes, there is no information
21
about complete avian or lizard Plasmodium spp. genomes. In Carlton et al's (2005)
22
review, the only avian malaria parasite listed to be in the process of being sequenced
23
was Plasmodium gallinaceum, however this attempt seems has come to a halt. The
24
lack of genomic resources from avian malaria and other haemosporidians has
25
prevented the construction of primers for amplification of microsatellites and other
3
1
rapidly evolving genomic regions. Therefore, studies on population genetics,
2
phylogeography, hybridization or molecular characterization of avian and reptilian
3
haemosporidians have so far been limited (Perkins, 2000; Austin and Perkins, 2006;
4
Beadell et al., 2006; Martinsen et al., 2008; Outlaw and Ricklefs, 2010; Valkiūnas et
5
al., 2012; Zehtindjiev et al., 2012).
6
In contrast to mammal blood, bird blood contains a high amount of host DNA,
7
which complicates preparation of template for genome sequencing. Random clone
8
sequencing is inefficient in haemosporidian parasites because the genome of
9
Plasmodium is 50 fold smaller than the genome of birds (Waltari and Edwards, 2002;
10
DeBarry and Kissinger, 2011). With genomes of an avian haemosporidians at hand it
11
would be possible to construct new markers for more variable nuclear genes for
12
phylogenetic investigations, including a detail comparison of genomes of mammalian
13
and bird malaria parasites. Until now, the majority of haemosporidian parasite studies
14
of birds have used primers of mitochondrial or other cytoplasmic genes, which are
15
maternal-inherited, thus not useful, for instance in investigations of within species
16
variation or determining mate-recognition signals in parasites (Valkiūnas et al.,
17
2008a, 2012).
18
Unique biology of haemosporidian parasites provides opportunities to
19
approach purified template preparation for analysis of their genomes. Mainly, the
20
sexual process of haemosporidians is oogamy (Garnham, 1966), i.e., fertilization
21
occurs extracellular and, therefore, development of gametes can be initiated and
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studied under controlled in vitro conditions (Valkiūnas et al., 2012). During
23
exflagellation, gametocytes leave erythrocytes within several seconds; the nucleus of
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microgametocyte divides to produce eight nuclei and then tiny thread-like
25
microgametes develop. This process naturally takes place in vectors immediately after
4
1
blood meal, but also can be easily induced in vitro (Garnham, 1966; Sinden, 1998;
2
Arai et al., 2001; Valkiūnas, 2005; Valkiūnas et al., 2012). The size and weight of
3
microgametes differ from blood cells thus the microgametes can be separated from
4
other cellular structures by gradient differences after centrifugation.
5
In the present study we describe a new method that generates large amount of
6
purified avian haemosporidian DNA. The method builds on in vitro manipulation of
7
mature haemosporidian gametocytes to enforce exflagellation and development of
8
numerous microgametes, which are separated from host blood cells by simple
9
centrifugation, followed by whole genome amplification. Our results reveal that this
10
simple method is suitable for preparation of purified template for next-generation
11
sequencing.
12 13
2. Materials and methods
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2.1. Study site and collection of blood samples
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The study was carried out at the Biological station “Rybachy” of the
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Zoological Institute of Russian Academy of Sciences on the Curonian Spit in the
19
Baltic Sea (55°05´N, 20°44´E) in May-July 2011. Based on previous work (Valkiūnas
20
et al., 2008a), we focused on the haemosporidian parasite Haemoproteus tartakovskyi
21
belonging to the family Haemoproteidae (cytochrome b lineage hSISKIN1). This
22
parasite is prevalent in common European songbirds, the siskin (Spinus spinus) and
23
crossbill (Loxia curvirostra), with parasitemia often >1% of infected red blood cells
24
in them. Importantly, single infections of H. tartakovskyi are often common in these
25
bird species (Valkiūnas, 2005).
5
1
In all, we captured 97 siskins and 23 crossbills for preliminary screening of
2
Haemoproteus infections by microscopy. The birds were caught opportunistically
3
using mist nets and ‘Rybachy’ type traps, and kept in cages for approximately 1-2 h
4
after capture. From each bird, the blood was collected into heparinized
5
microcapillaries by puncturing of the brachial vein. A drop of blood was used to make
6
2 blood films for microscopic investigation, and about 30-50 µl of blood was saved in
7
micro-tubes in non-lyses SET-buffer (Hellgren et al., 2004) for later molecular
8
analysis. The microtubes with blood in buffer were kept at -20°C until later analyses
9
in the laboratory.
10
Blood films were air dried, fixed with absolute methanol and stained as
11
described by Valkiūnas et al. (2008b). An Olympus BX51 light microscope equipped
12
with Olympus DP12 digital camera and imaging software DP-SOFT was used to
13
examine slides, prepare illustrations and to take measurements. About 100-150
14
microscopic fields were examined at low magnification (×400) and then at least 100
15
fields were studied at high magnification (×1000). Intensity of parasitemia was
16
estimated as a percentage by actual counting of the number of parasites per 1000
17
erythrocytes, as recommended by Godfrey et al. (1987). Blood parasites were
18
identified according to Valkiūnas (2005).
19
Uninfected birds and individuals with low parasitemia or co-infections with
20
other parasites were released immediately after microscopic examination of blood
21
films. Birds with > 1% of single H. tartakovskyi (hSISKIN1) were caged in a vector-
22
free room under controlled conditions (19 ± 1 °C; 50-60% RH; the natural light-dark
23
photoperiod). The birds were provided with canary seeds and water ad libitum. All
24
birds survived and were released after experiments.
25
6
1
2. 2. In vitro exflagellation and separation of microgametes
2 3
The purification assay of parasite cells is based on initiation of exflagellation
4
in vitro, followed by centrifugation resulting in separation of blood cells and
5
microgametes due to different weight of these cells.
6
Approximately 200 µl of blood was taken using heparinized microcapillaries
7
from the brachial vein of each individual donor bird with H. tartakovskyi parasitemia
8
≥1% (Table 1). The work was performed at 19 ± 1°C temperature. The blood was
9
placed immediately in a microtube containing 10µl of sodium citrate solution (3.7%),
10
gently mixed and exposed to air. Four minutes after exposure to air, the sample was
11
centrifuged for 5 min at 7000 rpm. After centrifugation, approximately 20-50µl of
12
supernatant (blood plasma) was stored in 150 µl SET-buffer and placed in -20°C
13
freezer for further processing in the laboratory.
14
For comparison of microscopy and PCR-based results, we prepared smears on
15
glass slides for microscopic examination; that was done 1) immediately after taking
16
blood from all donor birds, 2) 4 min after mixing the blood with sodium citrate and
17
exposure the mixture to air, and 3) after centrifugation when the supernatant of blood
18
plasma with microgametes was collected and placed in SET-buffer.
19
Number of microgametes in 1 µl of plasma was calculated as follows. First,
20
1µl of plasma with microgametes was placed using a micropipette on a glass slide,
21
resulting in a circular smear of approximately 3-5 mm in diameter. The smears were
22
air-dried, fixed with methanol and stained with Giemsa. Second, we calculated the
23
number of microscopic fields (× 1000 magnification) in each circular smear, and the
24
number of microgametes in each field, the summarized number of microgametes in all
7
1
microscopic fields is approximately equal to the total number of microgametes in 1µl
2
of plasma (Table 1).
3
Voucher specimens of microgamtes of H. tartakovskyi before and after
4
centrifugation were deposited in the Institute of Ecology, Nature Research Centre,
5
Vilnius, Lithuania (accession numbers 48664 – 48672 NS);
6 7
2. 3. DNA extraction and PCR of avian blood samples
8 9
For total DNA extraction from blood, we used innuPREP Blood DNA Mini Kit
10
(Analytikjena, Berlin, Germany) according to the manufacturer’s instructions. For
11
genetic analysis, we used a nested- PCR protocol amplifying part of the mitochondrial
12
cyt b gene (Bensch et al., 2000; Hellgren et al., 2004). In the first PCR we used the
13
primers HaemFNI and HaemNR3, general for haemosporidian parasites (Hellgren et
14
al., 2004). In the second PCR we used the primers specific to Haemoproteus and
15
Plasmodium spp., HAEMF and HAEMR2 (Bensch et al., 2000) and primers specific
16
to Leucocytozoon spp. HaemFL and HaemR3L (Hellgren et al., 2004). The
17
amplification was evaluated by running 1.5 µl of the final PCR product on a 2%
18
agarose gel.
19
Before amplification, we used samples with different DNA concentrations
20
(see below and Fig. 2A). After whole genome amplification, we expected to obtain
21
increased amounts of DNA correspondingly to the DNA concentrations in the initial
22
samples. The pattern of DNA concentration before and after whole genome
23
amplification was used as a control for possible contamination.
24 25
For sequencing we used the procedures as described by Bensch et al. (2000). Fragments for Haemoproteus spp. and Leucocytozoon spp. identification were
8
1
sequenced from the 5’ end with the primers HAEMF and HaemFL respectively. We
2
used dye terminator cycling sequencing (big dye) and loaded on an ABI PRISMTM
3
3100 capillary sequencing robot (Applied Biosystems, USA). Sequences were edited
4
and aligned using the program BioEdit (Hall, 1999). The ‘Basic Local Alignment
5
Search Tool’ (Blast) was used to determine the lineage of detected parasite sequences.
6 7
2. 4. Extraction and molecular evaluation of microgamete DNA
8 9
DNA of microgametes was extracted using DNeasy Blood & Tissue Kit
10
(Qiagen, Valencia, CA). Later, 3µl of total extracted DNA was evaluated by running
11
on 1% baby gel using a dilution series of lambda marker (100, 50, 25, 12.5, 6.25, and
12
3.125 ng). After evaluation of DNA concentration, samples with visible bands of
13
approximately 10 kb and approximate total quantities of 0.2-1 ng in 3 µl (Fig. 2, A)
14
were used for whole genome amplification (Illustra GenomiphiV2 DNA
15
Amplification Kit, GE Healthcare, Waukesha, WI). The amount of DNA post
16
amplification was evaluated by running 1 µl of amplified DNA on 1% baby gel using
17
a dilution series of lambda markers as described above.
18 19
3. Results
20
Haemoproteus tartakovskyi (the mitochondrial cyt b lineage hSISKIN1,
21
GenBank accession no. JX026908) was found in 22.7% of siskins and 52.2% of
22
crossbills (Fig. 1, A). The intensity of parasitemia was > 1% in 13 siskins and 4
23
crossbills. After microscopic examination and molecular analysis based on nested
24
PCR of mitochondrial cyt b gene, it was revealed that 10 siskins and 4 crossbills
9
1
contained co-infections with Leucocytozoon spp. For further analysis, we discarded
2
samples with co-infections and used 3 siskins with single H. tartakovskyi (SISKIN1)
3
infection (Table 1).
4
For further analysis we used 3 samples with approximately 38.5, 10.1 and 3.5
5
microgametes in each ×1000 magnification microscopic field determined by counting
6
the cells in the drop of plasma made after centrifugation (Fig. 1, D and E, and Table
7
1). For example, having 38.5 microgametes in one microscopic field, 1µl of plasma
8
contains approximately 3850 microgametes or 0.11 ng of DNA of H. tartakovskyi
9
(assuming that mass of 1000 malaria genomes is 0.0275ng).
10
The concentration of parasite DNA was increased several times by whole
11
genome amplification (Fig. 2 and Table 1). The DNA mass of samples 1 and 3 was
12
increased almost 200 times. The sample No 2 with initial mass of 0.13 ng/µl was
13
increased 50 times (Table 1).
14 15
4. Discussion
16 17
This study shows that separation of haemosporidian microgametes can be
18
efficiently initiated for genomic studies when the intensity of gametocytes is ≥ 2% in
19
donor birds. Importantly, the preparation of template is possible even if one individual
20
heavily infected bird with single haemosporidian infection is available. Interestingly,
21
exflagellation of Haemoproteus spp. is easy to initiate and it does not require using
22
additional media in vitro conditions; the exflagellation starts immediately after
23
exposure of blood containing mature gametocytes to air (Valkiūnas, 2005). Species of
24
Plasmodium (unlike many Haemoproteus spp.) require additional stimuli for initiation
25
of the sexual process in vitro, e.g., the presence of vector-derived xanthurenic acid
10
1
and blood-derived factors (Sinden, 1998; Arai et al., 2001); thus additional efforts will
2
be needed for collection of microgametes of malaria parasites. Additionally, intensity
3
of gametocyte parasitemia usually is lower during Plasmodium infections than
4
Haemoproteus infections (Valkiūnas, 2005); that also might complicate isolation of
5
microgametes of malaria parasites. This warrants additional investigation.
6
Recently, several methods to obtain purified avian Plasmodium spp. DNA
7
have been proposed by Graczyk et al. (1994) and Palinauskas et al. (2010). In the
8
former study, the authors used water-soluble cationic detergent EDTA-20 for
9
disruption of the red blood cell membranes and subsequent centrifugation technique
10
to separate haemosporidian parasites from the red blood cells (Graczyk et al., 1994).
11
It was shown that this method can be used for separation of avian blood cells and
12
haemosporidian cells. However, pure template for genomic studies has not been
13
obtained using this technique so far. An advantage of our method of purification of
14
haemosporidian parasites for genomic studies is that microgamete purification is done
15
directly without cell lyses, which is a case in the method described by Graczyk et al.
16
(1994). Supposedly, lysed blood cells would release huge amount of cytoplasmic and
17
nucleic genomic material to blood plasma and complicate purification process. In our
18
case, unlysed blood cells contain host DNA within the cells, which is suppressed to
19
the bottom of the microtube after centrifugation. Separation of haemosporidian
20
parasites from their host cells probably could be done using different sporogonic
21
stages (e.g. ookinetes, oocysts or sporozoites), however there is no information about
22
that so far.
23
The study by Palinauskas et al. (2010) revealed that it is possible to isolate
24
single parasite cells by using a laser micro-dissection techniques, and then to obtain
25
sequences from dissected parasites. With this technique one can collect hundreds of
11
1
single parasite cells into a single test tube. For instance, the total amount of DNA that
2
can be obtained from 500 malaria genomes is approximately 0.01375 ng (assuming a
3
25 MB genome has a mass of 2.75 x 10-14 g). However, for whole genome sequencing
4
the required amount of DNA is many folds larger, amounting to 50-2000 ng
5
depending on different protocols and sequencing platforms (Margulies et al., 2005).
6
From the present survey we can state that long lasting efforts to prepare the
7
purified template for sequencing the whole genome of haemosporidian parasites
8
developing in nucleated red blood cells finally is available. It is based on simple
9
separation of host DNA and parasite DNA based on natural exflagellation of
10
microgametes avoiding lyses of blood cells. Because exflagellation is a characteristic
11
feature of all haemosporidians and can be induced in vitro (Garnham, 1966; Sinden,
12
1998; Valkiūnas, 2005), our method is recommended for template preparation in
13
analysis of genomes of any other haemosporidian species inhabiting nucleated red
14
blood cells.
15 16
Acknowledgments
17
The experiments described herein comply with the current laws of Lithuania
18
and Russia. This article benefited from comments made by 2 anonymous reviewers.
19
The authors acknowledge the support of the Global Grant (VPI-3.1.-ŠMM-07-K-01-
20
047).
21 22
Figures
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Figure 1. Single Haemoproteus tartakovskyi (lineage hSISKIN1) infection in the
24
blood of siskin Spinus spinus: A – Gametocytes in the peripheral blood before
25
exposure to air; B – Exflagellation of 8 microgametes in vitro 4 min after exposure of
12
1
blood to air; C – Microgametes in blood smear before centrifugation; D, E – Purified
2
microgametes in blood plasma solution after centrifugation at high (D) and low (E)
3
magnification. Arrows – nuclei of parasites. Giemsa-stained thin blood films. Bar =
4
10 μm.
5 6
Figure 2. Agarose gel electrophoresis of microgametes’ DNA with Λ marker. A –
7
before whole genome amplification (3µl): line M – nucleotide size marker; lines 1, 2
8
and 3 – microgamete DNA samples used in analysis; lines N – microgamete DNA
9
samples with low concentration of genomic material not included in analysis; line C –
10
positive control from blood sample; lines Λ3.2 and 6.2 – markers for DNA
11
quantification. B – after whole genome amplification (1µl): lines M, N and Λ3.2-25.2
12
are the same as in (A); lines 1a, b, 2a, b and 3a, b – show the quantity of genomic
13
DNA after two different whole genome amplifications from the same sample.
14 15
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Janssen, C.S., Barrett, M.P., Lawson, D., Quail, M.A., Harris, D., Bowman, S., Phillips, R.S., Turner, C.M.R., 2001. Gene discovery in Plasmodium chabaudi by genome survey sequencing. Mol. Biochem. Parasitol. 113, 251–260.
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Perkins, S.L., 2000. Species concepts and malaria parasites: detecting a cryptic species of Plasmodium. Proc. R. Soc. Lond., B, Biol. Sci. 267, 2345–2350.
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Perkins, S.L., 2008. Molecular systematics of the three mitochondrial protein-coding genes of malaria parasites : Corroborative and new evidence for the origins of human malaria. Mitochondrial DNA 19, 471–478.
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Ricklefs, R.E., Fallon, S.M., Bermingham, E., 2004. Evolutionary relationships, cospeciation, and host switching in avian malaria parasites. Syst. Biol. 53, 111– 119.
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Ricklefs, R.E., Outlaw, D.C., 2010. A molecular clock for malaria parasites. Science 329, 226–229.
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Sinden, R.E., 1998. Gametogenesis and sexual development, in: Sherman, I.W. (Ed.), Malaria: Parasite biology, pathogenesis, and protection. American Society for Microbiology Press, Washington DC, pp. 25–48.
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Trager, W., 1950. Studies on the extracellular cultivation of an intracellular parasite (avian malaria). I. Development of the organisms in erythrocyte extracts, and the favoring effect of adenosinetriphosphate. J. Exp. Med. 92, 349–366.
18 19
Valkiūnas, G., 2005. Avian malaria parasites and other haemosporidia, America. CRC Press.
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Valkiūnas, G., Iezhova, T.A., Križanauskienė, A., Palinauskas, V., Bensch, S., 2008a. In vitro hybridization of Haemoproteus spp.: an experimental approach for direct investigation of reproductive isolation of parasites. J. Parasitol. 94, 1385–1394.
23 24 25
Valkiūnas, G., Iezhova, T. a, Križanauskienė, A., Palinauskas, V., Sehgal, R.N.M., Bensch, S., 2008b. A comparative analysis of microscopy and PCR-based detection methods for blood parasites. J. Parasitol. 94, 1395–1401.
26 27 28 29
Valkiūnas, G., Palinauskas, V., Asta Križanauskienė, A., Bernotienė, R., Rita Kazlauskienė, R., Iezhova, T.A., 2012. Further observations on in vitro hybridization of hemosporidian parasites: patterns of ookinete development in Haemoproteus spp. J. Parasitol. DOI: 10.1645/GE-3226.1.
30 31
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32 33
Warren, W.C., Clayton, D.F., Ellegren, H., Arnold, A.P., Hillier, L.W., et al., 2010. The genome of a songbird. Nature 464, 757–762.
34 35 36 37 38
Zehtindjiev, P., Križanauskienė, A., Bensch, S., Palinauskas, V., Asghar, M., Dimitrov, D., Scebba, S., Valkiūnas, G., 2012. A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction-based protocols for amplification of the cytochrome B gene. J. Parasitol. 98, 657–665.
39
16
1 2 3 4 5 6 7 8 9
Unique biology of haemosporidians provides approaching purified template for genomic studies. Separation of host and parasite DNA based on exflagellation avoids lyses of blood cells. Recommendation for De novo sequencing template preparation of avian and lizard haemosporidian parasites.
17
1 2 3
Table 1. Samples used for Haemoproteus tartakovskyi template preparation.
4 5 Experiment no.
Parasitemia in donor birds (%)
a
b
Amount of plasma (µl)
No of microgametes
c
d
The size of 1µl plasma drop (fields)
Approximate no of microgametes in 1µl of plasma
DNA before amplification (ng/µl)
DNA after6 amplification 7 (ng/µl)
e
8
1
5
40
38.5±26.1
100
3850
0.26
>30
2
2
20
10.1±9.8
160
1616
0.13
3
4
50
3.5±1.8
200
700
0.06
~6 10 ~12 11
9 a
- amount of plasma
12
discarded after centrifugation;
13
b
- number of microgametes in one microscopic field (×1000 magnification) after centrifugation (the arithmetic mean followed by and standard deviation);
14
c
- number of microscopic fields (×1000 magnification) in smears prepared using 1 drop (1µl) of plasma;
15
d
- amount of DNA before amplification in comparison to Λ marker;
16
e
- amount of DNA after whole genome amplification in comparison to Λ marker.
17
18
Figure 1
Figure 2
*Graphical Abstract (for review)
S0014-4894(12)00374-8 http://dx.doi.org/10.1016/j.exppara.2012.12.003 YEXPR 6575
To appear in:
Experimental Parasitology
Received Date: Revised Date: Accepted Date:
24 October 2012 11 December 2012 13 December 2012
Please cite this article as: Palinauskas, V., Križanauskienė, A., Iezhova, T.A., Bolshakov, C.V., Jönsson, J., Bensch, S., Valkiūnas, G., A new method for isolation of purified genomic DNA from haemosporidian parasites inhabiting nucleated red blood cells, Experimental Parasitology (2012), doi: http://dx.doi.org/10.1016/j.exppara.2012.12.003
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1
A new method for isolation of purified genomic DNA from haemosporidian parasites
2
inhabiting nucleated red blood cells
3 4
Vaidas Palinauskasa, Asta Križanauskienėa, Tatjana A. Iezhovaa, Casimir V.
5
Bolshakovb, Jane Jönssonc, Staffan Benschc and Gediminas Valkiūnasa
6 7
a
8
[email protected], tel: +37069887279, fax: +37052729352.
9
b
Nature Research Centre, Akademijos 2, Vilnius, LT-08412, Lithuania. E-mail:
Biological Station Rybachy of the Zoological Institute, Russian Academy of
10
Sciences, Rybachy, 238535, Kaliningrad Region, Russia.
11
c
12
University, Ecology Building, S-223 62 Lund, Sweden.
Department of Biology, Molecular Ecology and Evolution Laboratory, Lund
13 14 15 16 17 18 19 20 21 22 23 24 25
1
1
Abstract
2
During the last 10 years, whole genomes have been sequenced from an increasing
3
number of organisms. However, there is still no data on complete genomes of avian
4
and lizard Plasmodium spp. or other haemosporidian parasites. In contrast to
5
mammals, bird and reptile red blood cells have nuclei and thus blood of these
6
vertebrates contains high amount of host DNA; that complicates preparation of
7
purified template DNA from haemosporidian parasites, which has been the main
8
obstacle for genomic studies of these parasites. In the present study we describe a
9
method that generates large amount of purified avian haemosporidian DNA. The
10
method is based on a unique biological feature of haemosporidian parasites, namely
11
that mature gametocytes in blood can be induced to exflagellate in vitro. This results
12
in the development of numerous microgametes, which can be separated from host
13
blood cells by simple centrifugation.
14
Our results reveal that this straight forward method provides opportunities to collect
15
pure parasite DNA material, which can be used as a template for various genetic
16
analyses including whole genome sequencing of haemosporidians infecting birds and
17
lizards.
18 19
Key Words: Avian malaria, haemosporidians, Haemoproteus tartakovskyi,
20
hSISKIN1, template, DNA purification, next generation sequencing.
21 22
1. Introduction
23 24 25
The research of avian malaria parasites has a long history, starting in the end of XIXth century (Garnham, 1966). For a long time, these widespread parasites were
2
1
used as models to assist human malaria research (Trager, 1950; Davey, 1951; Coatney
2
et al., 1953; Ball and Chao, 1961; Ball, 1964; McGhee et al., 1977). The theoretical
3
importance of avian Plasmodium and related haemosporidian parasites remains
4
because of their astonishing diversity and phylogenetic relationships with the most
5
dangerous human malaria parasite Plasmodium falciparum (Pick et al., 2011).
6
Additionally, avian haemosporidian parasites continue to be of high interest in
7
evolutionary, ecological as well as in host-parasite interaction studies (Bensch et al.,
8
2000, 2004, 2009; Ricklefs et al., 2004; Palinauskas et al., 2008; Perkins, 2008; Levin
9
et al., 2009; Knowles et al., 2010; Ricklefs and Outlaw, 2010; Marzal et al., 2011).
10
During the last 10 years, whole genomes have been sequenced from an
11
increasing number of organisms. This innovative technique has progressively become
12
more accessible and the first genomes of mammals, plants and important pathogens
13
such as human malaria parasites have been determined (Arabidopsis Genome
14
Initiative, 2000; Lander et al., 2001; Gardner et al., 2002; Carlton et al., 2005; Pain et
15
al., 2008; Warren et al., 2010). The first malaria parasites to be sequenced was the
16
human parasite species Plasmodium falciparum (Gardner et al., 2002). Since then the
17
genomes of other malaria parasites, P. chabaudi, P. yoelii and P. knowlesi have been
18
completed and published (Janssen et al., 2001; Carlton et al., 2002; Pain et al., 2008).
19
Surprisingly, even though the cost of whole genome sequencing has been reduced to a
20
fraction of the costs of the first attempts to sequence genomes, there is no information
21
about complete avian or lizard Plasmodium spp. genomes. In Carlton et al's (2005)
22
review, the only avian malaria parasite listed to be in the process of being sequenced
23
was Plasmodium gallinaceum, however this attempt seems has come to a halt. The
24
lack of genomic resources from avian malaria and other haemosporidians has
25
prevented the construction of primers for amplification of microsatellites and other
3
1
rapidly evolving genomic regions. Therefore, studies on population genetics,
2
phylogeography, hybridization or molecular characterization of avian and reptilian
3
haemosporidians have so far been limited (Perkins, 2000; Austin and Perkins, 2006;
4
Beadell et al., 2006; Martinsen et al., 2008; Outlaw and Ricklefs, 2010; Valkiūnas et
5
al., 2012; Zehtindjiev et al., 2012).
6
In contrast to mammal blood, bird blood contains a high amount of host DNA,
7
which complicates preparation of template for genome sequencing. Random clone
8
sequencing is inefficient in haemosporidian parasites because the genome of
9
Plasmodium is 50 fold smaller than the genome of birds (Waltari and Edwards, 2002;
10
DeBarry and Kissinger, 2011). With genomes of an avian haemosporidians at hand it
11
would be possible to construct new markers for more variable nuclear genes for
12
phylogenetic investigations, including a detail comparison of genomes of mammalian
13
and bird malaria parasites. Until now, the majority of haemosporidian parasite studies
14
of birds have used primers of mitochondrial or other cytoplasmic genes, which are
15
maternal-inherited, thus not useful, for instance in investigations of within species
16
variation or determining mate-recognition signals in parasites (Valkiūnas et al.,
17
2008a, 2012).
18
Unique biology of haemosporidian parasites provides opportunities to
19
approach purified template preparation for analysis of their genomes. Mainly, the
20
sexual process of haemosporidians is oogamy (Garnham, 1966), i.e., fertilization
21
occurs extracellular and, therefore, development of gametes can be initiated and
22
studied under controlled in vitro conditions (Valkiūnas et al., 2012). During
23
exflagellation, gametocytes leave erythrocytes within several seconds; the nucleus of
24
microgametocyte divides to produce eight nuclei and then tiny thread-like
25
microgametes develop. This process naturally takes place in vectors immediately after
4
1
blood meal, but also can be easily induced in vitro (Garnham, 1966; Sinden, 1998;
2
Arai et al., 2001; Valkiūnas, 2005; Valkiūnas et al., 2012). The size and weight of
3
microgametes differ from blood cells thus the microgametes can be separated from
4
other cellular structures by gradient differences after centrifugation.
5
In the present study we describe a new method that generates large amount of
6
purified avian haemosporidian DNA. The method builds on in vitro manipulation of
7
mature haemosporidian gametocytes to enforce exflagellation and development of
8
numerous microgametes, which are separated from host blood cells by simple
9
centrifugation, followed by whole genome amplification. Our results reveal that this
10
simple method is suitable for preparation of purified template for next-generation
11
sequencing.
12 13
2. Materials and methods
14 15
2.1. Study site and collection of blood samples
16 17
The study was carried out at the Biological station “Rybachy” of the
18
Zoological Institute of Russian Academy of Sciences on the Curonian Spit in the
19
Baltic Sea (55°05´N, 20°44´E) in May-July 2011. Based on previous work (Valkiūnas
20
et al., 2008a), we focused on the haemosporidian parasite Haemoproteus tartakovskyi
21
belonging to the family Haemoproteidae (cytochrome b lineage hSISKIN1). This
22
parasite is prevalent in common European songbirds, the siskin (Spinus spinus) and
23
crossbill (Loxia curvirostra), with parasitemia often >1% of infected red blood cells
24
in them. Importantly, single infections of H. tartakovskyi are often common in these
25
bird species (Valkiūnas, 2005).
5
1
In all, we captured 97 siskins and 23 crossbills for preliminary screening of
2
Haemoproteus infections by microscopy. The birds were caught opportunistically
3
using mist nets and ‘Rybachy’ type traps, and kept in cages for approximately 1-2 h
4
after capture. From each bird, the blood was collected into heparinized
5
microcapillaries by puncturing of the brachial vein. A drop of blood was used to make
6
2 blood films for microscopic investigation, and about 30-50 µl of blood was saved in
7
micro-tubes in non-lyses SET-buffer (Hellgren et al., 2004) for later molecular
8
analysis. The microtubes with blood in buffer were kept at -20°C until later analyses
9
in the laboratory.
10
Blood films were air dried, fixed with absolute methanol and stained as
11
described by Valkiūnas et al. (2008b). An Olympus BX51 light microscope equipped
12
with Olympus DP12 digital camera and imaging software DP-SOFT was used to
13
examine slides, prepare illustrations and to take measurements. About 100-150
14
microscopic fields were examined at low magnification (×400) and then at least 100
15
fields were studied at high magnification (×1000). Intensity of parasitemia was
16
estimated as a percentage by actual counting of the number of parasites per 1000
17
erythrocytes, as recommended by Godfrey et al. (1987). Blood parasites were
18
identified according to Valkiūnas (2005).
19
Uninfected birds and individuals with low parasitemia or co-infections with
20
other parasites were released immediately after microscopic examination of blood
21
films. Birds with > 1% of single H. tartakovskyi (hSISKIN1) were caged in a vector-
22
free room under controlled conditions (19 ± 1 °C; 50-60% RH; the natural light-dark
23
photoperiod). The birds were provided with canary seeds and water ad libitum. All
24
birds survived and were released after experiments.
25
6
1
2. 2. In vitro exflagellation and separation of microgametes
2 3
The purification assay of parasite cells is based on initiation of exflagellation
4
in vitro, followed by centrifugation resulting in separation of blood cells and
5
microgametes due to different weight of these cells.
6
Approximately 200 µl of blood was taken using heparinized microcapillaries
7
from the brachial vein of each individual donor bird with H. tartakovskyi parasitemia
8
≥1% (Table 1). The work was performed at 19 ± 1°C temperature. The blood was
9
placed immediately in a microtube containing 10µl of sodium citrate solution (3.7%),
10
gently mixed and exposed to air. Four minutes after exposure to air, the sample was
11
centrifuged for 5 min at 7000 rpm. After centrifugation, approximately 20-50µl of
12
supernatant (blood plasma) was stored in 150 µl SET-buffer and placed in -20°C
13
freezer for further processing in the laboratory.
14
For comparison of microscopy and PCR-based results, we prepared smears on
15
glass slides for microscopic examination; that was done 1) immediately after taking
16
blood from all donor birds, 2) 4 min after mixing the blood with sodium citrate and
17
exposure the mixture to air, and 3) after centrifugation when the supernatant of blood
18
plasma with microgametes was collected and placed in SET-buffer.
19
Number of microgametes in 1 µl of plasma was calculated as follows. First,
20
1µl of plasma with microgametes was placed using a micropipette on a glass slide,
21
resulting in a circular smear of approximately 3-5 mm in diameter. The smears were
22
air-dried, fixed with methanol and stained with Giemsa. Second, we calculated the
23
number of microscopic fields (× 1000 magnification) in each circular smear, and the
24
number of microgametes in each field, the summarized number of microgametes in all
7
1
microscopic fields is approximately equal to the total number of microgametes in 1µl
2
of plasma (Table 1).
3
Voucher specimens of microgamtes of H. tartakovskyi before and after
4
centrifugation were deposited in the Institute of Ecology, Nature Research Centre,
5
Vilnius, Lithuania (accession numbers 48664 – 48672 NS);
6 7
2. 3. DNA extraction and PCR of avian blood samples
8 9
For total DNA extraction from blood, we used innuPREP Blood DNA Mini Kit
10
(Analytikjena, Berlin, Germany) according to the manufacturer’s instructions. For
11
genetic analysis, we used a nested- PCR protocol amplifying part of the mitochondrial
12
cyt b gene (Bensch et al., 2000; Hellgren et al., 2004). In the first PCR we used the
13
primers HaemFNI and HaemNR3, general for haemosporidian parasites (Hellgren et
14
al., 2004). In the second PCR we used the primers specific to Haemoproteus and
15
Plasmodium spp., HAEMF and HAEMR2 (Bensch et al., 2000) and primers specific
16
to Leucocytozoon spp. HaemFL and HaemR3L (Hellgren et al., 2004). The
17
amplification was evaluated by running 1.5 µl of the final PCR product on a 2%
18
agarose gel.
19
Before amplification, we used samples with different DNA concentrations
20
(see below and Fig. 2A). After whole genome amplification, we expected to obtain
21
increased amounts of DNA correspondingly to the DNA concentrations in the initial
22
samples. The pattern of DNA concentration before and after whole genome
23
amplification was used as a control for possible contamination.
24 25
For sequencing we used the procedures as described by Bensch et al. (2000). Fragments for Haemoproteus spp. and Leucocytozoon spp. identification were
8
1
sequenced from the 5’ end with the primers HAEMF and HaemFL respectively. We
2
used dye terminator cycling sequencing (big dye) and loaded on an ABI PRISMTM
3
3100 capillary sequencing robot (Applied Biosystems, USA). Sequences were edited
4
and aligned using the program BioEdit (Hall, 1999). The ‘Basic Local Alignment
5
Search Tool’ (Blast) was used to determine the lineage of detected parasite sequences.
6 7
2. 4. Extraction and molecular evaluation of microgamete DNA
8 9
DNA of microgametes was extracted using DNeasy Blood & Tissue Kit
10
(Qiagen, Valencia, CA). Later, 3µl of total extracted DNA was evaluated by running
11
on 1% baby gel using a dilution series of lambda marker (100, 50, 25, 12.5, 6.25, and
12
3.125 ng). After evaluation of DNA concentration, samples with visible bands of
13
approximately 10 kb and approximate total quantities of 0.2-1 ng in 3 µl (Fig. 2, A)
14
were used for whole genome amplification (Illustra GenomiphiV2 DNA
15
Amplification Kit, GE Healthcare, Waukesha, WI). The amount of DNA post
16
amplification was evaluated by running 1 µl of amplified DNA on 1% baby gel using
17
a dilution series of lambda markers as described above.
18 19
3. Results
20
Haemoproteus tartakovskyi (the mitochondrial cyt b lineage hSISKIN1,
21
GenBank accession no. JX026908) was found in 22.7% of siskins and 52.2% of
22
crossbills (Fig. 1, A). The intensity of parasitemia was > 1% in 13 siskins and 4
23
crossbills. After microscopic examination and molecular analysis based on nested
24
PCR of mitochondrial cyt b gene, it was revealed that 10 siskins and 4 crossbills
9
1
contained co-infections with Leucocytozoon spp. For further analysis, we discarded
2
samples with co-infections and used 3 siskins with single H. tartakovskyi (SISKIN1)
3
infection (Table 1).
4
For further analysis we used 3 samples with approximately 38.5, 10.1 and 3.5
5
microgametes in each ×1000 magnification microscopic field determined by counting
6
the cells in the drop of plasma made after centrifugation (Fig. 1, D and E, and Table
7
1). For example, having 38.5 microgametes in one microscopic field, 1µl of plasma
8
contains approximately 3850 microgametes or 0.11 ng of DNA of H. tartakovskyi
9
(assuming that mass of 1000 malaria genomes is 0.0275ng).
10
The concentration of parasite DNA was increased several times by whole
11
genome amplification (Fig. 2 and Table 1). The DNA mass of samples 1 and 3 was
12
increased almost 200 times. The sample No 2 with initial mass of 0.13 ng/µl was
13
increased 50 times (Table 1).
14 15
4. Discussion
16 17
This study shows that separation of haemosporidian microgametes can be
18
efficiently initiated for genomic studies when the intensity of gametocytes is ≥ 2% in
19
donor birds. Importantly, the preparation of template is possible even if one individual
20
heavily infected bird with single haemosporidian infection is available. Interestingly,
21
exflagellation of Haemoproteus spp. is easy to initiate and it does not require using
22
additional media in vitro conditions; the exflagellation starts immediately after
23
exposure of blood containing mature gametocytes to air (Valkiūnas, 2005). Species of
24
Plasmodium (unlike many Haemoproteus spp.) require additional stimuli for initiation
25
of the sexual process in vitro, e.g., the presence of vector-derived xanthurenic acid
10
1
and blood-derived factors (Sinden, 1998; Arai et al., 2001); thus additional efforts will
2
be needed for collection of microgametes of malaria parasites. Additionally, intensity
3
of gametocyte parasitemia usually is lower during Plasmodium infections than
4
Haemoproteus infections (Valkiūnas, 2005); that also might complicate isolation of
5
microgametes of malaria parasites. This warrants additional investigation.
6
Recently, several methods to obtain purified avian Plasmodium spp. DNA
7
have been proposed by Graczyk et al. (1994) and Palinauskas et al. (2010). In the
8
former study, the authors used water-soluble cationic detergent EDTA-20 for
9
disruption of the red blood cell membranes and subsequent centrifugation technique
10
to separate haemosporidian parasites from the red blood cells (Graczyk et al., 1994).
11
It was shown that this method can be used for separation of avian blood cells and
12
haemosporidian cells. However, pure template for genomic studies has not been
13
obtained using this technique so far. An advantage of our method of purification of
14
haemosporidian parasites for genomic studies is that microgamete purification is done
15
directly without cell lyses, which is a case in the method described by Graczyk et al.
16
(1994). Supposedly, lysed blood cells would release huge amount of cytoplasmic and
17
nucleic genomic material to blood plasma and complicate purification process. In our
18
case, unlysed blood cells contain host DNA within the cells, which is suppressed to
19
the bottom of the microtube after centrifugation. Separation of haemosporidian
20
parasites from their host cells probably could be done using different sporogonic
21
stages (e.g. ookinetes, oocysts or sporozoites), however there is no information about
22
that so far.
23
The study by Palinauskas et al. (2010) revealed that it is possible to isolate
24
single parasite cells by using a laser micro-dissection techniques, and then to obtain
25
sequences from dissected parasites. With this technique one can collect hundreds of
11
1
single parasite cells into a single test tube. For instance, the total amount of DNA that
2
can be obtained from 500 malaria genomes is approximately 0.01375 ng (assuming a
3
25 MB genome has a mass of 2.75 x 10-14 g). However, for whole genome sequencing
4
the required amount of DNA is many folds larger, amounting to 50-2000 ng
5
depending on different protocols and sequencing platforms (Margulies et al., 2005).
6
From the present survey we can state that long lasting efforts to prepare the
7
purified template for sequencing the whole genome of haemosporidian parasites
8
developing in nucleated red blood cells finally is available. It is based on simple
9
separation of host DNA and parasite DNA based on natural exflagellation of
10
microgametes avoiding lyses of blood cells. Because exflagellation is a characteristic
11
feature of all haemosporidians and can be induced in vitro (Garnham, 1966; Sinden,
12
1998; Valkiūnas, 2005), our method is recommended for template preparation in
13
analysis of genomes of any other haemosporidian species inhabiting nucleated red
14
blood cells.
15 16
Acknowledgments
17
The experiments described herein comply with the current laws of Lithuania
18
and Russia. This article benefited from comments made by 2 anonymous reviewers.
19
The authors acknowledge the support of the Global Grant (VPI-3.1.-ŠMM-07-K-01-
20
047).
21 22
Figures
23
Figure 1. Single Haemoproteus tartakovskyi (lineage hSISKIN1) infection in the
24
blood of siskin Spinus spinus: A – Gametocytes in the peripheral blood before
25
exposure to air; B – Exflagellation of 8 microgametes in vitro 4 min after exposure of
12
1
blood to air; C – Microgametes in blood smear before centrifugation; D, E – Purified
2
microgametes in blood plasma solution after centrifugation at high (D) and low (E)
3
magnification. Arrows – nuclei of parasites. Giemsa-stained thin blood films. Bar =
4
10 μm.
5 6
Figure 2. Agarose gel electrophoresis of microgametes’ DNA with Λ marker. A –
7
before whole genome amplification (3µl): line M – nucleotide size marker; lines 1, 2
8
and 3 – microgamete DNA samples used in analysis; lines N – microgamete DNA
9
samples with low concentration of genomic material not included in analysis; line C –
10
positive control from blood sample; lines Λ3.2 and 6.2 – markers for DNA
11
quantification. B – after whole genome amplification (1µl): lines M, N and Λ3.2-25.2
12
are the same as in (A); lines 1a, b, 2a, b and 3a, b – show the quantity of genomic
13
DNA after two different whole genome amplifications from the same sample.
14 15
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39
16
1 2 3 4 5 6 7 8 9
Unique biology of haemosporidians provides approaching purified template for genomic studies. Separation of host and parasite DNA based on exflagellation avoids lyses of blood cells. Recommendation for De novo sequencing template preparation of avian and lizard haemosporidian parasites.
17
1 2 3
Table 1. Samples used for Haemoproteus tartakovskyi template preparation.
4 5 Experiment no.
Parasitemia in donor birds (%)
a
b
Amount of plasma (µl)
No of microgametes
c
d
The size of 1µl plasma drop (fields)
Approximate no of microgametes in 1µl of plasma
DNA before amplification (ng/µl)
DNA after6 amplification 7 (ng/µl)
e
8
1
5
40
38.5±26.1
100
3850
0.26
>30
2
2
20
10.1±9.8
160
1616
0.13
3
4
50
3.5±1.8
200
700
0.06
~6 10 ~12 11
9 a
- amount of plasma
12
discarded after centrifugation;
13
b
- number of microgametes in one microscopic field (×1000 magnification) after centrifugation (the arithmetic mean followed by and standard deviation);
14
c
- number of microscopic fields (×1000 magnification) in smears prepared using 1 drop (1µl) of plasma;
15
d
- amount of DNA before amplification in comparison to Λ marker;
16
e
- amount of DNA after whole genome amplification in comparison to Λ marker.
17
18
Figure 1
Figure 2
*Graphical Abstract (for review)