Whatman fta card

A total of 244 unrelated animals of both sexes were randomly sampled from six provinces representing all the continental agroecosystems of Ecuador: Bolívar, Chimborazo, Cotopaxi, Guayas, Morona Santiago, and Tungurahua (Supplementary Material: Figure S1, Table S1). Specific sampling and geospatial locations are reported in Table S1, according to the 26 municipalities within the 6 provinces of precedence. Up to 200 μL of EDTA K3 (BD Vacutainer, Franklin Lakes, NJ, USA) anticoagulated blood was loaded in the collection circle of Whatman^® FTA^® Cards (GE healthcare Life Science, Little Chalfont, Buckinghamshire, UK). The cards were dried at 25 °C for a minimum of 3 h and then stored in paper envelopes until use. DNA was extracted for both mitochondrial DNA and STRs (short tandem repeats) analysis, following a modification of the method described by Walsh et al. [14 (link)]. Briefly, three circles were cut in the spotted cards using a 2 mm Harris Micro punch (GE healthcare Life Science, Little Chalfont, Buckinghamshire, UK), which was cleaned using 1% bleach solution between each sample. The circles were placed in a PCR plate and incubated in 100 µL of a 5% CHELEX 100 resin solution (Bio-Rad, Hercules, CA, USA) at 95 °C for 15 min, 60 °C for 15 min, and finally 99 °C for 3 min. The lysate was removed and frozen at −20 °C until use.

Genomic DNA was extracted from blood preserved on Whatman FTA cards (GE Healthcare, Chicago, IL, USA, CAT#WB120401). A total of 526 individuals that met concentration and DNA quality requirements were genotyped on the Axiom 600 K Genome-Wide Chicken Array Kit (Affymetrix, Santa Clara, CA, USA, CAT#902148) by Geneseek (Lincoln, NE, USA). SNPs were then filtered for a call rate greater than 95% and minor allele frequency of greater than 0.01. After quality filtering, 304,500 SNPs were used in the downstream analysis from 526 individuals. SNP positions were mapped to the galGal 5 reference genome.

Blood samples were collected on Whatman FTA cards (GE Healthcare Life Sciences, IL, USA) from 400 unrelated Kuwaiti (253 males and 147 females). DNA was amplified directly, without quantification, from a 1.2 mm FTA card punch, according to the directions in the PowerPlex Fusion 6C manual, using a SureCycler 8800 thermal cycler (Agilent Technologies, CA, USA). Detection and separation of the DNA fragments were carried out using an Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific) with the internal lane standard WEN ILS 500 and allelic ladder provided with the PowerPlex Fusion 6C kit. Genotype determination and allele calling for only the 23 autosomal loci were carried out using GeneMapper ID-X software version 1.4 (Thermo Fisher Scientific).

To infer the evolutionary history of the haplotypes at the Red locus we constructed gene genealogies and compared the level of evolutionary divergence between the black and red haplotypes with those of orthologous sequences in other species. First, blood samples of 14 species in the family Estrildidae including red-faced (E. pealii, E. psittacae) or blue-faced (E. tricolor and E. trichroa) Erythrura species were obtained from an aviculturist. Blood samples were collected and stored using Whatman FTA cards (GE Healthcare, Little Chalfont, UK) and DNA was extracted following the manufacturer’s instructions. Sequences of the fragments amplifiable using the M1–M3 primers (Supplementary Table 2) were obtained using Sanger sequencing. For zebra finch, the sequences from the reference genome assembly were used.
We tested alternative nucleotide substitution models using the R package, phangorn v2.3.1^{78 (link)}, which suggested GTR+Γ+I to be the best model for the sequence of the Red locus. Maximum-likelihood trees were then constructed using this model and tested with 1000 bootstraps using phangorn and visualised using ggtree v1.4.20^{79 (link)}. To examine if the molecular clock hypothesis applied to the Red locus, we used Tajima’s nonparametric relative rate test implemented in pegas v0.10^{80 (link)}.

This descriptive study included a total of 400 blood samples from the same number of Honduran malaria patients and collected onto Whatman FTA^® cards (GE Healthcare). Patients had been routinely assessed for malaria by the Honduran Ministry of Health laboratory network both by standard microscopy and subsequent confirmation by molecular analysis. Cards had been stored in the molecular biology laboratory at the National Autonomous University of Honduras (UNAH), and archived by malaria species diagnosis and patient’s provenance, but devoid of patients’ personal information. Other than malaria infection, no other clinical information (e.g. health status, haemolytic anaemia, etc.) was associated with the samples. According to the place of origin, samples were from 22 municipalities located in five malaria-endemic departments (second-level political jurisdictions) of Honduras: Atlántida (n = 35), Colón (n = 45), Gracias a Dios (n = 133), Islas de la Bahía (n = 107), and Olancho (n = 78) (Fig. 1).Fig. 1

Map of Honduras and number of samples collected in five departments showing the parasite species causing the malaria infection.

Malaria-naturally exposed subjects who received parasitological diagnosis using Giemsa-stained thick blood smear microscopy under 1000x magnification were referred to our study. Whole blood specimens from subjects who were diagnosed with the presence of Pf asexual parasitaemia (parasites counted per 200 leukocytes and parasite density calculated as the number of parasites per microliter by assuming a fixed leukocyte count of 8000 cells/μL of blood) were sampled as dried blood spots (DBSs) on Whatman FTA cards (GE Healthcare) as recommended by the manufacturer. Genomic DNA was extracted [using the QIAGEN DNeasy Blood & Tissue Kit (Qiagen), according to the manufacturer’s instructions] from DBSs and monospecies infection was confirmed by polymerase chain reaction (PCR). Ten clinical samples with high parasitaemia (parasite density > 50000/μL), and qualitatively and quantitatively good enough, were selected to ensure the integrity of sequencing.

To examine the use of Whatman FTA cards as a convenient method for the capture, transport, and storage of DNA, pus and blood samples from case horses were placed onto classic Whatman FTA indicating cards (GE Healthcare). Aliquots (0.5 ml) of pus diluted with 0.5 ml of sterile saline were inactivated by heat treatment at 95°C for 30 min in a water bath prior to application of 200 μl of sample to the card (45 (link)). Two 200-μl blood spots and two heat-inactivated pus samples from each horse were placed onto separate sample zones, and the loaded FTA cards were air dried overnight before being microwaved at full power (800 W) for 30 s, left to stand for 1 min, and microwaved for a further 30 s. A glass beaker containing >200 ml of water was placed alongside the cards in the microwave to dissipate heat. Cards with heat-inactivated samples were individually placed into sealable plastic bags and dispatched to the University of Liverpool, where they were stored at 4°C upon arrival.