Nucleospin 96 tissue kit

A total of 483 C. imicola individuals from 16 localities in North Africa and south-western Europe were used for microsatellite analyses, and a portion of the mitochondrial genes Cytochrome oxydase subunit I (COI) and Cytochrome b (CytB) were sequenced for 132 successful genotyped individuals (Table 1, Fig. 1). Microsatellite data as well as COI and CytB sequences from eight of the localities were previously published in16 (link) (see details in Table 1). Genomic DNA was extracted from single adult C. imicola using a NucleoSpin96 Tissue Kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions. Nuclear genotyping was conducted at 9 microsatellite markers previously developed for C. imicola by Mardulyn et al.17 (link) (Supplementary Table 7) and following the protocol described in16 (link). Insects were sequenced for the mitochondrial genes COI and CytB using the primers C1J1718/C1N2191 and CytB_12329F/CytB_13038R, respectively, as described in16 (link).

Frozen cultures of the desired populations and time points were thawed and dilutions were plated and grown overnight. Ten colonies from each culture were used to inoculate 4 ml of medium in a test tube and were grown until they reached an OD of ∼1. This was done to reduce the time these colonies can evolve and accumulate mutations. An aliquot of 1 ml of the culture was centrifuged at 10,000 × g for 5 min and the pellet was used for DNA extraction. The remainder of each culture was then archived by freezing in 50% glycerol in a −80°C freezer. DNA was extracted using the MACHEREY-NAGEL Nucleo-Spin 96 tissue kit. Library preparation followed the protocol outlined in reference 35 (link). Sequencing was carried out at the Technion Genome Center using an Illumina HiSeq 2500 machine. All clones were sequenced using paired-end 150-bp reads.

Fish DNA was extracted from tissue samples of three specimens using the NucleoSpin 96 tissue kit (Macherey-Nagel) following the manufacturer’s instructions. The 5′ region of the cytochrome oxidase I (COI) mitochondrial gene was amplified using the primers FishF1 (5′-TCAACCAACCACAAAGACATTGGCAC-3′) and FishR1 (5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′) (Ward et al. 2005 [36 ]). The PCR reactions were performed in a 20-μL solution, containing 1 ng of DNA, 1 × CoralLoad PCR buffer, 3 mM MgCl2, 66 μM of each dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was: 4 min at 94 °C, followed by 40 cycles of 94 °C for 30 s, 48 °C for 40 s, 72 °C for 50 s, with a final extension at 72 °C for 7 min. PCR products were purified and sequenced in both directions on 3730xl DNA Analyser 96-capillary sequencer (Applied Biosystems). Sequences were edited using CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA), compared with the GenBank database content using BLAST and deposited in GenBank under accession numbers KJ192344, KJ192345 and KJ192346. Species identification was confirmed using the BOLD identification engine [35 (link)]. The fish nomenclature adopted follows FishBase [3 ].

Frozen cultures were thawed and dilutions were plated on solid LB-agar plates and grown over night. Colonies to be sequenced were used to inoculate 4 ml of LB medium in a test tube and were grown until they reached an OD of 1. One milliliter of the culture was centrifuged at 5,000 g for 5 min and the pellet was used for DNA extraction. The remainder of each culture was then archived by freezing in 50% of glycerol at -80°C. DNA was extracted using the Macherey-Nagel NucleoSpin 96 Tissue Kit. Library preparation followed the protocol outlined in Baym et al [48 (link)]. Sequencing was carried out at Admera Health (New Jersey USA) using an Illumina HiSeq machine. Clones were sequenced using paired end 150 bp reads. In order to call mutations, the reads obtained for each clone were aligned to the E. coli K12 MG1655 reference genome (accession NC_000913). Mutations were recorded if they appear within a clone’s genome, but not within the ancestral genome. Alignment and mutation calling were carried out using the Breseq platform, which allows for the identification of point mutations, short insertions and deletions, larger deletions, and the creation of new junctions [49 (link)].

Standards were prepared and the samples were lysed and DNA extracted using the NucleoSpin^® 96 Tissue kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions, following the protocols described below.

Genomic DNA was extracted from each single midge using the NucleoSpin96 Tissue Kit (Macherey-Nagel, Duren, Germany), according to the manufacturer’s instructions, starting with an additional step where each individual midge was ground in 200 μL of 1X PBS buffer. Each insect (~32 individuals per site) was genotyped at nine microsatellite markers previously developed for C. imicola [8 (link)] (Additional file 1: Table S1) following the protocol described in [7 (link)].

Echinococcus multilocularis specimens were collected from gastrointestinal (GI) tracts of red foxes and coyotes of either road-killed or trap-harvested animals (trapped for purposes independent of this study), collected between 2012 and 2017 in Western Canada. Trapped animals were obtained from licensed trappers with the collaboration of the Alberta Trappers Association. GI tracts were screened using a modification of the scraping, filtration and counting technique, to identify and collect Echinococcus spp. worms [35 (link),36 (link)]. We analysed Em worms from 70 coyotes and 13 foxes from northern, central and southern Alberta (AB); four coyotes from north-west British Columbia (BC); and 10 coyotes from southeast Saskatchewan (SK). Extraction of DNA was performed on up to five individual worms per host using the Nucleospin 96 Tissue Kit (Macherey-Nagel, Germany) for samples processed in France (Anses Nancy Laboratory for Rabies and Wildlife) and the E.Z.N.A. MicroElute Genomic DNA Kit (Omega Bio-tek, US) for samples processed in Canada (University of Calgary, Faculty of Veterinary Medicine). Extraction was performed following the manufacturer's instructions, and DNA was stored at −20°C until processed.