Abi sequencer 3730 dna analyzer

DNA was isolated from flash-frozen individuals using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer’s instructions. The mitochondrial COI gene was amplified with the primers LCO1490 and HCO2198 [53 ]. The resulting PCR products were sequenced from both strands on an ABI sequencer 3730 DNA analyzer (Applied Biosystems, Foster City, USA) by the laboratory center of the Senckenberg Biodiversity and Climate Research Centre Frankfurt (SBiK-F) or by LGC Genomics (Berlin, Germany) with the primers used for the PCR. COI sequences of additional species were downloaded from GenBank and aligned with MUSCLE [54 (link)] in AliView v1.26 [55 (link)] and inspected manually. IQ-TREE v1.6.12 with the implemented ModelFinder was used to build a maximum likelihood (ML) phylogeny [56 (link),57 (link),58 (link)] in order to identify the closest relative for each species and reconstruct a phylogenetic tree. Due to the short length of the partial COI gene and deep divergences of the included species, ML analyses were carried out for each of the three nemertean groups separately. The resulting trees were inspected and used to manually build a consensus phylogeny that conforms to previously published hypotheses based on larger datasets [10 (link),15 (link)]. The topology was rooted at Palaeonemertea and consistent with previous phylogenies [10 (link)].

For samples that were positive in both qRT-PCR kits, NoV GII ORF1-ORF2 junction (also known as the polymerase-capsid region) was amplified using method designed by the US CDC [20 (link)]. QIAxcel capillary electrophoresis was run with QIAxcel DNA Screening Kit (Qiagen, Hilden, Germany) to determine whether the specimen was successfully amplified and showed an expectant product size of 570 bp. All specimens positive with the target fragment size were sequenced by ABI sequencer 3730 DNA analyzer with BigDye™ Terminator v3.1 kit (Applied Biosystems, California, US). The resulting sequences were spliced using Sequncher software v4.1.4 (Gene Codes, US) [21 ] and then genotyped by RIVM online Norovirus genotyping tool (http://www.rivm.nl/mpf/norovirus/typingtool,RIVM,MA Bilthoven, Netherlands). Sequences representative of the main variants of the recombinant strains focused in this study were deposited in GenBank (accession numbers MK779279-MK779304; MK789447–MK789463—Additional file data) (Additional file 1).