Abi prism bigdye terminator v 3

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The ABI PRISM BigDye Terminator v. 3.1 is a fluorescent dye-labeled terminator chemistry used for DNA sequencing. It is designed to work with Applied Biosystems DNA sequencing instruments.

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17 protocols using abi prism bigdye terminator v 3

Molecular identification of plant species

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The samples of individual plants were taken from Lomonosov Moscow State University Herbarium [41 ,42 (link)] and from Lomonosov Moscow State University Botanical Garden. The list of samples is given in the Supplementary Table S2. The DNA extraction and amplification conditions were the same as those for the products. The PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter) and were sequenced using ABI PRISM BigDye Terminator v. 3.1 reagents and the Applied Biosystems 3730 DNA Analyzer (Waltham, MA, USA). The obtained sequences were analysed using BLAST. The results of sequencing were considered positive if the best BLAST hits of the query sequence fell within the same genus as the specimen used for sequencing.

Omelchenko D.O., Speranskaya A.S., Ayginin A.A., Khafizov K., Krinitsina A.A., Fedotova A.V., Pozdyshev D.V., Shtratnikova V.Y., Kupriyanova E.V., Shipulin G.A, & Logacheva M.D. (2019). Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products. Genes, 10(2), 122.

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Viral RNA Extraction and Sequencing Protocol

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Viral RNA from tick suspensions, mosquito suspensions and infected cell culture supernate was isolated with TRI Reagent LS (Sigma-Aldrich, MO, USA) according to the manufacturer’s protocols. Reverse transcription was performed with random hexamer primers (R6) and M-MLV reverse transcriptase (Promega, Madison, WI, USA), according to the manufacturer’s protocols. Viral genomic cDNA was amplified by PCR using primers for the genus Flavivirus [33 (link)] and specific primers for ALSV: Alongshan_seg1_5 and Miass_NS5_1R for segment 1 (Table S3) and Miass_gly_3F and Miass_gly_3R for segment 2 [7 (link)]. Sequencing was carried out in both directions directly from PCR-amplified DNA on the ABI PRISM 3730 (Applied Biosystems, Foster City, CA, USA) sequencer using ABI PRISM^® BigDye™ Terminator v. 3.1. Genomic sequences were assembled using SeqMan software (DNAstar, Madison, WI, USA).

Kholodilov I.S., Belova O.A., Morozkin E.S., Litov A.G., Ivannikova A.Y., Makenov M.T., Shchetinin A.M., Aibulatov S.V., Bazarova G.K., Bell-Sakyi L., Bespyatova L.A., Bugmyrin S.V., Chernetsov N., Chernokhaeva L.L., Gmyl L.V., Khaisarova A.N., Khalin A.V., Klimentov A.S., Kovalchuk I.V., Luchinina S.V., Medvedev S.G., Nafeev A.A., Oorzhak N.D., Panjukova E.V., Polienko A.E., Purmak K.A., Romanenko E.N., Rozhdestvenskiy E.N., Saryglar A.A., Shamsutdinov A.F., Solomashchenko N.I., Trifonov V.A., Volchev E.G., Vovkotech P.G., Yakovlev A.S., Zhurenkova O.B., Gushchin V.A., Karan L.S, & Karganova G.G. (2021). Geographical and Tick-Dependent Distribution of Flavi-Like Alongshan and Yanggou Tick Viruses in Russia. Viruses, 13(3), 458.

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Genotyping of TNF Gene Polymorphisms

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Two TNF gene polymorphisms, previously associated with tuberculosis in European, Mexican American, Hispanic, and Latino populations, were selected [19 (link), 28 (link)–31 (link)]. TNF –308G/A and –238G/A gene polymorphisms were genotyped by polymerase chain reaction and restriction fragment length polymorphisms (PCR-RFLP) [32 (link)]. The –308G/A homozygous AA genotype is identified by a 107 bp fragment, the GG genotype has a combination of 87 and 20 bp bands, and the heterozygous GA has three bands (107, 87, 20 bp). The –238 AA polymorphic genotype is identified by a 152 bp fragment, the GG wild type has two bands (133, 19 bp), and the heterozygous GA has three fragments. Genotypes were confirmed by Sanger sequencing with an ABI Prism 3130 genetic analyser and ABI Prism BigDye Terminator^® v3.1 (Applied Biosystems, USA); each genotype was confirmed twice in a bidirectional way by PCR (Figure 1).

de Arellano I.T., Lara C.S., Espíndola L.M., de Jesús Castillejós López M., Prado A.J., Cruz R.V., Camacho J.L., Patiño N., Bojórquez J.D, & Suck M.L. (2020). Exposure to biomass smoke, cigarettes, and alcohol modifies the association between tumour necrosis factor (–308G/A, –238G/A) polymorphisms and tuberculosis in Mexican carriers. Archives of Medical Science : AMS, 16(3), 672-681.

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Multilocus Sequence Analysis of Fungal Samples

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Four gene regions were amplified for inclusion in the multi-locus sequence data analysis, i.e. rDNA internal transcribed spacer (ITS), and the partial genes calmodulin (CAL), translation elongation factor-1 (TEF1) and -3 (TEF3). Primers used for amplification and sequencing of CAL were CL1 and CL2a (O’Donnell et al. 2000 ). TEF primers were developed by B. Stielow (unpubl. data). PCR reactions were performed in a mixture containing 1.25 μL 10× PCR buffer, 6.7 μL ddH₂O, 1 μL dNTP mix (2.5 mM), 0.25 μL of each primer (10 pmol), 0.06 μL Taq DNA polymerase (5 U/μL), 0.625 μL DMSO (Sigma), and 2.5 μL template DNA (100 ng/μL). PCR reactions were performed in a Hybaid Touchdown PCR machine (Hybaid, Middlesex, UK); the used annealing temperatures are listed in Table 2. PCR products were visualized by electrophoresis on a 1 % (w/v) agarose gel. Amplicons were purified using exoSAP-IT (Affymetrix, Santa Clara, CA, USA). The ABI Prism BigDye Terminator v. 3.1 (Applied Biosystems, Foster City, CA, USA) was applied according to the instructions provided by the manufacturer. Reactions were purified by using Sephadex G-50 ultrafine (GE Healthcare Bio-Sciences, Uppsala, Sweden) and sequencing was performed by using an ABI 3730xL automatic sequencer (Applied Biosystems).

Zhang Y., Hagen F., Stielow B., Rodrigues A.M., Samerpitak K., Zhou X., Feng P., Yang L., Chen M., Deng S., Li S., Liao W., Li R., Li F., Meis J.F., Guarro J., Teixeira M., Al-Zahrani H.S., Pires de Camargo Z., Zhang L, & de Hoog G.S. (2015). Phylogeography and evolutionary patterns in Sporothrix spanning more than 14 000 human and animal case reports. Persoonia : Molecular Phylogeny and Evolution of Fungi, 35, 1-20.

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Multilocus PCR for Toxoplasma Clonal Types

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To investigate the clonal type of B1 gene-positive samples, multilocus PCR was carried out using nine markers: SAG1, 3′SAG2, 5′SAG2, SAG3, BTUB, GRA6, altSAG2, C29-2, and L358, based on the method described by Su et al. (2010 (link)).
Nested and multilocus PCR were carried out in a C1000 Thermal Cycler (Bio-Rad). Real-time PCR amplification was performed in a thermal cycler CFX-96 (Bio-Rad). As positive controls, RH (type I), ME49 (type II), and C56 (type III) DNA isolates of T. gondii strains, and as a negative control nuclease-free water, were used.
DNA sequencing of amplicons was performed using ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA), with the use of Abi Prism Big Dye Terminator v. 3.1. Cycle Sequencing Kits and Big Dye XTerminator Purification Kit (Applied Biosystems). Sequences were analyzed using Geneious v. 11.1.4. software (Geneious Co., Wellington, New Zealand) and compared with the sequences deposited in NCBI database using Blast.

Sroka J., Bilska-Zając E., Wójcik-Fatla A., Zając V., Dutkiewicz J., Karamon J., Piotrowska W, & Cencek T. (2019). Detection and Molecular Characteristics of Toxoplasma gondii DNA in Retail Raw Meat Products in Poland. Foodborne Pathogens and Disease, 16(3), 195-204.

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HMPV Gene Sequencing Protocols

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Both partial F and G genes were retrospectively sequenced from all HMPV laboratory-confirmed samples. The amplification was performed using the One-Step RT-PCR kit (Qiagen, Hilden, Germany), conditions in Table 1. PCR products were purified using Exo-SAP-IT (USB, Affymetrix Inc., Cleveland, USA) and sequenced by the ABI Prism BigDye Terminator v3.1 (Applied Biosystems, Carlsbad, USA) on the ABI PRISM 3130xl Genetic Analyzer (Thermo Fisher Scientific, Waltham, USA). Nucleotide sequences were edited and assembled using MEGA v6.0 [12 (link)]. A collapse to haplotypes was done with ALTER server [13 (link)]. The best fit substitution model was determined by MEGA v6.0, and the lowest Bayesian information criterion score model was used and evaluated with 1,000 bootstrap resamplings.Table 1

Primers and PCR conditions.

Table 1

Gene	Primers	CAN97-83		PCR conditions	Fragment length (bp)	Reference
Gene	Primers	Initial position	Final position	PCR conditions	Fragment length (bp)	Reference
G	GF:GAGAACATTCGRRCRATAGAYA	6,247	6,268	50ºC×30’ – 95 °C×15’ – 45x (95 °C×30” – 59 °C×30” – 72 °C×1’) – 72ºc×10’	924	Ludewick HP et al., 2005
G	GR:AGATAGACATTRACAGTGGATT	7,149	7,170		924	Ludewick HP et al., 2005
F	FF:CAATGCWGGRATAACACCAGC	3,693	3,713	50ºC×30’ – 95 °C×15’ – 45x (95 °C×30” – 55 °C×30” – 72 °C×1’) – 72 °C×10’	745	Designed for this study
F	FR:ATTGAAYTGATCYTCAGGAAAC	4,416	4,437		745	Designed for this study

Piñana M., Vila J., Maldonado C., Galano-Frutos J.J., Valls M., Sancho J., Nuvials F.X., Andrés C., Martín-Gómez M.T., Esperalba J., Codina M.G., Pumarola T, & Antón A. (2020). Insights into immune evasion of human metapneumovirus: novel 180- and 111-nucleotide duplications within viral G gene throughout 2014-2017 seasons in Barcelona, Spain. Journal of Clinical Virology, 132, 104590.

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Viral RNA Isolation and Virus Detection Protocol

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Viral RNA from tick suspensions and infected cell culture supernate was isolated with TRI Reagent LS (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s protocols. Reverse transcription was performed with random hexamer primer (R6) and M-MLV reverse transcriptase (Promega, Madison, WI, USA), according to the manufacturer’s protocols. To detect the virus, newly obtained viral genomic cDNA was amplified by PCR using primers for the genus Flavivirus [52 (link)], and specific primers for ALSV: Miass_gly_3F and Miass_gly_3R [33 (link)]. In addition, previously described [23 (link)] and newly obtained viral genomic cDNA were amplified by PCR using pan-phlebovirus primers PhlP2 and PhlM2 [53 (link)]; specific primers for YGTV: Yanggou_gly_1F and Yanggou_gly_1R (Table S2); and for TBEV: Kgg19 and Kgg31 [54 ]. To sequence the complete or partial genome, viral genomic cDNA was amplified by PCR using specific primers for ALSV [23 (link)], YGTV (Table S2), TBEV [51 (link)], and phenuiviruses [53 (link)]. The PCR product was gel-purified and then sequenced in both directions on the ABI PRISM 3500 (Applied Biosystems, Foster City, CA, USA) sequencer using ABI PRISM^® BigDye™ Terminator v. 3.1. Genomic sequences were assembled using SeqMan software (DNAstar, Madison, WI, USA).

Kholodilov I.S., Belova O.A., Ivannikova A.Y., Gadzhikurbanov M.N., Makenov M.T., Yakovlev A.S., Polienko A.E., Dereventsova A.V., Litov A.G., Gmyl L.V., Okhezin E.V., Luchinina S.V., Klimentov A.S, & Karganova G.G. (2022). Distribution and Characterisation of Tick-Borne Flavi-, Flavi-like, and Phenuiviruses in the Chelyabinsk Region of Russia. Viruses, 14(12), 2699.

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Listeria monocytogenes Strain Sequencing

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Individual PCR product sequencing was performed using a set of reagents ABI PRISM ^® BigDye ^™ Terminator v. 3.1, followed by the analysis of the reaction products on an automatic sequencer, Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Electrophoretic DNA separation was performed in 50-cm capillaries with POP7 polymer.
All the “house-keeping gene”-derived sequences were deposited in the Institute Pasteur MLST BIGSdb-Lm database (https://bigsdb.pasteur.fr/listeria/primers_used.html, the access numbers: 76379-76383, accessed on 22 March 2021).
The identification of obtained sequences was executed with the help of the BIGSdb-Lm database (https://bigsdb.pasteur.fr/listeria/primers_used.html, accessed on 22 March 2021) and included a search for allele numbers, relevant sequence type (ST), clonal complex (CC), and phylogenetic lineage for each L. monocytogenes isolate.
The determination of allelic profiles for each consensus gene sequence of internalins derived from Serbia-2019 strains was carried out by means of comparing the relevant sequences against loci lmo0433 (inlA), lmo0434 (inlB), lmo1786 (inlC), and lmo0264 (inlE) of the reference nucleotide sequences of L. monocytogenes strains that were available in the BIGSdb-Pasteur MLST database (https://bigsdb.pasteur.fr/listeria/listeria.html, accessed on 4 April 2021).

Bespalova T.Y., Mikhaleva T.V., Meshcheryakova N.Y., Kustikova O.V., Matovic K., Dmitrić M., Zaitsev S.S., Khizhnyakova M.A, & Feodorova V.A. (2021). Novel Sequence Types of Listeria monocytogenes of Different Origin Obtained in the Republic of Serbia. Microorganisms, 9(6), 1289.

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Isolation and Sequencing of TBEV

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TBEV was isolated and sequenced as previously described [40 (link)]. Briefly, tick suspensions were tested by RT-PCR for the presence of TBEV RNA. Pig embryo kidney (PEK) cells were infected with PCR-positive samples. Two-day-old ICR mice (FSBSI Scientific Center of Biomedical Technologies of Federal Medical Biological Agency, "Stolbovaya" branch, Moscow Oblast, Russian Federation) were injected intracerebrally with 10 μl of virus-containing fluid (infected cell culture supernate, tick suspension). After second passage, viral RNA were isolated from the 10% suspension of suckling mice brain with TRI Reagent LS (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Reverse transcription was performed with virus-specific primers (Kgg30, Kgg32). Viral genomic cDNA was amplified by PCR using TBEV-specific primers Kgg 35, Kgg26, Kgg 16, and Kgg30 [40 (link)]. Sequencing was carried out in both directions directly from PCR-amplified DNA on the ABI PRISM 3730 (Applied Biosystems, Waltham, MA, USA) sequencer using ABI PRISM® BigDye™ Terminator v. 3.1. Genomic sequences were assembled using SeqMan software (DNAstar, Madison, WI, USA).

Deviatkin A.A., Kholodilov I.S., Belova O.A., Bugmyrin S.V., Bespyatova L.A., Ivannikova A.Y., Vakulenko Y.A., Lukashev A.N, & Karganova G.G. (2020). Baltic Group Tick-Borne Encephalitis Virus Phylogeography: Systemic Inconsistency Pattern between Genetic and Geographic Distances. Microorganisms, 8(10), 1589.

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Mitochondrial DNA Sequencing of Stink Bug

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DNA was extracted from the abdomen of each insect using QIAamp DNA Mini Kit (QIAGEN). We designed PCR primers (Table S2) to amplify mitochondrial DNA fragments with reference to a mitochondrial genome sequence of M. cribraria from the introduced population (accession no. JF288758) [13] (link). The 1.0–1.4 kb PCR products were purified, directly subjected to cycle sequencing reactions with ABI PRISM BigDye Terminator v3.1 (Applied Biosystems), and analyzed by ABI PRISM 3130×l Genetic Analyzer (Applied Biosystems).

Hosokawa T., Nikoh N, & Fukatsu T. (2014). Fine-Scale Geographical Origin of an Insect Pest Invading North America. PLoS ONE, 9(2), e89107.

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