The full-length genome of HQ-22 was amplified using an overlapping RT-PCR method (primers are shown in Table 2 ). The 50 μL reaction mixtures were set up with 25 μL 2 × Phanta Max Master Mix (Vazyme, Nanjing, China), 1 μL of each 10-μM primer, 2 μL cDNA, and 21 μL ddH2O. The PCR products were purified and cloned into pCE2 TA/Blunt-Zero vector (Vazyme, Nanjing, China), and clones were obtained for sequencing (Tsingke, Nanjing, China) to determine the correct genome information. We used DNAMAN v.6.0 software (Lynnon Biosoft, San Ramon, CA, USA) and Lasergene.v7.1 software (DNAStar, Madison, WI, USA) to splice different fragments with the Clustal W method to obtain the correct full-length genome of HQ-22. The E gene of HQ-22 was aligned with known TMUV strains and the phylogenetic trees of these TMUV E genes were constructed using the neighbor-joining method using MEGA v.11.0 software with bootstrap values calculated from 1000 replicates. The homology analysis of the nucleotide and amino acid sequences of the coding structural protein genes of HQ-22 and other TMUV strains was conducted using Lasergene.v7.1 software (DNAStar) with the Clustal W method.
Lasergene v7
Manufactured by DNASTAR
Sourced in United States
Lasergene v7.1 is a software suite for DNA and protein sequence analysis and visualization. It provides a range of tools for sequence alignment, primer design, and sequence manipulation. The core function of Lasergene v7.1 is to enable users to analyze and manage biological sequence data.
Automatically generated - may contain errors
Lab products found in correlation
Fungus genomic dna extraction kit, by Sangon (4 mentions)
Tprofessional standard thermocycler, by Analytik Jena (3 mentions)
Abi prism 3730 genetic analyzer, by Thermo Fisher Scientific (3 mentions)
Dream taqtm green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Dreamtaqtm green pcr master mix 2, by Thermo Fisher Scientific (2 mentions)
Pce2 ta blunt zero vector, by Vazyme (1 mentions)
Phanta max master mix, by Vazyme (1 mentions)
Dnaman v6, by Lynnon Biosoft (1 mentions)
Abi prism 377 sequencer, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Pmdtm18 t vector cloning kit, by Takara Bio (1 mentions)
Trelieftm 5α chemically competent cells, by Tsingke (1 mentions)
Spss 22, by IBM (1 mentions)
Prism v5, by GraphPad (1 mentions)
Sequencher v5, by Gene Codes (1 mentions)
Abi prism 3730 genetic analyser, by Thermo Fisher Scientific (1 mentions)
Seqman, by DNASTAR (1 mentions)
Big dye terminator kit v3, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Rna extraction kit, by Fastagen (1 mentions)
33 protocols using lasergene v7
1
Full-Genome Amplification and Phylogenetic Analysis of TMUV
2
Comparative Genomic Analysis of Porcine Epidemic Diarrhea Virus
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The raw sequence fragments were imported to SeqMan in DNAStar Lasergene V 7.10 (DNAStar, Inc., Madison, WI) for assembly and annotation. Nucleotide and deduced amino acid (aa) sequences of both CH/JX-1/2013 and CH/JX-2/2013 and 30 reference PEDV sequences retrieved from GenBank were comparatively analyzed. A summary of the background information of PEDVs used in this study is shown in Table 1 . The complete genome sequences of CH/JX-1/2013 and CH/JX-2/2013 were deposited into GenBank. Phylogenetic trees based on the entire genomes, and deduced aa sequences of S, ORF3, E, M, and N genes were constructed using the neighbor-joining method of MEGA 5.2.2 (http://www.megasoftware.net/ ) with a bootstrap of 1,000 replicate datasets. Primary sequences of 5´-proximal region of 5´-UTRs (nt 42 to 133) were pairwise compared between the two Jiangxi strains (CH/JX-1/2013 and CH/JX-2/2013) and the reference strains. Antigenicity and hydrophilicity analyses based on the aa sequence from 1 to 350 at N-terminal of the S proteins were carried out by Protean software of DNAStar Lasergene V7.10 (DNAStar, Inc., Madison, WI).
3
Amplification of (GT)n Repeat Polymorphism
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Genomic DNAs were extracted from peripheral venous blood leucocytes using the QIAamp DNA Blood Mini Kit (Qiagen NV, Venlo, the Netherlands). The (GT)n repeat polymorphism was amplified by polymerase chain reaction (PCR) using the forward primer 5′-ACG CCT GGG GTG CAT CAA GTC-3′ and the reverse primer 5′-GTG GGG TGG AGA GGA GCA GTC ATA-3′, which were designed according to the published sequence in our previous study.25 (link) PCR was performed over 30 cycles of 20 seconds at 94°C, 10 seconds at 60°C, and 20 seconds at 72°C using a fluorescently labeled deoxycytidine triphosphate (Applied Biosystems Inc., Waltham, MA, USA) as described by the manufacturer’s protocol. The PCR products were electrophoresed on a denaturing polyacrylamide gel using ABI PRISM 377 sequencer (Applied Biosystems Inc.). The results were analyzed with DNASTAR.Lasergene v7.1 (DNAStar Inc., Madison, WI, USA) software package.
4
Phylogenetic Analysis of Pseudorabies Virus gE Gene
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The full-length gE gene was amplified by PCR from viral DNA extracted from the isolates as described previously [23 (link)]. The PCR product was purified using the V-ELUTE Gel Mini Purification Kit (Beijing Zoman Biotechnology Co., Ltd., Beijing, China), then ligated into the pMDTM 18 T Vector Cloning Kit (Takara, Dalian, China), and finally transformed into TreliefTM 5α Chemically Competent Cells (Tsingke, Beijing, China). The positive monoclonal clones were verified by PCR and sequenced by Wuhan AuGCT DNA-SYN Biotechnology Co., Ltd. (Wuhan, China), in triplicate.
Twenty-eight PRV strains were retrieved from the NCBI database and served as the reference strains. The nucleotide sequences and the corresponding amino acid variations in the gE gene between PRV isolates sequenced in our study and the reference strains were analyzed using DNASTAR Lasergene.v7.1 (DNASTAR, Inc., Madison, WI, USA). The phylogenetic tree was constructed by the neighbor-joining method using the MEGA 7.0 software (www.megasoftware.net ) with a bootstrap of 1000 replicates [24 (link)].
Twenty-eight PRV strains were retrieved from the NCBI database and served as the reference strains. The nucleotide sequences and the corresponding amino acid variations in the gE gene between PRV isolates sequenced in our study and the reference strains were analyzed using DNASTAR Lasergene.v7.1 (DNASTAR, Inc., Madison, WI, USA). The phylogenetic tree was constructed by the neighbor-joining method using the MEGA 7.0 software (
5
Comparative Analysis of Rhizopogon Mitogenomes
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Base composition of the two mitogenomes was analyzed using the DNASTAR Lasergene v7.1 (http://www.dnastar.com/ ). Strand asymmetry of the two mitogenomes was assessed according to the following formulas: AT skew = [A − T] / [A + T], and GC skew = [G − C] / [G + C] [65 (link)]. Genetic distances between each pair of the 15 core PCGs (atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, and rps3) were calculated with MEGA v6.06 [66 (link)], using the Kimura−2-parameter (K2P) model. We used the DnaSP v6 [67 (link)] to calculate the nonsynonymous substitution rate (Ka) and the synonymous substitution rate (Ks) for all the 15 core PCGs in the two Rhizopogon mitogenomes. Codon usage analysis was conducted using the Sequence Manipulation Suite [68 (link)], based on the genetic code 4. Genome synteny of the two Rhizopogon mitogenomes and representative species from other genera were analyzed using the Mauve v2.4.0 [69 (link)].
6
Comparative Analysis of Boletales Mitogenomes
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Base compositions of the two Coniophora mitogenomes and other Boletales mitogenomes were calculated using the DNASTAR Lasergene v7.1 (http://www.dnastar.com/ ). Strand asymmetries of the 6 Boletales mitogenomes were assessed based on the following formulas: AT skew = [A - T] / [A + T], and GC skew = [G - C] / [G + C] [37] (link). The pairwise genetic distances between each pair of the 15 core PCGs (atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, and rps3) in the 6 Boletales mitogenomes were calculated using MEGA v6.06 [38] based on the Kimura-2-parameter (K2P) substitution model. The DnaSP v6.10.01 software [39] (link) was used to calculate synonymous (Ks) and nonsynonymous substitution rates (Ka) for core PCGs in the 6 Boletales mitogenomes. Gene collinearity analysis of 6 Boletales species was conducted by using Mauve v2.4.0 [40] (link).
7
Analysis of Ganoderma Mitogenomes
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The base compositions of the four Ganoderma mitogenomes were analyzed using DNASTAR Lasergene v7.1 (http://www.dnastar.com/ (accessed on 1 January 2022)). We determined the four mitogenomes’ strand asymmetry using the formulas as follows: AT skew = [A − T]/[A + T] and GC skew = [G − C]/[G + C] [53 (link)]. We determined the genetic distances between the pairs among the 15 core protein-coding genes (PCGs)—atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, and rps3—using MEGA v6.06 [54 (link)] following the Kimura-2-parameter (K2P) model. We used DnaSP v6 to compute the nonsynonymous substitution rate (Ka) and the synonymous substitution rate (Ks) for the 15 core PCGs [55 (link)]. The Sequence Manipulation Suite [56 (link)] following the genetic code 4 conducted the codon usage analysis. BLASTn searching of the four mitogenomes against themselves at an E value of <10−10 was carried out in order to establish whether there were intra-genomic duplications or interspersed repeats (>30 bp) in the four Ganoderma mitogenomes. In addition, tandem repeats (>10 bp) in the four mitogenomes were identified with Tandem Repeats Finder [57 (link)].
8
Comparative Analysis of Agaricales Mitogenomes
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The DNASTAR Lasergene v7.1 (http://www.dnastar.com/ ) was used to calculate base compositions of the C. fumosa mitogenome and other Agaricales mitogenomes. Strand asymmetries of the Agaricales mitogenomes tested were calculated based on the following formulas: AT skew = [A - T] / [A + T], and GC skew = [G - C] / [G + C]. Sequence Manipulation Suite (Stothard 2000 (link)) was used to analyze codon usage within the C. fumosa mitogenome, based on the genetic code 4. The nonsynonymous substitution rates (Ka) and synonymous substitution rates (Ks) for core PCGs in the C. fumosa mitogenome and other Agaricales mitogenomes reported were calculated using the DnaSP v6.10.01 (Rozas et al. 2017 (link)). The genetic distances between each pair of the 15 core PCGs were calculated using MEGA v6.06 (Caspermeyer 2016 (link)) based on the Kimura-2-parameter (K2P) substitution model.
9
Comprehensive Bioinformatic Analysis Pipeline
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Sequencher v 5.0 (Gene Codes Corporation, Ann Arbor, MI, USA), Snapgene v 2.3.2 (GSL Biotech LLC, Chicago, IL, USA), and DNASTAR Lasergene v 7.1 (DNASTAR, Madison, WI, USA) were applied to analyze all of the plasmids and DNA sequences. Evolutionary protein conservation analysis was performed using the ClustalW2 program (EMBL-EBI, United Kingdom). SgRNA was designed using the http://crispr.mit.edu website. Data were presented as the mean ± standard deviation. Differences between two groups were analyzed by Student's t test. Comparisons among three or more groups were analyzed by one-way analysis of variance. A P value less than 0.05 was considered statistically significant. Expression graphs were analyzed using GraphPad Prism v 5.04 software (GraphPad Software, San Diego, CA, USA) and statistical analyses were performed by using SPSS 22.0 (IBM, Armonk, New York, USA).
10
Comparative Mitogenomic Analysis of Paxillus
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Base compositions of the two Paxillus mitogenomes were calculated using DNASTAR Lasergene v7.1 (http://www.dnastar.com/ ). We then used the following formulas to assess strand asymmetries of the two mitogenomes: AT skew = [A - T] / [A + T], and GC skew = [G - C] / [G + C] (Wang et al. 2017 (link)). The synonymous (Ks) and nonsynonymous (Ka) substitution rates for core PCGs in the two mitogenomes were calculated with DnaSP v6.10.01(Rozas et al. 2017 (link)). MEGA v6.06 (Caspermeyer 2016 (link)) was used to calculate the overall mean genetic distances between each pair of the 15 core PCGs (atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, and rps3), using the Kimura-2-parameter (K2P) substitution model.
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