The positive PCR amplicons of ten randomly selected samples obtained from domestic and wild goats were further sequenced to gain information about the individual N gene structure. PCR products were purified using QIAquick PCR purification kit (Qiagen, GmbH) according to the manufacturer’s instructions. Quality and concentration of purified PCR products were confirmed as mentioned in the above section. The purified PCR products were sequenced at Seqlab-Sequence Laboratories GmbH (Göttingen, Germany), and the sequences were analyzed using FinchTV (version, 1.4.0). For confirming sequence identity, sequence data was subjected to Nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) against the global database. Additionally, all sequences were further analyzed and compared by Clustal-V and Clustal-W pairwise multiple sequence alignment using MegAlign (DNASTAR Inc., Madison, WI, USA). The phylogenetic tree was constructed where bootstrapping was performed by creating 1000 trials. Similarly, the nucleotide sequences were converted to amino acid sequences using the translate DNA step of the EditSeq program (DNASTAR Inc.) and the phylogenetic tree was constructed using the MegAlign program.
Megalign
Manufactured by DNASTAR
Sourced in United States
MegAlign is a multiple sequence alignment software tool. It aligns DNA, RNA, or protein sequences and displays the alignment results. MegAlign supports various alignment algorithms and provides visualization options to analyze the alignment.
Automatically generated - may contain errors
Lab products found in correlation
Editseq, by DNASTAR (25 mentions)
Seqman, by DNASTAR (17 mentions)
Seqman pro, by DNASTAR (6 mentions)
Pmd18 t, by Takara Bio (3 mentions)
Genomic walking kit, by Takara Bio (3 mentions)
Qiaquick pcr purification kit, by Qiagen (3 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (3 mentions)
Seqman software, by DNASTAR (3 mentions)
Restriction enzyme, by Takara Bio (2 mentions)
Phyml v3, by DNASTAR (2 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (2 mentions)
3130 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator cycle sequencing kit v3, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator chemistry v 3, by Thermo Fisher Scientific (2 mentions)
Abi 3130xl capillary sequencer, by Thermo Fisher Scientific (2 mentions)
Snapgene, by GSL Biotech (2 mentions)
Clustalw, by DNASTAR (2 mentions)
Clustalx, by DNASTAR (2 mentions)
Patho gene spin dna rna extraction kit, by iNtRON Biotechnology (2 mentions)
Pmd18 t vector, by Takara Bio (2 mentions)
202 protocols using megalign
1
Genetic Characterization of Goat N Gene
2
Phylogenetic Analysis of EHDV Strains
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The sequences of 20 strains of distinct serotypes EHDV downloaded from GenBank were aligned with the corresponding sequences of JC13C644 and JC13C673 viruses using Align in MEGA 7.0 (http://www.megasoftware.net/ , accessed on 3 June 2017) [20 (link)]. The detailed information for the analysis of virus strain is shown in Table S1 . Homology analysis was performed using MegAlign (DNASTAR Inc.). Sequence assembly and homology analysis of nucleotide and amino acid sequences were performed using SeqMan and MegAlign in DNAStar software (version 4.0; DNASTAR Inc., Madison, WI, USA). The construction of a phylogenetic tree was carried out using MEGA 7.0, employing the maximum likelihood (ML) method [20 (link)] with a bootstrap value of 1000 replications.
3
Phylogenetic Analysis of D6DES Genes
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The sequences of D6DES genes registered in GenBank were collected by carrying out BLASTN with the PcD6DES gene sequence as a query. The phylogenetic relationship of these D6DES genes was analyzed using DNASTAR MegAlign (Ver. 7.2.1) via the ClustalW method. A phylogenetic tree was constructed from the alignment data using TreeView (Ver. 1.6.6). The transmembrane domain was predicted with TOPCONS (http://topcons.cbr.su.se/ ).
4
Ebola Virus Glycoprotein Sequences
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The template plasmid for EBOV was a clinical isolation and was kindly provided by Professor Shengqi Wang, Beijing Institute of Radiation Medicine, China. Artificial plasmids spanning the glycoprotein (GP) were synthesized for other species of the Ebola virus by Sangon Biotech (shanghai, China). The sequences were determined by evaluating the GP genes of SUDV (GenBank accession no. NC006432), BDBV (GenBank accession no. NC014373), and TAFV (GenBank accession no. NC014372). All related RNA sequences, available in the National Center for Biotechnology Information GenBank (Bethesda, MD, USA), were aligned by using DNAstar MegAlign (DNASTAR, Madison, USA) to identify conversed sequences.
5
Identifying SATB1 Promoter Variants
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PCR products from DNA of 10 healthy, unrelated volunteers were used to identify new polymorphisms within the promoter region. Using available reference sequences of human SATB1, primer pairs were designed to amplify overlapping PCR products of the assumed promoter region from -3807 up to -2828 bp upstream of ATG.
DNA sequencing was performed by a third party (MWG Eurofins Medigenomix, Ebersberg, Germany). Reference sequences and sequenced fragments were analyzed using DNASTAR MegAlign® (DNASTAR, Inc., Madison, WI, USA) for Windows®.
DNA sequencing was performed by a third party (MWG Eurofins Medigenomix, Ebersberg, Germany). Reference sequences and sequenced fragments were analyzed using DNASTAR MegAlign® (DNASTAR, Inc., Madison, WI, USA) for Windows®.
6
Screening Linezolid Resistance Mechanisms in LRE
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The genomic DNA of each LRE isolate was extracted using the HiPure Bacterial DNA Kit (Magen, Guangzhou, China), according to the manufacturer's instructions and stored at −20℃ until use. The possible mechanisms of linezolid resistance, were screened by PCR using previously described primers and conditions: the 23S rRNA domain [17 (link)], ribosomal protein (L3 and L4) domain [6 (link)], the methyltransferase gene cfr [18 (link)], and ABC-type transporter gene optrA [7 (link)]. All positive PCR products were sequenced and blasted against the wild-type sequences from E. faecalis ATCC 29212 (GenBank Accession No. CP008816.1) and the complete optrA gene sequence from plasmid pE349 (GenBank Accession No. NG_048023.1). Nucleotide and deduced amino acid (AA) sequences were aligned using the multiple alignment algorithm in the MegAlign package (version 7.1.0; DNASTAR, Madison, WI) with Clustal W [19 (link)].
7
Molecular Biology Techniques for DNA Analysis
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Manipulation of genomic DNA and plasmids and DNA cloning were performed as previously described (Sambrook & Russell, 2001 ). Restriction enzymes (TaKaRa) were used for DNA digestion and modification. DNA sequencing was performed by Macrogen (Seoul). DNA sequences were analyzed using the BLAST program at the National Center for Biotechnology Information (Gish & States, 1993 ), MEGALIGN (DNASTAR, Madison, WI, USA), and GENETYX‐WIN software (Genetyx).
8
H7N9 Hemagglutinin DNA Vaccine Development
9
Phylogenetic Analysis of Ehrlichia Sequences
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Ehrlichia sequences (16S rRNA, groEL and gltA sequences) obtained in this study, as well as those retrieved from GenBank (Table S1), were aligned using the Clustal W method implemented in the MEGA program, version 6.0 [31] . Nucleotide sequence identities were calculated by MegAlign program available within the DNASTAR Lasergene package, version 7.0 (DNASTAR, Inc., Madison, WI).
Phylogenetic trees of basing on these three genes were constructed using the maximum likelihood (ML) method in the PhyML v3.0 package [32] based on the best-t GTR + I + Γ4 model of nucleotide substitution as determined by jModeltest. Bootstrap values higher than 70% were considered signi cant. The best-t evolutional model was determined using jModel Test version.
Phylogenetic trees of basing on these three genes were constructed using the maximum likelihood (ML) method in the PhyML v3.0 package [32] based on the best-t GTR + I + Γ4 model of nucleotide substitution as determined by jModeltest. Bootstrap values higher than 70% were considered signi cant. The best-t evolutional model was determined using jModel Test version.
10
Phylogenetic Analysis of Sequence Assemblies
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Initial sequence assembly and analyses were conducted using the DNAStar software package (ver. 4.0; DNASTAR Inc., Madison, WI, USA). Homology and alignment analysis was performed using Clustal X (ver. 2.1) [9 (link)] (http://www.directoryofshareware.com/preview/clustalx/ ) and MegAlign (DNASTAR Inc.). Phylogenetic and evolutionary analyses were conducted using MEGA 5.1 (http://www.megasoftware.net/ ) based on the neighbor-joining technique and 1,000 bootstrap replications [9 (link)].
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