The PCR products were purified by PCRquick-spin (iNtRON) according to the manufacturer's instructions, and the purified products were sequenced by Genotech. The DNA sequences of the ribosomal 45S region obtained in the sequencing experiments were compiled using SeqMan software (SeqMan 2, DNASTAR, Inc., 3801, Regent St, Madison, WI, 53705, USA) and edited using the BioEdit program (BioEdit 7.2.5, Ibis Biosciences, 2251 Faraday Avenue, Carlsbad, CA 92008, USA) [21] . Multiple sequence alignments were performed using the online ClustalW2 program (http://www.ebi.ac.uk/Tools/clustalw2/ ).
Seqman 2
Manufactured by DNASTAR
Sourced in United States
SeqMan II is a powerful DNA sequence analysis software developed by DNASTAR. It provides advanced tools for assembling, editing, and analyzing DNA sequences. The software's core function is to facilitate the processing and interpretation of DNA sequence data.
Automatically generated - may contain errors
Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (5 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (3 mentions)
Qiaexii pcr purification kit, by Qiagen (2 mentions)
Editseq, by DNASTAR (2 mentions)
Chromas 2, by Technelysium (2 mentions)
Bigdye terminator kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 sequencing system, by Illumina (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Mega 7, by Mega Software (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Dneasy kit, by Qiagen (1 mentions)
Emeraldamp master mix, by Takara Bio (1 mentions)
Icycler, by Bio-Rad (1 mentions)
Abi prism big dye terminator cycle sequencing reaction kit, by Thermo Fisher Scientific (1 mentions)
Gfx pcr dna and gel band purification kit, by Cytiva (1 mentions)
Abi prism 310, by Thermo Fisher Scientific (1 mentions)
Takara ex taq, by Takara Bio (1 mentions)
Universal dna purification kit, by Tiangen Biotech (1 mentions)
Abi 3130xl genetic analyser, by Thermo Fisher Scientific (1 mentions)
Qiaamp stool mini kit, by Qiagen (1 mentions)
43 protocols using seqman 2
1
Ribosomal 45S Region Sequencing and Analysis
2
Purification and Sequencing of trnL-F Region
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The PCR products were purified using a total fragment DNA purification kit (iNtRON Biotechnology, Seongnam, Korea), following the manufacturer's instructions. The purified products were sequenced by Genotech Co. (Daejeon, Korea) using the designated primers. The base sequence was decoded by cycle sequencing, using the BigDye Terminator Kit (Thermo Fisher Scientific, WA, MA, USA). On completion of the PCR, the purified product was dissolved in sterile water or HDF (Hi-Di Formamide) and mounted on the device (ABI 3730xl DNA analyzer) for electrophoresis using capillary method. The sequences of the trnL-F region were compiled using Seq-Man software (SeqMan 2, DNASTAR, Inc., WI, USA), and BioEdit program (BioEdit 7.2.5, Ibis Biosciences, CA, USA) was applied [8] (link). ClustalW2 (http:// www. ebi. ac. uk/ Tools/ clust alw2/) was used to perform multiple sequence alignments.
3
Viral Capsid Coding Region Sequencing
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The sequences of the entire capsid coding region (P1) of selected viruses were generated. RNA extraction from the cell culture grown viruses and reverse transcription (RT) were performed as described [16] (link). PCR was carried out using the “KOD hot-start DNA polymerase” kit (Novagen) as recommended by the manufacturer, using the forward primer L463F (5′-ACCTCCRACGGGTGGTACGC-3′) and one of the reverse primers NK72 (5′-GAAGGGCCCAGGGTTGGACTC-3′) or EUR2B52R (5′-GACATGTCCTCCTGCATCTGGTTGAT-3′). PCR products were purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions and sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) using the PCR primers and additional internal sequencing primers (sequences available on request). Sequences (from the ABI 3730 machine) were assembled and analysed using SeqMan II (DNAStar Lasergene 8.0). Nucleotide sequences of the viruses were aligned using the CLUSTAL X multiple sequence alignment program [17] (link) and the predicted aa sequences were translated using BioEdit 7.0.1 [18] . Alignments were used to construct distance matrices using the Kimura 2-parameter nucleotide substitution model [19] (link) as implemented in the programme MEGA 4.0 [20] (link).
4
Characterizing eae Gene Subtypes
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Each of the sequenced ~3.2 kb LEE region that contained the complete eae gene was checked and assembled by SeqMan II (DNASTAR Inc., USA). The eae subtypes reference sequences were downloaded from GenBank. The MEGA 7 software (www.megasoftware.net )50 (link) was used to align the complete eae sequences obtained in this study and the reference eae sequences. A Neighbor-Joining tree was constructed with maximum composite likelihood model. Bootstrap analyses were performed (1,000 replicates) to estimate the stability and genetic distances were calculated by the maximum composite likelihood method. A novel subtype was defined by a cutoff value of 95% nucleotide sequence identity as described previously51 (link).
5
Sequence-Based Genotype Identification Protocol
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Discrepancies in genotype identification were resolved by sequencing. Briefly, the PCR amplicons were purified using a QIAQuick PCR purification kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and were inspected for purity by electrophoresis on 2% agarose gels as well as by measuring the absorbance at 260 and 280 nm. The purified PCR products were adjusted to concentrations of 3–5 ng/µl and direct sequencing was performed at Macrogen, Inc. (Rockville, MD) using the PCR primers.
Sequence data were edited using the Lasergene software program Seqman II (DNASTAR, Inc. Madison, WI) then searched against the Genbank database using the BLASTn protocol at the National Center for Biotechnology (NCBI) website (http://www.ncbi.nlm.nih.gov/BLAST ). Phylogenetic analysis was performed using the MEGA software version 5.063 (link). Genetic distances were calculated using the Kimura 2 parameter64 (link), and the phylogenetic tree was constructed by the neighbor-joining method.
Sequence data were edited using the Lasergene software program Seqman II (DNASTAR, Inc. Madison, WI) then searched against the Genbank database using the BLASTn protocol at the National Center for Biotechnology (NCBI) website (
6
Mitochondrial Genome Assembly and Analysis
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Total genomic DNA was extracted from the whole body using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Subsequent high-throughput sequencing was performed by the Illumina NextSeq 500 Sequencing System (Illumina, CA, United States). The mitogenome was assembled using the SeqMan II program from the Lasergene software package (DNAStar Inc., Madison, United States). The beginning and stop codons of the protein-coding genes (PCGs) were determined by ORF Finder using invertebrate mitochondrial genetic codes, which was carried out by the NCBI website (https://www.ncbi.nlm.nih.gov/orffinder/ ). To acquire the base organization of nucleotide sequences, we determined composition skewness as depicted by Junqueira AT skew = [A−T]/[A + T], GC skew = [G−C]/[G + C]. The relative synonymous codon usage (RSCU) values were determined utilizing Codon W. The overlapping regions and intergenic spacers between genes were counted by manual means.
7
Insect Interactors of SRBSDV-P10
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Plasmids were recovered from the positive interactors and inserted into E. coli strain DH5-α, which was then cultured on LB agar plates supplemented with 100 mg/mL ampicillin. Colonies were checked by PCR and sent for sequencing to determine the putative insect proteins that interacted with SRBSDV-P10. The resultant sequences were assembled by the SeqMan II program (DNAstar) to exclude duplicated sequences. The different sequences were then used as queries in a BLAST search of the NCBI database (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi ), and the tentative interacting proteins were identified and then annotated using Gene Ontology (GO) for molecular function, biological process and cellular component using the UniProt KB database (http://www.uniprot.org/ ).
8
Nucleotide Sequence Analysis of I. exustus
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The nucleotide sequences were checked for errors and manually edited in the software program SeqMan II (DNASTAR, Madison, WI, USA). All of the nucleotide sequences were aligned by using Clustal W in the MEGA Version 7.0 program [31 (link)]. Species identification of I. exustus and cercariae was supported by a BLASTN search to identify similarities to sequences deposited in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi ).
9
Phylogenetic Analysis of ehxA Subtypes
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The sequenced ~2997-bp ehxA was assembled using SeqMan II (DNASTAR Inc., USA). The representatives of reference ehxA sequences of six ehxA subtypes A to F were downloaded from GenBank10 (link),24 (link). The ehxA sequences obtained in this study and the reference sequences were aligned using MEGA 7 software (www.megasoftware.net )33 (link). Phylogenetic trees were constructed with two algorithms, neighbour-joining (NJ), and maximum-likelihood (ML), using MEGA 7. The stability was estimated by bootstrap analysis (1000 replications), and genetic distances were calculated by the maximum composite likelihood method. A sequence was designated to a specific ehxA group based on its phylogenetic placement.
10
Analyzing FMDV VP1 Sequence Variability
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A number of sequences (Supplementary Table 1 ) of serotypes O and A FMDV circulating in Bangladesh in 2012–13 (KC795948–KJ175182) and reference sequences (NC011450 and NC004004) from NCBI database were used to predict the amino acid sequence variability of VP1 region. EBI, EMBOSS Bioinformatics Tools (Transeq) were used to translate the nucleic acid sequences into respective amino acid sequences. VP1 amino acid sequences and reference sequences were aligned by ClustalX (1.81) and analyzed using SeqMan II (Lasergene 8.0; DNAStar Inc., WI, USA) for discerning amino acids sequence variability. Protein variability server (PVS) was used to calculate protein variability index using Wu–Kabat variability coefficient.19 (link) The variability coefficient is computed using the following formula: variability = N*k/n, where, N is the number of sequences in the alignment, k is the number of different amino acids at a given position, and n is the times that the most common amino acid at that position is present.20 (link)
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