Gene polymorphisms of ABCB1, ABCG2, CYP3A, UGT1A, and OR2B11 were examined by Sanger sequencing. PCR conditions were 95°C for 5 minutes; 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; and 72°C for 10 minutes. PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), cycle-sequenced using a BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol, and resolved on an ABI 3500 × L sequencer (Applied Biosystems). Sequences were analyzed using Sequence Scanner Software v2.0 (Applied Biosystems). The primers used are shown in Supplementary Table 1 .
Sequence scanner software v2
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sequence Scanner Software v2.0 is a software application designed for the analysis and management of DNA sequence data. The software provides tools for visualizing, editing, and annotating DNA sequences, as well as for performing various sequence analysis tasks.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator version 3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Mastercycler pro, by Eppendorf (1 mentions)
Abi 3500 genetic analyser, by Thermo Fisher Scientific (1 mentions)
Lasergene genomics suite of software, by DNASTAR (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Lasergene 6, by DNASTAR (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Caliper labchip gx system, by PerkinElmer (1 mentions)
Abi 3730, by Thermo Fisher Scientific (1 mentions)
9 protocols using sequence scanner software v2
1
Genetic Polymorphism Analysis of Key Pharmacokinetic Genes
2
Phylogenetic Analysis of 16S rRNA Gene
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Genomic DNA was isolated using the phenol chloroform method and confirmed in 1% agarose gel electrophoresis. Polymerase chain reaction (PCR) amplification of full-length 16S rRNA gene with 8F and 1492R was carried out at 55 °C annealing temperature. The amplicons were sequenced at Eurofins Scientific (Bangalore, India). Raw Sanger sequences were checked for their quality using Sequence Scanner Software v2.0 (Applied Biosystems, Foster City, CA, USA). Low quality reads at the 5’ and 3’ ends were trimmed and sequences below 500 base pairs after trimming were excluded. The 16s rRNA gene sequences generated in this study are available in the National Center for Biotechnology Information (NCBI) gene bank under the accession number MN629977–MN630016. The resultant sequences were further processed for top hit taxonomy similarity by considering “valid name only” criteria in EzTaxon. Sequences in FASTA format of the top hit against each isolate were retrieved from EzTaxon and multiple sequence alignment (MSA) was performed using ClustalW [13 (link)] in Molecular Evolutionary Genetics Analysis X (MEGA) [14 (link)]. The phylogenetic tree was constructed using maximum-likelihood algorithm with 1000 bootstraps.
3
PANK2 Exons Amplification and Sequencing
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Polymerase chain reaction (PCR) was performed to amplify PANK2 exons 1 to 7 as well as their exon/intron boundaries using Mastercycler® pro, Eppendorf AG, Hamburg, Germany. We used the mRNARefSeq NM_153638.3 as a reference sequence for designing our primers. All the primer sequences used in this study are available on request. The PCR amplicons were purified using illustraTM GFXTM PCR DNA and Gel Band Purification Kit, Cat. No. 28–9034–70, GE Healthcare. For genetic evaluation, purified PCR amplicons were sequenced in both forward and reverse directions using dye terminator chemistry (BigDye® Terminator v3.1 Cycle Sequencing Kit) on an automated capillary sequencer (ABI 3500 Genetic Analyser, Applied Biosystems®, Foster City, California, USA). Sequence electrophoregrams were analyzed using the Sequence Scanner Software v2.0 (Applied Biosystems®) and Chromas v2.5.1 (Technelysium Pty Ltd, South Brisbane, Australia). Lasergene Genomics Suite of DNASTAR® software, DNASTAR Inc., USA was used for sequence alignments and for evaluation of the genetic variations.
4
Viral Sequence Confirmation via Sequencing
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To confirm the identity of the dirRT-qPCR products, we sent the 143 bp DNA bands for the 5’UTR gene of chicken CoV obtained to Bioneer corporation (Daejeon, Korea) for nucleotide sequencing. The chromatograms for both forward and reverse primers were read and assessed for their quality using Sequence Scanner software v2.0 (Applied Biosystems, Foster, CA, USA), and the consensus sequences were assembled using BioEdit software v7.2. The nucleotide identity of the viral sequences was verified using BLASTn search against the NCBI GenBank database.
5
Multilocus Sequence Typing of NDM-producing Isolates
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MLST of isolates was performed using seven conserved housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) according to protocols available at the MLST Pasteur website (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html ) for NDM-producing isolates. Products of the above genes in MLST were sequenced by Bioneer, Co. Sequences of each housekeeping gene in both directions were analyzed by Sequence Scanner Software v.2.0 (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA) and assembled by Lasergene 6 software (DNASTAR). The sequence types (STs) of each isolate were determined based on the seven studied loci described at http://bigsdb.pasteur . fr/klebsiella/klebsiella.html.
6
H1N1 Viral Genome Sequencing Protocol
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A total of 4 DNA bands (101 bp) for the M gene of the H1N1 strain obtained, were sent to Bioneer Corporation (Daejeon, Republic of Korea) for nucleotide genome sequencing. The chromatograms for both forward and reverse primers were read and assessed for their quality using Sequence Scanner software v2.0 (Applied Biosystems, Foster, CA, USA), and the consensus sequences were assembled using BioEdit software v7.2. The nucleotide identity of the viral sequences was verified using BLASTn search against the NCBI GenBank database.
7
Heterogeneous Mutant Cell Isolation
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The genomic region of approximately 300–600 kb centered on the position of the mutation was amplified by PCR with PrimeSTAR HS DNA Polymerase (TaKaRa Bio, Japan) and the corresponding primers (Supplementary Table 7 ). Amplicons were purified using a MinElute PCR Purification Kit (Qiagen, United States), and Sanger sequencing was conducted by Eurofins Genomics K. K. (Tokyo, Japan). The resulting electropherogram was analyzed using Sequence Scanner Software v2.0 (Thermo Fisher Scientific, United States), and the ratio of the mutants within the cell population was calculated according to the peak values, as described previously (Kishimoto et al., 2015 (link)). Stored cell cultures with an interval of ∼100 generations were analyzed to identify the heterogeneity of the cell population. Single-colony isolation was performed from the heterogeneous population to isolate the homogeneous mutants. The cell culture was spread on LB agar plates, and 10∼30 single colonies per plate were subjected to Sanger sequencing. The colonies of the homogeneous mutant were stored for the fitness assay as described above.
8
Sequencing Mitogenome Control Region of Horses
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The sequences of the 246 bp mitogenome control region fragment for modern horses were obtained using Sequencing Analysis Software v7.0 (primary analysis tool, Thermo Fisher Scientific) and Sequence Scanner Software v2.0 (viewer, Thermo Fisher Scientific).
For ancient and medieval horses, the sequences of the 246 bp mitogenome control region fragment were obtained using PALEOMIX BAM Pipeline v1.3.2 [28 (link)]. Removal of adapter sequences and collapse of reads was performed using AdapterRemoval v2.2.2 [29 (link)]. Sequence aligner bwa v0.7.15 [30 (link)] was used to perform collapsed read alignment against the reference sequence of the horse mitogenome (GenBank accession №: NC_001640.1), with a minimum read mapping quality of 25. The MapDamage v2.2.0 computational framework [31 (link)] helped recalculate base quality scores according to the probability of post-mortem DNA damage at each position in the sequence. The bioinformatics software platform Geneious Prime v2020.2.4 (https://www.geneious.com ; Biomatters Ltd., Auckland, New Zealand) was used to visualize the resulting alignment and obtain a consensus sequence of the studied fragment of the mitogenome control region in which the quality of each base would be higher than 60% of the total adjusted base quality.
For ancient and medieval horses, the sequences of the 246 bp mitogenome control region fragment were obtained using PALEOMIX BAM Pipeline v1.3.2 [28 (link)]. Removal of adapter sequences and collapse of reads was performed using AdapterRemoval v2.2.2 [29 (link)]. Sequence aligner bwa v0.7.15 [30 (link)] was used to perform collapsed read alignment against the reference sequence of the horse mitogenome (GenBank accession №: NC_001640.1), with a minimum read mapping quality of 25. The MapDamage v2.2.0 computational framework [31 (link)] helped recalculate base quality scores according to the probability of post-mortem DNA damage at each position in the sequence. The bioinformatics software platform Geneious Prime v2020.2.4 (
9
DNA Amplification and Sanger Sequencing
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Primers were designed using Primer3 Plus software [17] . DNA was amplified on a GeneAmp ® PCR System 9700 (Thermo Fisher, MA, USA).
We checked the quantity and quality of PCR products, including product size and off-target amplification, using the Caliper ® LabChip GX System and its related software (PerkinElmer, MA, USA) according to the manufacturer's protocol. Sanger sequencing was then done at the labs of Eurofins Genomics (Germany) using the Big Dye Chemistry in an ABI3730 automated sequencer (Applied Biosystems, Thermo Fisher Scientific, USA) using the procedures recommended by the manufacturer on the PCR product. Sequencing files (ABI format) were then visualized and analyzed using Sequence Scanner Software ® v2.0 (Thermo Fisher Scientific, USA).
We checked the quantity and quality of PCR products, including product size and off-target amplification, using the Caliper ® LabChip GX System and its related software (PerkinElmer, MA, USA) according to the manufacturer's protocol. Sanger sequencing was then done at the labs of Eurofins Genomics (Germany) using the Big Dye Chemistry in an ABI3730 automated sequencer (Applied Biosystems, Thermo Fisher Scientific, USA) using the procedures recommended by the manufacturer on the PCR product. Sequencing files (ABI format) were then visualized and analyzed using Sequence Scanner Software ® v2.0 (Thermo Fisher Scientific, USA).
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