PCR was performed in 15 μL volume containing approximately 60 ng of genomic DNA of ‘Fukushima-1’ (Table 1 ), 1 × Multiplex PCR Master Mix (Qiagen, Hilden, Germany), and 0.2 μM of Primer_F3 and Primer_R (Figure S3 and Table S2 ). The thermal profile for the PCR was as follows: an initial denaturing step of 15 min at 95 °C, followed by 40 cycles of 30 s at 94 °C, 90 s at 63 °C and 90 s at 72 °C, before a final elongation step at 72 °C for 10 min using a GeneAmp 9700 PCR System (Applied Biosystems, PE Corp., Foster City, CA, USA). PCR products were purified using ExoSAP-IT (Affymetrix, Inc., Santa Clara, CA, USA). Direct sequencing was performed using the ABI PRISM BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI 3130 Genetic Analyser (Applied Biosystems). For sequencing, we also used additional internal primers (Primer_F2, Primer_F2-2, Primer_R2, Primer_R2-2, Primer_R2-3, Primer_R2-4) developed in this study (Figure S3 and Table S2 ). Sequence alignment was performed using CodonCode Aligner v.8.0.2 software (CodonCode Corporation, Dedham, MA, USA), followed by manual editing.
Aligner v8
Manufactured by CodonCode
Sourced in United States
CodonCode Aligner v8.0.2 is a DNA sequence alignment software. It provides tools for analyzing, editing, and comparing DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Multiplex pcr master mix, by Qiagen (6 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (6 mentions)
Abi prism bigdye terminator version 3.1 cycle sequencing kit, by Thermo Fisher Scientific (6 mentions)
Abi 3130 genetic analyser, by Thermo Fisher Scientific (6 mentions)
Exosap it, by Thermo Fisher Scientific (3 mentions)
Abi 3730xl, by Thermo Fisher Scientific (3 mentions)
Genemapper v4, by Thermo Fisher Scientific (1 mentions)
7 protocols using aligner v8
1
Sequencing of chloroplast DNA in Fukushima-1
2
PCR Amplification and Sequencing of Fukushima-1
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PCR was performed in 15 μL volume containing approximately 60 ng of genomic DNA of Fukushima-1 (Table 1), 1× Multiplex PCR Master Mix (Qiagen, Hilden, Germany), and 0.2 μM of Primer_F3 and Primer_R (Figure S2 and Table S2). The thermal profile for the PCR was as follows: an initial denaturing step of 15 min at 95 °C, followed by 40 cycles of 30 s at 94 °C, 90 s at 63 °C and 90 s at 72 °C, before a final elongation step at 72 °C for 10 min using a GeneAmp 9700 PCR System (Applied Biosystems, PE Corp., Foster City, CA, USA). PCR products were purified using ExoSAP-IT (Affymetrix, Inc., Santa Clara, CA, USA). Direct sequencing was performed using the ABI PRISM BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI 3130 Genetic Analyser (Applied Biosystems). For sequencing, we also used additional internal primers (Primer_F2, Primer_F2-2, Primer_R2, Primer_R2-2, Primer_R2-3, Primer_R2-4) developed in this study (Figure S2 and Table S2). Sequence alignment was performed using CodonCode Aligner v.8.0.2 software (CodonCode Corporation, Dedham, MA, USA), followed by manual editing.
3
Phylogenetic Analysis of Pangasiidae Catfish
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The resulting DNA sequences were assembled and edited using CodonCode Aligner v.8.0.2 (CodonCode Corporation, Dedham, MA, USA). Sequences (usually at both ends) with low quality or problematic base calls (below Q [phred quality value] 20) were verified by eye and trimmed. For RAG1 gene sequences, observed heterozygous sites were coded following the International Union of Pure and Applied Chemistry (IUPAC) nucleotide code. Edited sequences were aligned and trimmed to a common length in MEGA7 using the MUSCLE algorithm [56 (link)]. Alignment was subsequently checked by eye and adjusted manually when necessary.
The datasets compiled and used for molecular analyses included (1) a complete cytb dataset comprising the cytb sequences from all collected 316 samples, plus 8 and 6 samples of P. argentata and P. pawak, respectively, obtained from Lo et al. [48 (link),52 (link)]; (2) individual datasets for the cyt b sequences of P. anea, P. ovata, and P. macrocephalus; and (3) a combined (or multi-gene) dataset (cytb, COI, and RAG1) comprising the sequences from representative samples of each species/lineage based on the reconstructed cytb gene tree and biogeographic regions.
The datasets compiled and used for molecular analyses included (1) a complete cytb dataset comprising the cytb sequences from all collected 316 samples, plus 8 and 6 samples of P. argentata and P. pawak, respectively, obtained from Lo et al. [48 (link),52 (link)]; (2) individual datasets for the cyt b sequences of P. anea, P. ovata, and P. macrocephalus; and (3) a combined (or multi-gene) dataset (cytb, COI, and RAG1) comprising the sequences from representative samples of each species/lineage based on the reconstructed cytb gene tree and biogeographic regions.
4
Phylogenetic Sequence Alignment and Analysis
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Both strands of all sequences were assembled into contigs using CodonCode Aligner v8.0.2 (CodonCode Corporation, Centerville, Massachusetts, USA), and multiple sequence alignment for each gene was performed using ClustalW algorithm in MEGA v. 7 [33 (link)]. The saturation substitution index (Iss) was estimated for each gene to evaluate if the Iss was significantly lower than the critical value of Iss (Iss.c). This test is necessary to ascertain whether sequences are phylogenetically informative [34 (link)]. Iss and the proportions of conserved and polymorphic sites were computed using DAMBE v.5.5.1 [35 (link)].
5
Sanger Sequencing and Assembly of Genomic Contig
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Individual amplicons were sequenced by the Sanger sequencing method. For this, primer walking was performed using additionally designed strain-specific primers (Supplementary Table 1 ). Sequencing was performed using an ABI 3730xl (Applied Biosystems). Sequences that satisfied the conditions of Phred score > 20 and length ≥ 700 bp were filtered and used for assembly. The filtered sequences were assembled using CodonCode Aligner v8.0.2 (CodonCode Corporation). The assembled single contig (27,273 bp) was further curated manually by observing the sequencing chromatograms.
6
Genetic Diversity Analysis of Heleobia ascotanensis
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The DNA samples were extracted and amplified by PCR following the protocol of Valladares et al. [30 (link), 47 (link)]. For the COI marker, both strands of the amplification product were sequenced by Macrogen Inc. (Seoul, Korea). The nucleotide sequences were edited in the Codon Code Aligner v.8.0.1 program (CodonCode Corporation, Dedham, MA, USA) and were then aligned using the MAFFT v7.505 algorithm using the web server [57 (link), 58 ].
For microsatellite markers, seven specific primers were developed for H. ascotanensis following the protocol of Fabres et al. [59 (link)], and three other primers previously reported for the congeneric species Heleobia atacamensis (Philippi, 1860) were used [59 (link)]. The amplification products were analyzed by the sequencing service of the Pontificia Universidad Católica de Chile (Santiago, Chile). Then, fragment analysis was performed using Gene Mapper v4.0 software (Applied Biosystems, Waltham, MA). Subsequently, the excess of homozygotes or heterozygotes was checked with Micro -Checker v2.2.3 [60 (link)], and for each locus, the presence of stutters and null alleles were searched. The details of the mitochondrial and microsatellites are indicated in Table S3 .
For microsatellite markers, seven specific primers were developed for H. ascotanensis following the protocol of Fabres et al. [59 (link)], and three other primers previously reported for the congeneric species Heleobia atacamensis (Philippi, 1860) were used [59 (link)]. The amplification products were analyzed by the sequencing service of the Pontificia Universidad Católica de Chile (Santiago, Chile). Then, fragment analysis was performed using G
7
ITS2 Sequence Annotation and Alignment for Schisandra
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Raw ITS2 trace files generated from our study were assembled using CodonCode Aligner V.8.01 (CodonCode Co., Dedham, MA, USA). All the ITS2 sequences of Schisandra species, also including those downloaded from GenBank, were annotated as well as trimmed through a hidden Markov Model-based method to obtain the complete ITS2 regions [34 (link)]. The haplotypes of Schisandra species were selected by CodonCode Aligner and then aligned with MEGA 7.0 software [35 (link)]. The unique region was finally verified by BLAST analysis.
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