Applied biosystems 3130 dna analyzer

Sequencing was performed in both directions using a Big Dye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA) and the same set of PCR primers. The sequencing products were purified using Sephadex G50 columns (GE Healthcare, Chicago, IL, USA) and analyzed in an Applied Biosystems 3130 DNA Analyzer (PE Applied Biosystems). Sequences were edited in Sequencher v.4.9 software (Genes Codes Corporation, Ann Arbor, MI, USA) and the primer regions removed. Sequences from GenBank or those previously processed in the Systematics Molecular Laboratory (FSP) were also included to serve as references in the analyses. The COI gene sequences were aligned by nucleotide using the muscle algorithm [51 (link)], implemented in SeaView [52 (link)], and then by amino acid using TranslatorX [53 (link)].

Sequencing reactions proceeded in both directions using a Big Dye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA) and Applied Biosystems 3130 DNA Analyzer (Applied Biosystems). Sequencing reactions were carried out with the same set of PCR primers. The sequencing products were purified using Sephadex G50 columns (GE Healthcare) and analysed in an Applied Biosystems 3130 DNA Analyser (PE Applied Biosystems). Sequences were edited in Sequencher v.4.9 software (Genes Codes Corporation, Ann Arbor, MI, USA), and the primer regions removed. In addition to these novel sequences, 162 sequences from GenBank were also included, to serve as references in the phylogenetic and species delimitation analysis. The COI gene sequences were aligned first by nucleotides using the Muscle algorithm [48 (link)] implemented in SeaView [49 (link)], and then by amino acid using TranslatorX [50 (link)].

Candidate variants from whole-exome sequencing were validated by Sanger sequencing. Along with this validation process, segregation analyses were performed on the family members. Primer3 v0.4.0, a web-based tool, was used to designed primers for amplification and sequencing [24 (link)]. Sequencing reactions were performed using Applied Biosystems 3130 DNA Analyzer (Life Technologies, Carlsbad, CA, USA).