Seqman pro

Colonies grown on MEA for 7 days were used to perform total DNA extraction using the Wizard^®® Genomic DNA Purification Kit (Promega, Madison, WI, USA) standard protocol. The primer pair ITS4/ITS5 [51 ] was used to amplify the ITS. The primer sets EF1-728F/EF2 [52 (link),53 (link)] and Bt2a/Bt2b [54 (link)] were used to amplify partial fragments of the TEF1 and TUB2 genes, respectively. Amplification by PCR was conducted as described by Yang et al. [16 (link)]. The PCR products were sequenced in both directions using the BigDye^®® Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems Life Technologies, Carlsbad, CA, USA), after which amplicons were purified through Sephadex G-50 Fine columns (GE Healthcare, Freiburg, Germany) in MultiScreen HV plates (Millipore, Billerica, MA, USA). Purified sequence reactions were analyzed on an Applied Biosystems 3730xl DNA Analyzer (Life Technologies, Carlsbad, CA, USA). The DNA sequences generated were analyzed and consensus sequences were computed using SeqMan Pro (DNASTAR, Madison, WI, USA). Sequences obtained in this study were deposited in GenBank https://academic.oup.com/nar/article/49/D1/D92/5983623 (accessed on 30 January 2021) (Table 2).

Two mitochondrial genes, cytochrome oxidase c subunit 1 (COI) and 16S ribosomal RNA (16S rRNA), were used. DNA was extracted from a dorsal fin clip of each specimen using Gentra Puregene Tissue Kit (QIAGEN, USA), according to the manufacturer’s instructions. Total DNA concentration and quality was quantified using BioPhotometer Plus spectrophotometer (Eppendorf, Germany). Both mitochondrial cytochrome COI and 16S rRNA genes were amplified using primer pairs (Table 2) to validate the species identity of each specimen. PCR reaction and condition for COI gene was performed PageBreakaccording to Abdul Kadir et al. (2015) , whereas PCR amplification for 16S rRNA gene was conducted according to Arai and Wong (2016) . PCR amplicons were purified using QIAquick® PCR Purification Kit (QIAGEN, USA), labeled with BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems Inc., USA), and sequenced bi-directionally on an ABI PRISM 3730xl Genetic Analyzer. Generated sequence trace files were manually edited and assembled using SeqMan Pro application in DNASTAR version 6.0 (DNASTAR Inc., USA). The contig sequences were compared for percentage similarity with the reference sequences in the GenBank using BLAST search. The sequences for both specimens were deposited to GenBank with accession numbers as listed in Table 3.

The Zp and BZLF1 gene sequences were aligned with their respective reference sequences using the B95-8 prototype (GenBank number NC_007605) as a reference. Visualization, quality assessment, and editing were performed using the SeqMan Pro (DNASTAR, version 11.1.0; Madison, WI, USA). For the classification of the Zp promoter, the sequences were aligned with the reference sequences through the ClustalW algorithm using the MEGA 11 program [34 (link)]. After alignment, the sequences were classified following preestablished nucleotide substitution criteria [33 (link)] using the AliView v1.28 program.
The Zp-V1 variant is the sequence present in the B95-8 prototype. The V3 variant differs in three positions from the prototype sequence [−141 (A>G), −106 (A>G), and −100 (T>G)], and the positions of these changes are related to the BZLF1 transcription start site [13 (link)]. The analysis and recognition of polymorphisms in BZLF1 were compared with those of the B95-8 prototype using the AliView v1.28 program, and the results were described according to the functional domain of the protein [13 (link)].

Whole genome sequencing was carried out by SEQ-IT (Kaiserslautern, Germany) as previously described [35 (link)]. The sequence data were analyzed using SeqManPro (DNASTAR, Madison, WI, USA). The contigs were assembled and aligned to the wild-type C. ljungdahlii genome (GenBank Accession Number: CP001666.1), the plasmid, and a genomic integration strain created in silico.

Direct Sanger sequencing was performed to validate the variant identified by whole exome sequencing. Genomic DNA was amplified by PCR using GoTaq 2X master mix (AB gene; Thermo Scientific, Epsom, UK) and CRYAA, CRYBA1, CRYBB1, CRYGA, CRYGC and CRYGD -specific primers designed with https://bioinfo.ut.ee/primer3-0.4.0/PCR conditions were as follows: 94 °C for 5 min of initial denaturation followed by 30 cycles of amplification of 30 s at 94 °C denaturing, 30 s at 60 °C annealing, and 45 s at 72 °C for extending. After cleaning, the PCR products were reacted with BigDye Terminator v3.1, they were run on ABI 3730 Genetic Analyzer (both from Applied Biosystems, Foster City, CA, USA) and analysed using SeqMan Pro (version 8.0.2 from DNASTAR) sequence analysis. After validating the variant, segregation was performed in all the available family members.

Sequence reads were trimmed by excluding low-quality regions using the NGen module of the DNAStar Lasergene software suite. Subsequently, assembly was performed with SeqMan pro (DNASTAR, USA) using high stringency de novo transcriptome assembly (100% identity between reads with 50 nucleotide sequence overlap). Similar 454 sequence reads were assembled into contigs using CLC Genomics Workbench 3 with its default parameters. Raw reads and contigs were uploaded in a proprietary web-based searchable database. As mentioned previously, such long sequence reads are likely to contain the full nucleic sequences of toxin precursors. Both raw reads and assembled contigs were identified as transcripts. Peptide sequences were identified from the transcript data using tBlastn. The E-value threshold of e ≤10⁻⁵ with a bit score >40 was recorded as a significant match for each query sequence. We analyzed the Blast results and used a home-made PERL script to classify representative sequences into five categories (‘Toxin-like’, ‘Putative toxin’, ‘Cellular Proteins’, ‘Unknown function’, ‘No Hit’). The identified peptide sequences were aligned using ClustalX 2.0.