A single colony of P. agglomerans KM1 was grown in LB broth (Conda, Spain) overnight at 37°C. The genomic DNA was isolated using the LaboPass™ Tissue Genomic DNA mini kit (Cosmo Genetech, Seoul, South Korea) according to the manufacturer’s protocol. The identity of the isolate was verified through 16S rRNA gene sequencing. The 16S rRNA gene was amplified from the extracted genomic DNA using 16S universal primers 27F (5’-AGA GTTTGATCMTGGCTCAG-3’ ) and 1492R (5’-GGTTACCTTGTTACGACTTC-3’ ) and sequenced using an automated ABI3730XL capillary DNA sequencer (Applied Biosystems, USA) for taxonomic identification at Cosmo Genetech (Seoul, South Korea). The 16S rRNA sequences were confirmed through BLASTn search against the NCBI microbial 16S database.
Abi 3730xl capillary dna sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 3730XL capillary DNA Sequencer is a high-throughput automated DNA sequencing system. It uses capillary electrophoresis technology to analyze and detect fluorescently labeled DNA samples. The system is designed to generate DNA sequence data efficiently and accurately.
Automatically generated - may contain errors
Lab products found in correlation
Accuprep pcr purification kit, by Bioneer (3 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (2 mentions)
G dex iic genomic dna extraction kit, by iNtRON Biotechnology (2 mentions)
Megaquick spin total fragment dna purification kit, by iNtRON Biotechnology (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Pgem t vector, by Promega (2 mentions)
Lb broth, by Condalab (1 mentions)
Labopass tissue genomic dna mini kit, by Cosmogenetech (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Pgemt t easy vector systems, by Promega (1 mentions)
8 protocols using abi 3730xl capillary dna sequencer
1
Identification of P. agglomerans KM1 by 16S rRNA sequencing
2
Phylogenetic Analysis of Sarcocystis spp.
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Positive PCR products were directly sequenced by dideoxy chain termination with an automatic sequencer (ABI 3730 xl capillary DNA sequencer, Applied Biosystems, Foster City, California, USA) using above primers and additional 17 (5′-AGAATTTCACCTCTG-3′) primers. All of sequence data were linked with single sequence. The resulting sequences were aligned and then subjected to phylogenetic analyses of the SSU rRNA genes against previously sequenced Sarcocystis species as well as with other registered sequences of Sarcocysits spp. from intermediate hosts, retrieved from GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST ). A score of sequences were aligned by DNASTAR (DNASTAR Inc.), Clustal W (www.clustal.org ), and MEGA 6.0 [12 (link)]. Neighbor-joining [13 (link),14 (link)] methods were based on a guide tree as pairwise and multiple alignment parameters. The final alignment comprised a score of sequences with 36 taxa.
3
Bisulfite Sequencing of Genomic DNA
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Genomic DNA was isolated from cells using G-DEX IIc Genomic DNA Extraction Kit (iNtRON Biotechnology, Gyeonggi-do, Korea) according to the manufacturer’s protocol. Bisulfite conversion was performed using the EZ DNA Methylation—Gold Kit (ZYMO RESEARCH, Orange, CA, USA) according to the manufacturer’s protocol. Bisulfite-specific PCR reactions were carried out on a GeneAmp PCR System 9700 (Applied Biosystems) using the following protocol: 95°C for 15 minutes, 50 cycles of 95xC for 20 seconds, 55°C for 40 seconds, 72°C for 30 seconds, and extension at 72°C for 10 minutes. The primer sequences used for PCR are listed in S2 Table . PCR products were purified using the MEGAquick-spin Total Fragment DNA Purification Kit (iNtRON Biotechnology), cloned into pGEM T vector (Promega, Madison, WI, USA), and sequenced using an ABI 3730XL Capillary DNA sequencer (Applied Biosystems). Methylated or unmethylated states of CpG sites were determined from the sequence data by using QUMA (QUantification tool for Methylation Analysis) software [53 (link)].
4
Phylogenetic Analysis of Methanogen Isolates
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PCR products were purified with the AccuPrep PCR purification kit (Bioneer, Daejeon, Korea). Sequencing of PCR products was performed using the BigDye terminator cycle sequencing kit on ABI 3730XL capillary DNA Sequencer (Applied Biosystems, Thermo Fisher Scientific Inc., Carlsbad, CA, USA). 16S rRNA and mcrA gene sequences from the isolated strains were compared to that of similar sequences obtained from GenBank using the BLAST program. Phylogenetic analysis was conducted using MEGA 4.0 [17 (link)]. We examined nine additional 16S rRNA sequences (M. mazei [NR041956], M. acetivorans [NR044724], Methanoculleus receptaculi [NR043961], Methanomicrobium mobile [NR 044726], Methanosphaera stadtmanae [M59139], Methanobacterium alcaliphilum [NR028228], Methanobacterium subterraneum [NR028247], Methanobacterium formicicum [JQ973735], and Methanobacterium formicicum [AF169245]). Phylogeny was further confirmed by mcrA gene and mcrA protein sequences (M. mazei [AB703645], M. barkeri [Y00158], M. lacustris [AY260439], Methanopyrus kandleri [U57340], Methanobacterium kanagiense [AB551869], Methanobacterium formicicum [EF465103], and Methanobacterium formicicum [JX141395]).
5
Genome-Wide DNA Methylation Analysis
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Genomic DNA was isolated from cells using a G-DEX IIc Genomic DNA Extraction Kit (iNtRON Biotechnology, Gyeonggi-do, Korea) according to the manufacturer’s protocol. Bisulfite conversion was performed using an EZ DNA Methylation-Gold Kit (ZYMO RESEARCH, Orange, CA, USA) according to the manufacturer’s protocol. Bisulfite-specific PCR reactions were performed on a GeneAmp PCR System 9700 (Applied Biosystems) using the following protocol: 95 °C for 15 min, 50 cycles of 95 °C for 20 s, 55 °C for 40 s, 72 °C for 30 s, and extension at 72 °C for 10 min. The primer sequences used for PCR are listed in Supplementary Table 2 . PCR products were purified using a MEGAquick-spin Total Fragment DNA Purification Kit (iNtRON Biotechnology), cloned into pGEM T vector (Promega, Madison, WI, USA), and sequenced using an ABI 3730XL capillary DNA sequencer (Applied Biosystems). The methylation state of the CpG sites was determined from the sequence data by using QUMA (QUantification tool for Methylation Analysis) software60 (link).
6
Molecular Cloning and Phylogenetic Analysis
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The amplified PCR products were purified using a Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. After purification, the PCR products were cloned into the pGEMt-T Easy Vector Systems (Promega, Madison, Wisconsin), followed by transformation into Escherichia coli DH5a cells, and then the cells were plated onto LB agar containing 100 lg/ml of ampicillin. Recombinant clones were selected by blue-white screening. Plasmid DNA for sequencing was purified using the MGe Plasmid SV Miniprep kits (Macrogen, Seoul, Korea). Purified recombinant plasmid DNA was sequenced using a T7 and SP6 promoter primer set by dideoxy termination with an automatic sequencer (ABI 3730xl capillary DNA sequencer, Applied Biosystems, Foster City, California). The obtained sequences were evaluated with Chromas software (Ver. 2.6.2, http://technelysium.com.au/wp/chromas/) and aligned by Clustal X2 (Ver 2.0, http://www.clustal.org/). Phylogenetic trees were constructed using the maximum-likelihood method in the Kimura 2-parameter model with the MEGA 7 program (1,000 bootstrap replicates) (Kimura, 1980; Kumar et al., 2016) . The nucleotide sequences generated were deposited into GenBank under accession numbers MK389598-MK389641, MK605995-MK606024, MK606031-MK606060, and MK613790-MK613833 for the 16S rRNA gene and MK836055-MK836059 and MT040624 for COI.
7
Phylogenetic Analysis of Methanogenic Archaea
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PCR products were purified with the AccuPrep PCR purification kit (Bioneer, Daejeon, Korea). Sequencing of PCR products was performed using the BigDye terminator cycle sequencing kit on ABI 3730XL capillary DNA Sequencer (Applied Biosystems, Thermo Fisher Scientific Inc., Carlsbad, CA, USA). 16S rRNA and mcrA genes sequences of strain KOR-1 were compared with the other similar sequences obtained from GenBank using the BLAST program. Phylogenetic analysis was conducted in MEGA 4.0 (Tamura et al., 2007 (link)). Eight additional 16S rRNA sequences (Methanobacterium formicicum [NR025028]; Methanobacterium subterraneum [NR028247]; Methanobacterium palustre [NR041713]; Methanobacterium alcaliphilum [NR028228]; Methanobacterium bryantii [NR042781]; Methanobacterium oryzae [NR028171]; Methanobacterium ivanovii [NR041716]; Methanosphaera stadtmanae [NR028236]) representing methanogens were included in phylogenetic analysis. Phylogeny was further confirmed by mcrA gene and mcrA protein sequences (Methanobacterium formicicum [EF465103]; Methanobacterium palustre [AB542760]; Methanobacterium kanagiense [AB551870]; Methanobacterium bryantii [AF313806]; Methanobacterium aarhusense [AY386125]; Methanopyrus kandleri [U57340]).
8
Phylogenetic Analysis of Methanogenic Archaea
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Purification of PCR products was performed with the AccuPrep PCR purification kit (Bioneer, Daejeon, Korea). PCR products were sequenced using the BigDye terminator cycle sequencing kit on ABI 3730XL capillary DNA Sequencer (Applied Biosystems, Thermo Fisher Scientific Inc., Carlsbad, CA, USA). 16S rRNA and mcrA gene sequences from the isolated strain were compared to the similar sequences obtained from GenBank using the BLAST program. Phylogenetic analysis was conducted using MEGA 4.0 [24 (link)]. We examined eight additional 16S rRNA sequences (M. bourgensis MS2 [HE964772], M. palmolei [Y16382], M. receptaculi ZC-2 [DQ787476], M. taiwanensis CYW4 [KM111599], Methanofollis tationis [AF 095272], Methanoregula formicica SMSP [CP003167], Methanogenium marinum AK-1 [DQ177344], and Methanoplanus limicola DSM 2279 [CM001436]). Phylogeny was further confirmed with mcrA gene sequences (M. bourgenisis MS2 [AB AB300787], M. bourgensis RC/ER [AB300785], M. bourgensis CB1 [AB300786], M. bourgensis MAB1 [KJ708788], M. bourgensis MAB2 [KJ708789], M. chikugoensis NBRC 101202 [AB 703634], M. chikugensis [AB300779], M. thermophius DSM 2624 [AF313804], and M. thermophiles [AB300783]).
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