Complete genome sequencing of Nakamurella sp. PAMC28650 was obtained by PacBio sequel single-molecule real-time (SMRT) sequencing technology (Pacific Biosciences, Menlo Park, CA, USA). SMRTbell library inserts (20 kb) were sequenced using SMRT cells. Results were generated from raw sequence data of 5,629,850 bp that were assembled de novo by using the hierarchical genome assembly process (HGAP) protocol (Chin et al. 2013 (link)) and HGAP4 assembly using SMRT analysis software (ver. 2.3; Pacific Biosciences, https://github.com/PacificBiosciences/SMRT-Analysis ). The complete genome sequence was deposited in the GenBank database under the GenBank accession number CP060298.1.
Pacbio sequel single molecule real time sequencing technology
Manufactured by Pacific Biosciences
Sourced in United States
The PacBio Sequel single-molecule real-time (SMRT) sequencing technology enables long-read DNA sequencing. It utilizes zero-mode waveguides to detect the incorporation of fluorescently labeled nucleotides during DNA synthesis in real-time at the single-molecule level.
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4 protocols using pacbio sequel single molecule real time sequencing technology
1
Complete Genome Sequencing of Nakamurella sp.
2
PacBio Sequel SMRT Genome Sequencing
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Genome sequencing was performed using PacBio sequel single-molecule real-time (SMRT) sequencing technology (Pacific Biosciences, Menlo Park, CA, USA). SMRTbell library inserts (20 kb) were sequenced using SMRT cells. Raw sequence data were generated from 7,698 reads and 4,170,970 bp that were assembled de novo by using the hierarchical genome-assembly process (HGAP v.4) protocol [56 (link)] and HGAP4 assembly using SMRT analysis software (ver. 2.3; Pacific Biosciences, https://github.com/PacificBiosciences/SMRT-Analysis ). The complete genome sequence was deposited in the GenBank database under the GenBank accession number NZ_CP039290.1.
3
PacBio SMRT Sequencing of Polar Bacterial Genomes
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Genomic DNA was extracted and purified from cultures grown in R2A media at 15 °C using the Qiagen (Venlo, The Netherlands) Blood & Tissue kit, following the manufacturer’s recommended protocol for Gram-positive bacteria. The extracted DNA was confirmed using 1% agarose gel electrophoresis. It was then sequenced using the PacBio Sequel single-molecule real-time (SMRT) sequencing technology (Pacific Biosciences, Menlo Park, CA, USA) with SMRTbell library inserts (20 kb) and SMRT cells. Raw sequence data were generated for both PAMC22086 and PAMC21962 and were assembled de novo using the hierarchical genome-assembly process (HGAP v.4) protocol and HGAP4 assembly using SMRT analysis software (ver. 2.3; Pacific Biosciences, https://github.com/pacificbiosciences/SMRT-Analysis accessed on 28 June 2023) [24 (link)]. The circular map of PAMC22086 and PAMC21962 was obtained using Artemis [25 (link)].
4
Complete Genome Sequencing and Annotation of PAMC25284
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Genome sequencing was performed using PacBio sequel single-molecule real-time (SMRT) sequencing technology (Pacific Biosciences, Menlo Park, CA, USA). SMRTbell library inserts (20 kb) were sequenced using SMRT cells. Raw sequence data were generated from 77,075 reads and 821,081,934 bp that were assembled de novo using the hierarchical genome assembly process (HGAP) protocol [22 (link)] and HGAP4 assembly using SMRT analysis software (ver. 2.3; Pacific Biosciences, https://github.com/PacificBiosciences/SMRT-Analysis ). The complete genome sequence was deposited in the GenBank database under the GenBank accession number NZ_CP080382.1 (Bio project number PRJNA748195).
The PAMC25284 genome was annotated using the rapid annotation subsystem technology (RAST) server [23 (link)]. The predicted gene sequences were translated and searched in the National Center for Biotechnology Information (NCBI) nonredundant database, the Cluster of Orthologous Groups (COG) from the eggnog v.4.5.1 database [24 (link)], and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A circular map of the PAMC25284 genome was prepared using the CGViewBETA comparison tool [25 (link)].
The PAMC25284 genome was annotated using the rapid annotation subsystem technology (RAST) server [23 (link)]. The predicted gene sequences were translated and searched in the National Center for Biotechnology Information (NCBI) nonredundant database, the Cluster of Orthologous Groups (COG) from the eggnog v.4.5.1 database [24 (link)], and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A circular map of the PAMC25284 genome was prepared using the CGViewBETA comparison tool [25 (link)].
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