Genomic DNA of E. coli 50579417 was prepared from an overnight culture using the GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. The DNA library was prepared using the Pacific Biosciences 20-kb library preparation protocol and size selection with a 9-kb cutoff using BluePippin (Sage Sciences, Beverly, MA). Sequencing was performed using the Pacific Biosciences RSII instrument using P6-C4 chemistry with a 360-min movie time and one single-molecule real-time sequencing (SMRT) cell. The sequences were assembled and polished at The Norwegian Sequencing Centre (https://www.sequencing.uio.no/ ) using HGAP v3 (Pacific Biosciences, SMRT Analysis Software v.2.3.0). Minimus2 from AMOS was used to circularize contigs, and RS_Resequencing.1 software (Pacific Biosciences, SMRT Analysis Software v.2.3.0) was used for correction of bases after circularization.
Smrt analysis software v2
Manufactured by Pacific Biosciences
SMRT Analysis Software v2.3.0 is a bioinformatics software developed by Pacific Biosciences. It provides tools for the analysis of data generated by Pacific Biosciences' SMRT sequencing technology.
Automatically generated - may contain errors
Lab products found in correlation
Genelute bacterial genomic dna kit, by Merck Group (1 mentions)
Rsii instrument, by Pacific Biosciences (1 mentions)
Hgap v3, by Pacific Biosciences (1 mentions)
Smrt link 4, by Pacific Biosciences (1 mentions)
Hgap4, by Pacific Biosciences (1 mentions)
Rs 2 sequencing instrument, by Pacific Biosciences (1 mentions)
Smrt portal, by Pacific Biosciences (1 mentions)
Bluepippin, by Sage Science (1 mentions)
5 protocols using smrt analysis software v2
1
High-Quality E. coli Genome Sequencing
2
PacBio Transcript Isoform Profiling
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Subread filtering was performed using SMRT analysis software (v2.3.0) (Pacific Biosciences, Menlo Park, CA). Full-length read information was gathered using the RS_ReadsOfInsert.1 protocol. The reads corresponding to spliced isoforms from each device were demultiplexed, using Pacific Biosciences’ protocols and tutorials (https://github.com/PacificBiosciences/cDNA_primer/ ), and classified into CCS (Circular Consensus Sequences) and non-CCS subreads by ToFu54 (link). The isoform-level clustering algorithm ICE (Iterative Clustering for Error Correction) was run and the results were polished using Quiver. Transcripts lacking the 5′ end of exon 1 or the 3′ end of exon 4 were removed. Reads were mapped to the ASD to generate spliced isoform profiles and relative abundance estimates. Only transcripts accounting for 1% or more of the total number of reads are reported. The number of reads and the sequence corresponding to each spliced isoform generated by an ASD is available in the Supplementary Data 1 file.
3
Long-read SMRT Sequencing and De Novo Genome Assembly
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The single-molecule real-time (SMRT) sequencing technology developed by PacBio [44 (link)] was used for generating long sequencing reads. A total of 285,082 pre-filtered reads were obtained on a PacBio RS II sequencing instrument. The Norwegian Sequencing Centre (www.sequencing.uio.no 5 October 2017) provided the sequencing service.
A hierarchical genome-assembly process (HGAP) [45 (link)], using the HGAP3 (Pacific Biosciences, SMRT Analysis Software v2.3.0) and HGAP4 (Pacific Biosciences, SMRT Link 4.0.0) protocols, was carried out. Three strategies in the de novo genome assemblies were followed: (i) and (ii) HGAP3 and expected genome sizes of 34 and 35 Megabases (Mb), respectively, and (iii) HGAP4 and an expected genome size of 35 Mb. Equivalent assemblies were obtained in all three strategies. PacBio contigs having low coverage (< 40×) or short length (<15-Kb) were considered spurious and discarded.
A hierarchical genome-assembly process (HGAP) [45 (link)], using the HGAP3 (Pacific Biosciences, SMRT Analysis Software v2.3.0) and HGAP4 (Pacific Biosciences, SMRT Link 4.0.0) protocols, was carried out. Three strategies in the de novo genome assemblies were followed: (i) and (ii) HGAP3 and expected genome sizes of 34 and 35 Megabases (Mb), respectively, and (iii) HGAP4 and an expected genome size of 35 Mb. Equivalent assemblies were obtained in all three strategies. PacBio contigs having low coverage (< 40×) or short length (<15-Kb) were considered spurious and discarded.
4
Genome-wide Analysis of DNA Methylation
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The RS_Modification_and_Motif_Analysis pipeline analysis platform SMRT Portal (Pacific Biosciences, SMRT Analysis Software v2.3.0, smrtanalysis_2.3.0.140936.p5.167094) was used for genome-wide analysis of modified motifs with quality value (QV) limit > 60. DNA methyltransferase genes associated with the different methyltransferase recognition motifs identified were searched using SEQWARE routines as described previously29 (link), and deposited in the Restriction Enzyme Database REBASE30 (link). Motif summary files have been deposited in the NCBI submission PRJNA386981 (see Supplementary Table 4 for a summary).
5
Genome Sequencing of Yersinia ruckeri
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Initial database searches for invasin homologues were performed using pBLAST with the protein sequence of Y. pseudotuberculosis invasin (GenBank accession number: AAA27632.1) as a search model.
After failing to amplify and clone the identified genes for structural and functional analyses using standard molecular biology procedures, we decided to sequence the genome of NVH_3758. Genomic DNA was isolated from 10 ml overnight culture of Y. ruckeri NVH_3758 using a large genomic DNA extraction protocol (Sun et al., 2015) (link). Genome sequencing was performed by the Norwegian Sequencing Centre (Oslo, Norway) using the Pacific Biosciences RS II platform. A DNA library was prepared following the Pacific Biosciences 20 kb library preparation protocol. The final library was selected based on a 8 kb cut-off using BluePippin (Sage Science). The library was sequenced using P6-C4 chemistry with 360 min movie time (time of fluorescent nucleotides incorporation). The generated reads were assembled using the Hierarchical Genome Assembly Process (HGAP) v3 (Pacific Biosciences, SMRT Analysis Software v2.3.0) comprising preassembly, assembly and consensus polishing.
After failing to amplify and clone the identified genes for structural and functional analyses using standard molecular biology procedures, we decided to sequence the genome of NVH_3758. Genomic DNA was isolated from 10 ml overnight culture of Y. ruckeri NVH_3758 using a large genomic DNA extraction protocol (Sun et al., 2015) (link). Genome sequencing was performed by the Norwegian Sequencing Centre (Oslo, Norway) using the Pacific Biosciences RS II platform. A DNA library was prepared following the Pacific Biosciences 20 kb library preparation protocol. The final library was selected based on a 8 kb cut-off using BluePippin (Sage Science). The library was sequenced using P6-C4 chemistry with 360 min movie time (time of fluorescent nucleotides incorporation). The generated reads were assembled using the Hierarchical Genome Assembly Process (HGAP) v3 (Pacific Biosciences, SMRT Analysis Software v2.3.0) comprising preassembly, assembly and consensus polishing.
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