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Smrt analysis 2

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SMRT Analysis 2.3 is a software suite for data analysis and visualization of PacBio sequencing data. It provides tools for tasks such as genome assembly, mapping, and variant calling.

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Lab products found in correlation

Smrtbell template prep kit 1, by Pacific Biosciences (4 mentions) G tube, by Covaris (2 mentions) Rsii sequencer, by Pacific Biosciences (1 mentions) Qiacube extraction system, by Qiagen (1 mentions) Bluepippin size selection system, by Sage Science (1 mentions) Qiaamp dna mini kit, by Qiagen (1 mentions) Bcl2fastq, by Illumina (1 mentions) Pacbio rs 2 system, by Pacific Biosciences (1 mentions) Genomic tip 100 g, by Qiagen (1 mentions) Smrt cell v3, by Pacific Biosciences (1 mentions) Bluepippin size selection system protocol, by Pacific Biosciences (1 mentions) Agilent 2200 tapestation, by Thermo Fisher Scientific (1 mentions) Qubit dsdna br assay, by Thermo Fisher Scientific (1 mentions) Nucleobond buffer set 3, by Takara Bio (1 mentions) Nucleobond axg column, by Takara Bio (1 mentions)

9 protocols using smrt analysis 2

1

Whole Genome Sequencing of Bacteria

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Bacteria were grown in tryptic soy broth as previously described [17 (link)]. Bacterial DNA was extracted from 2 mL of bacterial cell culture using the automated QIAcube extraction system (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions for Gram-negative bacteria.
Sequencing was carried out using single molecule real time (SMRT) DNA sequencing on the Pacific Bioscience RS II Sequencer (PacBio, Menlo Park, CA) as previously described [17 (link)]. Briefly, template DNA was sheared to ≥10-kbp using g-TUBEs (Covaris, Inc., Woburn, MA). Whole genome libraries were prepared according to the PacBio 10-kbp insert library protocol using DNA Template Kit 1.0 and afterwards size-selected using the BluePippin size-selection system (Sage Science, Inc., Beverly, MA) according to the manufacturer’s instruction. Libraries were sequenced using P4/C2 Chemistry Kits on five SMRT cells with a 180-min collection protocol. The obtained sequence reads were analyzed by SMRT Analysis 2.3.0 (PacBio, Menlo Park, CA) and de novo assembled using the PacBio hierarchical genome assembly process 3 (HGAP3.0)/Quiver software package.
Lorenz S.C., Gonzalez-Escalona N., Kotewicz M.L., Fischer M, & Kase J.A. (2017). Genome sequencing and comparative genomics of enterohemorrhagic Escherichia coli O145:H25 and O145:H28 reveal distinct evolutionary paths and marked variations in traits associated with virulence & colonization. BMC Microbiology, 17, 183.
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2

Whole-Genome Sequencing of Probiotic Strain

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The probiotic strain was inoculated into MRS broth at a ratio of 0.1%. Cells were obtained at the late exponential phase of growth. After washing three times with PBS, gDNA was extracted using a QIAamp DNA Mini Kit (Qiagen). PacBio library construction and whole-genome sequencing were performed by Chunlab. Genomic DNA was cut into 10 kb using a g-tube (Covaris, USA) and purified. Ends were repaired, and SMRTbell adapters were ligated to the blunt end using the SMRTbell Template Prep Kit 1.0 (PacBio, USA). The library was then sequenced using PacBio P6C4 chemistry in an 8-well-SMART Cell v3 of PacBio RSII (PacBio). PacBio sequencing data were assembled with PacBio SMRT Analysis 2.3.0 using the HGAP2 protocol. The genome was then circularized using a Circlator 1.4.0 (Sanger Institute, UK). Protein coding sequences (CDSs) were predicted with Prodigal 2.6.2 [27 (link)] and grouped according to roles regarding orthologous groups (EggNOG; http://eggnogdb.embl.de). Genes encoding tRNAs were searched using tRNAscan-SE 1.3.1 [28 (link)]. rRNAs and other noncoding RNAs were searched by covariance model searches using the Rfam 12.0 database [29 (link)]. For comparison of prokaryotic genome sequences, OrthoANIu algorithm-based Average Nucleotide Identity (ANI) calculator (https://www.ezbiocloud.net/tools/ani) was used [30 (link)].
Yoon Y., Seo H., Kim S., Lee Y., Rahim M.A., Lee S, & Song H.Y. (2021). Anti-Tuberculosis Activity of Pediococcus acidilactici Isolated from Young Radish Kimchi against Mycobacterium tuberculosis. Journal of Microbiology and Biotechnology, 31(12), 1632-1642.
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3

PacBio Sequencing and Genome Assembly

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PacBio sequencing and analysis were conducted by OE Biotech Co. Ltd. (Shanghai, China). In brief, genomic DNA was extracted from bacteria using the CTAB method [22 (link)] according to the manufacturer’s instructions. The genomic DNA was subjected to quality control by agarose gel electrophoresis and quantified by Qubit (Invitrogen, Waltham, MA, USA). The library was constructed with the SMRTbell Template Prep Kit 1.0 (PacBio, Menlo Park, CA, USA). Single-molecule real-time (SMRT) sequencing was performed on the PacBio Sequel II platform using DNA/Polymerase Binding Kit 3.0 (New England Biolabs, Ipswich, MA, USA). Genome assembly was performed using SMRT Analysis 2.3.0 (Pacific Biosciences, Menlo Park, CA, USA).
Liu Q., Guo Z., Zhu G., Li N., Bai G, & Jiang M. (2023). Genomic Characteristics and Phylogenetic Analyses of a Multiple Drug-Resistant Klebsiella pneumoniae Harboring Plasmid-Mediated MCR-1 Isolated from Tai’an City, China. Pathogens, 12(2), 221.
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4

Comprehensive Genomic Analysis Pipeline

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Analysis of PacBio sequence reads was implemented using SMRT Analysis 2.3.0 and the SMRT Portal 2.0 platform (Pacific Biosciences; Menlo Park, CA). De novo assembly was performed using the Hierarchical Genome Assembly Process (HGAP) with default parameters (Bjedov et al., 2003 (link)), as described previously (Pirone-Davies et al., 2015 (link)). Genomes were annotated using the NCBI (National Center for Biotechnology Information) Prokaryotic Genomes Automatic Annotation Pipeline (Goodman, 2002 (link)).1 Adapters were removed from MiSeq reads using bcl2fastq (Illumina, 2017 ) and reads were trimmed to phred 20 with Trimmomatic v. 0.36 (Bolger et al., 2014 (link)), assembled with SPAdes v3.13.0 (Bankevich et al., 2012 (link)), and annotated with Prokka (Seemann, 2014 (link)).
Davies C.P., Jurkiw T., Haendiges J., Reed E., Anderson N., Grasso-Kelley E., Hoffmann M, & Zheng J. (2022). Changes in the genomes and methylomes of three Salmonella enterica serovars after long-term storage in ground black pepper. Frontiers in Microbiology, 13, 970135.
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5

Long-read transcriptome profiling using PacBio

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Libraries of ~20 kb SMRTbell Templates were prepared for the DSN-treated (normalized) or non-treated cDNA. Libraries were checked for the quality and quantified, and sequenced using SMRT sequencing on the PacBio RS II System (Pacific Biosciences, USA). For each sample, normalized or non-normalized, two cells were sequenced. ROIs were called and corrected from the raw polymerase reads with SMRT analysis 2.3.0 (Pacific Biosciences, USA). More than one full pass was required to build the ROIs. The library preparation, sequencing and preprocessing of raw data were performed in Macrogen, Korea. SMRT Portal IsoSeq analysis was further performed with default parameters to filter the artifacts, classify and cluster the ROIs to obtain the full-length reads, consensus isoforms and high-quality isoforms respectively, which were used for further analysis. The criteria of consensus isoforms and high-quality isoforms conformed to PacBio SMRTLink default definition, that is, a consensus isoform should contain at least one full-length ROI (with 3′-polyA tail, 5′- and 3′-primers) and the predicted accuracy of sequences reached 95–99%, whereas a high-quality isoform should be supported by at least two full-length ROIs and the predicted sequence accuracy should not be lower than 99%.
Hu Y., Shu X.S., Yu J., Sun M.A., Chen Z., Liu X., Fang Q., Zhang W., Hui X., Ying Y., Fu L., Lu D., Kumar R, & Wang Y. (2020). Improving the diversity of captured full-length isoforms using a normalized single-molecule RNA-sequencing method. Communications Biology, 3, 403.
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6

H. pylori Genome Sequencing Protocol

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Genomic DNA from various deletions and the complemented strains was prepared by CTAB reagent followed by QIAGEN genomic-tip 100/G. SMRTbell template libraries were prepared from total H. pylori genomic DNA following the 10 kb Template Preparation Procedure (Pacific Biosciences, Menlo Park, CA, USA). One SMRT cell was run on an RSII sequencer using P6/C4 chemistry, producing between 800 and 1100 Mb of raw sequence (two SMRT cells for the dR_dM1_dM2 strain). The genome sequence was assembled for each strain using Pacific Biosciences SMRT Analysis 2.3 program ‘RS_HGAP_Assembly.3′, with coverage between 411X and 513X, and the assembly was closed to a single circular genome manually. SMRT sequencing and methylation detection was performed as described earlier in (8 (link),18 (link)). PacBio motif files and the sequencing data was submitted to NCBI database (Table 1).
Kumar S., Karmakar B.C., Nagarajan D., Mukhopadhyay A.K., Morgan R.D, & Rao D.N. (2018). N4-cytosine DNA methylation regulates transcription and pathogenesis in Helicobacter pylori. Nucleic Acids Research, 46(7), 3429-3445.
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7

PacBio Transcriptome Sequencing Analysis

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PacBio sequences were analyzed using Pacific Biosciences command line SMRTAnalysis 2.3 package for analyzing transcriptome data generated using the PacBio(R) Single Molecule, Real-Time (SMRT) technology. Analysis included PacBio’s RS IsoSeq, a SMRTPortal/SMRTPipe protocol, and pbtranscript, a PacBio Bioinformatics tool. Pbtranscript.py “classify” tool was used to identify full-length reads with a 5’ primer, poly-A tail and a 3’ primer. The pbtranscript.py “cluster” tool was used to obtain consensus isoforms. The clustering step used graph algorithms to cluster reads that came from the same isoform. Finally, the consensus sequences were aligned to hg19 and redundant isoforms were removed.
Merrick B.A., Phadke D.P., Bostrom M.A., Shah R.R., Wright G.M., Wang X., Gordon O., Pelch K.E., Auerbach S.S., Paules R.S., DeVito M.J., Waalkes M.P, & Tokar E.J. (2020). KRAS-retroviral fusion transcripts and gene amplification in arsenic-transformed, human prostate CAsE-PE cancer cells. Toxicology and applied pharmacology, 397, 115017.
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8

Single Molecule Sequencing and Genome Assembly

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Single Molecule, Real-Time (SMRT) sequencing libraries were prepared using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences) and 20 kb Template Preparation Using BluePippin Size-Selection System protocol (Pacific Biosciences). Library quality and quantity were determined using an Agilent 2200 TapeStation and Qubit dsDNA BR Assay (Life Technologies), respectively. Sequencing was conducted using P5-C3 chemistry and a v3 SMRT Cell (Pacific Biosciences) at Weill Cornell Medical College. Genome assembly was conducted using the Hierarchical Genome Assembly Process 2.0 (HGAP 2.0) (Chin et al. 2013 (link)). Raw sequencing reads were filtered for length and quality such that the minimum polymerase read score was 0.8, the minimum subread length was 500 bp, and the minimum polymerase read score was greater than 100. The assembly was generated using CeleraAssembler v1 with the default parameters and was polished using the Quiver algorithm (Chin et al. 2013 (link)). To identify methylated DNA bases, we used the RS Modification and Motif Analysis module of Pacific Biosciences’ SMRT Analysis 2.3 with a Quality Value (QV) threshold of 30. This analysis uses an in silico kinetic reference and a t-test-based kinetic score to detect modified bases (Flusberg et al. 2010 (link)).
Kurylo C.M., Alexander N., Dass R.A., Parks M.M., Altman R.A., Vincent C.T., Mason C.E, & Blanchard S.C. (2016). Genome Sequence and Analysis of Escherichia coli MRE600, a Colicinogenic, Nonmotile Strain that Lacks RNase I and the Type I Methyltransferase, EcoKI. Genome Biology and Evolution, 8(3), 742-752.
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9

Fungal Genome Assembly and Annotation

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The genomic DNA of the fungal isolate was extracted from a one-day-old culture using phenol-chloroform extraction and Nucleobond AXG column (Takara) with Nucleobond buffer set III (Takara) . Genome sequencing was performed on a Pacific Biosciences RS II (Pacific Biosciences, Menlo Park, CA, USA) using libraries prepared with the SMRT-bell template prep kit 1.0 (Pacific Biosciences) . A draft genome of the fungal isolate was assembled using SMRT Analysis 2.3 (Pacific Biosciences) . The sequencing runs and assembly of the libraries were conducted by Takara Bio (Mie, Japan) . Genome annotation was performed by using the Funannotate (v.1.7.4) pipeline (Palmer, 2016) . The soft-masked genome by RepeatMasker (v.4.0.7) (Smit et al., 2018) and RepeatModeler (v.1.0.11) (Smit and Hubley, 2008) was used for generating an ab initio gene model with the options --busco_seed_species "fusarium_ graminearum" and --busco_db "sordariomycetes". The completeness of the assembly and gene prediction was assessed using BUSCO v.4.0.5 with 3817 Sordariomycetes ortholog genes (Seppey et al., 2019) .
Yamamoto T., Alimu Y., Takahashi H., Kusuya Y., Hosoya K., Shigemune N., Nagai S, & Yaguchi T. (2021). Isolation and Characterization of the Polyhexamethylene Biguanide Hydrochloride-Resistant Fungus, Purpureocillium lilacinum. Biocontrol science, 26(3).
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