Cultures for RNA-sequencing were grown to an OD600 of ~0.7. 1 mL of culture was pelleted and resuspended in 1 mL of RNAlater (Invitrogen). RNA extraction, library building, and sequencing were contracted with Genewiz, Inc Reads were mapped to the published ATCC17978 genome using CLC Genomic Workbench software (Qiagen). After local alignment, reads per kilobase of transcript per million reads (RPKM) was calculated for each coding sequence. A log2 fold-change of > |2| with an FDR p-value<0.05 were considered statistically significant. The PCA plot was generated using ClustVis (55). For whole-genome sequencing, cells were pelleted and gDNA extracted using the Easy gDNA Extraction Kit (Invitrogen). Libraries were built using the Nextera DNA Flex Kit (Illumina) per manufacturer instructions and sequenced on an iSeq100. Reads were trimmed, locally realigned, and variants mapped using CLC Genomic Workbench software (Qiagen).
Clc genomic workbench software
Manufactured by Qiagen
Sourced in Germany, Denmark
CLC Genomic Workbench is a powerful software application for the analysis and visualization of biological sequence data. It offers a suite of tools for tasks such as genome assembly, read mapping, variant calling, and gene expression analysis. The software provides a user-friendly interface and supports a wide range of file formats, enabling researchers to efficiently manage and analyze their genomic data.
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Lab products found in correlation
Kapa library quantification kit, by Avantor (3 mentions)
Monarch genomic dna purification kit, by New England Biolabs (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Genome analyzer 2, by Illumina (1 mentions)
Nextera xt, by Illumina (1 mentions)
Masterpure dna purification kit, by Illumina (1 mentions)
Isogen 2, by Nippon Gene (1 mentions)
Genomicprep mini spin kit, by GE Healthcare (1 mentions)
Nextera dna sample preparation kit, by Illumina (1 mentions)
Miseq system, by Illumina (1 mentions)
Dnbseq g400, by MGI Tech (1 mentions)
Miseq reagent nano kit v2, by Illumina (1 mentions)
Agilent 2200 tapestation system, by Agilent Technologies (1 mentions)
D1000 screentape, by Agilent Technologies (1 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Nextseq 500, by Illumina (1 mentions)
318 chip kit v2, by Thermo Fisher Scientific (1 mentions)
Ion pgm next generation sequencer, by Thermo Fisher Scientific (1 mentions)
31 protocols using clc genomic workbench software
1
Transcriptomic and Genomic Analysis of A. baumannii
2
Genome sequencing of Streptomyces pactum
3
Whole-genome Sequencing Protocol
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Genomic DNA was prepared using the MasterPure DNA purification kit (Epicentre Biotechnologies, USA). To create libraries of paired-end fragments, the Nextera XT sample preparation kit (Illumina, USA) was used according to the instructions from the manufacturer. Sequencing was performed on the Illumina MiSeq instrument, generating 250-bp paired-end reads. Whole-genome sequencing data were analyzed using the CLC Genomic Workbench software (CLC bio, Denmark).
4
Bovine Transcriptome Analysis by RNA-Seq
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Short sequence reads of 75 bp in the XSQ file created by the SOLiD5500 system were
assembled and mapped with CLC Genomic Workbench software (ver. 8.5.1, CLC Bio, Aarhus,
Denmark) using Bos_taurus UMD3.1 (National Center for Biotechnology Information [NCBI]
assembly accession GCA_000003055.3) as a reference sequence. RNA-Seq analysis was carried
out using parameters as below. For trimming of reads, the ambiguous limit was two, the
minimum number of nucleotides in reads was 25, and the quality limit was 0.05. For reads
mapping, the number of mismatch was two, number of insertion was three, number of deletion
was three, and number of color error was three. The RPKM value was used to represent the
expression level for each gene.
assembled and mapped with CLC Genomic Workbench software (ver. 8.5.1, CLC Bio, Aarhus,
Denmark) using Bos_taurus UMD3.1 (National Center for Biotechnology Information [NCBI]
assembly accession GCA_000003055.3) as a reference sequence. RNA-Seq analysis was carried
out using parameters as below. For trimming of reads, the ambiguous limit was two, the
minimum number of nucleotides in reads was 25, and the quality limit was 0.05. For reads
mapping, the number of mismatch was two, number of insertion was three, number of deletion
was three, and number of color error was three. The RPKM value was used to represent the
expression level for each gene.
5
Filtering Genetic Variants in PAX2
6
Sequencing of Viral Genomes from Insect Hosts
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H. magnanima female adults of the male-killing strain (hereinafter referred to as the late strain) were homogenized in PBS as above and passed through a 0.45 μm filter. The filtrates were treated with RNase A and DNase I as described previously (Fujita et al., 2016 (link)); then RNA was extracted using Isogen II (Nippon Gene). Construction of a cDNA library and sequencing analysis was carried out according to the method described in Fujita et al. (2016 (link)). Reads were analyzed with CLC Genomic Workbench software (CLC bio) or the SPAdes algorithm (Bankevich et al., 2012 (link)). Complete OGV genomic sequences were determined by the FLDS sequencing method as described previously (Urayama et al., 2016 (link)). Complete OGV nucleotide sequences were submitted to the DDBJ/GenBank/EMBL database under accession numbers LC383810 -LC383814 and LC597875 -LC597896 .
7
Plasmid Screening for Cry1 Genes in Bacillus Strains
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In total, a set of 406 complete plasmids from Bacillus thuringiensis and other Bacillus cereus group members deposited in GenBank were screened for the presence of cry1 genes using the B. thuringiensis Toxin_scanner (Ye etal. 2012 (link)) (supplementary table S1 , Supplementary Material online). A genetic environment of cry1 was visualized with Easyfig tool (Sullivan etal. 2011 (link)). In addition, ISfinder (Siguier etal. 2006 (link)) and PHASTER (Arndt etal. 2016 (link)) website tools were used in order to identify mobile elements and prophage regions, respectively. DNA sequence alignment and analysis were performed with CLC Genomic Workbench software (CLC Bio), and Blast2GO software was used for functional annotation of proteins (Conesa etal. 2005 (link)).
8
Genome-Wide Identification of Inactivating Insertions
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FASTQ data files were analyzed using CLC Genomic Workbench software version 7.0.4 (CLC Bio) according to a previously described method (Carette et al., 2011a (link), b (link)). Briefly, after quality trimming and removal of the common LTR sequence, the 50–base pair reads were mapped onto the human genome (hg19). To exclude ambiguous alignments, mismatch reads were not allowed, and all nonspecific matched reads were ignored. To eliminate PCR amplification bias and determine the unique insertion sites, duplicate reads were removed and counted as one read (a unique insertion site). The independent insertion sites were further classified as being in the sense or antisense orientation compared with the gene. The total number of inactivating insertions, which consisted of all the sense or antisense orientations in the exons of the genes and the number of inactivating insertions per individual gene, were counted. The amount of enrichment of a particular gene in the screen was calculated by comparing the selected with the unselected population. For each gene, a p value and a p value corrected for the false-discovery rate (FDR) were calculated by the one-sided Fisher exact test using R software. A bubble plot was created using R software.
9
Identifying Mutations in Bradyrhizobium Genomes
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Total DNA was extracted from a stationary-phase culture of Bradyrhizobium cells by using an illustra bacteria genomicPrep Mini Spin Kit (GE Healthcare UK Ltd., Buckinghamshire, UK) and was processed using a Nextera DNA Sample Preparation Kit (Illumina, San Diego, CA, USA) to generate a shotgun library with unique index adapters. The library from each Bradyrhizobium strain was sequenced on a MiSeq system (Illumina), yielding ~250 bp paired-end reads. The raw reads from each Bradyrhizobium genome were trimmed and assembled de novo in CLC Genomic Workbench software (CLC bio, Inc., Aarhus, Denmark). The mutations in spontaneous mutants were identified by comparing the obtained MiSeq short reads and the reference B. diazoefficiens USDA 122 genome sequence (DDBJ/EMBL/GenBank accession number CP013127) using the “missing k-mer” tool of ShortReadManager53 (link),54 (link).
10
Genomic DNA Sequencing of H. formosensis
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Genomic DNA from strains of interest was purified using a Monarch Genomic DNA Purification Kit (NEB) from an overnight LB culture. Afterwards, 1 μg of DNA was used for library preparation using the NEBNext Ultra™ II DNA Library Prep Kit for Illumina (NEB). The library was evaluated by qPCR using the KAPA library quantification kit (Peqlab, Erlangen, Germany). Afterwards, normalization for pooling was done and paired‐end sequencing with a read length of 2 × 150 bases was performed on a MiSeq (Illumina). The reads of demultiplexed fastq files as the sequencing output (base calls) were trimmed and quality‐filtered using the CLC Genomic Workbench software (Qiagen Aarhus A/S, Aarhus, Denmark). Then, the filtered reads were used for de novo assembly using the CLC Genomic Workbench software. Sequencing data are stored in the NCBI Sequence Read Archive under the BioProject number PRJNA987411 under accession number SRR25019772. The annotated genome of H. formosensis FZJ can be accessed via the accession number JAVRDO000000000.
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