The whole genome of MA was sequenced using Illumina Nova Seq PE150 and Pac Bio Sequel, and FM using Illumina Nova Seq PE150. All of the above were sequenced at the Beijing Novogene Bioinformatics Technology Co., Ltd., China.
Novaseq pe150
Manufactured by Illumina
Sourced in China, United States
The NovaSeq PE150 is a high-throughput sequencing system designed for large-scale genomic research projects. It can generate paired-end reads of up to 150 base pairs in length, enabling the sequencing of a wide range of sample types and applications.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (17 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (13 mentions)
Nebnext ultra dna library prep kit, by Illumina (5 mentions)
Nebnext ultra dna library prep kit, by New England Biolabs (4 mentions)
Promethion, by Illumina (3 mentions)
Promethion platform, by Illumina (3 mentions)
Agilent 2100, by Agilent Technologies (3 mentions)
Ultra dna library prep kit, by New England Biolabs (2 mentions)
Phage dna isolation kit, by Norgen Biotek (2 mentions)
Smarter stranded total rna seq kit v2 pico input mammalian, by Takara Bio (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Miniamp thermal cycler, by Thermo Fisher Scientific (2 mentions)
Dnasecure plant kit, by Tiangen Biotech (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Sureselectxt mouse methyl seq library prep kit, by Agilent Technologies (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Tianamp bacteria dna kit, by Illumina (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
141 protocols using novaseq pe150
1
Whole Genome Sequencing of MA and FM
2
Comparative Genomics of Bacterial Mutants
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Bacteria from a single colony were cultured at 37 °C in CAMHB broth and collected at OD600 of 0.4. The cells were collected and genomic DNA was extracted using TIANamp Bacteria DNA Kit. The whole genome of wild type and Mut-S were sequenced respectively using Illumina NovaSeq PE150 and PacBio Sequel platform by Beijing Novogene Bioinformatics Technology Co., Ltd. For Illumina NovaSeq PE150 data, SAMTOOLS software (V0.1.18, http://www.htslib.org/ )31 (link) was used to detect the individual SNPs and insertion and deletion of small fragments (<50 bp), as well as the variation analysis of SNP/InDel in the functional regions of the genome. The variation map of the whole genome was created by Circos (V0.64, http://circos.ca/ )32 (link) to show reads coverage and the distribution of SNP and InDel informations. The insertion (INS), deletion (DEL), inversion (INV), intra-chromosomal translocation (ITX), and inter-chromosomal translocation (CTX) between the mutant and wild type strains were analyzed by BreakDancer software (V1.4.4, http://breakdancer.sourceforge.net/ )33 (link). For PacBio Sequel data, Genome Assembled with SMRT Link software (V5.0.1, https://www.pacb.com/support/software-downloads/ ) was used. Genomic alignments were performed using the MUMmer (V3.23)34 (link) and LASTZtools (V1.03.54)27 ,35 . Genomic synteny was analyzed based on the alignment results.
3
Comprehensive Whole Genome Sequencing Approach
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PacBio Sequel and Illumina NovaSeq PE150 platforms were used for whole genome sequencing at the Beijing Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) Library for single-molecule real-time (SMRT) sequencing in the PacBio Sequel platform was constructed with an insert size of 20 kb using the SMRTbell™ Template kit version 1.0. The library quality was assessed by Qubit® 2.0 Fluorometer (Thermo Scientific) and the insert fragment size was detected by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Regarding the Illumina NovaSeq PE150 platform, sequencing library of 350 bp was generated from 1 μg DNA using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following manufacturer’s recommendation. The library was analyzed for size distribution by Agilent 2100 Bioanalyzer (Agilent Technologies).
After filtering low-quality reads (less than 500 bp), the clean data from the PacBio Sequel platform were preliminary assembled using SMRT Link version 5.0.1. The long reads were selected (more than 6 kb) as the seed sequence, and the other shorter reads were aligned to the seed sequence using BLASR version 5.3.5 [24 (link)]. Finally, the arrow algorithm was used to correct and count the variant sites in the initial genome sequence using the variant Caller module of the SMRT Link.
Regarding the Illumina NovaSeq PE150 platform, sequencing library of 350 bp was generated from 1 μg DNA using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following manufacturer’s recommendation. The library was analyzed for size distribution by Agilent 2100 Bioanalyzer (Agilent Technologies).
After filtering low-quality reads (less than 500 bp), the clean data from the PacBio Sequel platform were preliminary assembled using SMRT Link version 5.0.1. The long reads were selected (more than 6 kb) as the seed sequence, and the other shorter reads were aligned to the seed sequence using BLASR version 5.3.5 [24 (link)]. Finally, the arrow algorithm was used to correct and count the variant sites in the initial genome sequence using the variant Caller module of the SMRT Link.
4
Comprehensive Genome Sequencing of Streptococcus salivarius
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The genomic DNA was extracted using the SDS method (Wilson, 2001 (link)). Subsequently, the extracted DNA was subjected to agarose gel electrophoresis for detection, and its concentration was quantified using a Nanodrop spectrophotometer (Thermo Scientific). To sequence the entire genome of Streptococcus salivarius, a combination of the Nanopore PromethION platform and Illumina NovaSeq PE150 sequencing was employed. The sequencing services were provided by Beijing Novogene Bioinformatics Technology Co., Ltd. The obtained sequencing data, including the PE150 data and Nanopore data, were used for assembly. The software tool Unicycler (Wick et al., 2017 (link)) was utilized to perform the assembly process. Unicycler integrates the data from both sequencing platforms to generate a comprehensive assembly. Subsequently, the reads were compared to the assembled sequence, and the distribution of sequencing depth was analyzed. Based on the sequence length and alignment, the assembled sequence was evaluated to distinguish between a chromosomal sequence and a plasmid sequence. Additionally, the circularity of the assembled genome was checked.
5
Genomic DNA Extraction and Sequencing
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The genomic DNA of the isolates was extracted using TIANamp Bacteria DNA Kit (Beijing Tiangen, China) and then sequenced using Illumina NovaSeq PE150 at Beijing Novogene Bioinformatics Technology Co., Ltd. Sequencing libraries of ∼ 350 bp were prepared using NEBNext® Ultra™ DNA Library Prep Kits and analysed for size distribution using an Agilent 2100 Bioanalyzer and quantified using real-time polymerase chain reaction. SOAP denovo [23 (link)] was used to assemble paired reads. All of the genomic sequences are available at the National Center for Biotechnology Information (NCBI; accession nos. SAMN21988700, SAMN22062944-49). Further, as VFBJ05 and VFBJ07 were found to own transposon islands, the extracted DNA of them were further subjected to 250-bp paired-end whole-genome sequencing with 150× coverage using the Nanopore sequencer. The filtered subreads were assembled using Canu v1.5 [24 (link)], and then Circlator v1.5.5 (https://github.com/sanger-pathogens/circlator ) was used to cyclise the assembled genomes. Coding gene prediction was performed using Prodigal v2.6.3 [25 (link)]. Genome annotation was performed using the RAST server (https://rast.nmpdr.org/rast.cgi ). The whole-genome sequences of VFBJ05 and VFBJ07 are available at the NCBI (accession nos. SAMN35555507 and SAMN35555508).
6
Genome Sequencing and Phylogenetic Analysis of Bacterial Strain K1
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Genomic DNA of K1 was extracted using a bacterial genomic DNA isolation kit (Biotech Corporation, Beijing, China). The harvested DNA was detected via the agarose gel electrophoresis, and it was quantified using a Qubit® 2.0 Fluorometer (Thermo Scientific, USA). 16S rRNA was amplified using the universal primers 27F and 1492R as previously described (Gao et al., 2017 (link)). The 16S rRNA sequence was blasted at NCBI (https://www.ncbi.nlm.nih.gov ), and sequences of related strains were downloaded. A phylogenetic tree was then built according to the neighbor-joining method using Mega-X software (Kumar et al., 2018 (link)). The neighbor-joining tree was constructed based on bootstrap values with 1,000 replications. To further distinguish the K1 strain, the whole genome of K1 was sequenced using the Nanopore PromethION platform and Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd.
A total of 5,378,358,199-bp paired end reads were generated. These reads were assembled using Unicycler v0.4.7 (https://github.com/rrwick/Unicycler , Wick et al., 2017 (link)). K1 genome sequences were uploaded to the Type Strain Genome Server (TYGS) for genome-based taxonomic classification (Meier-kolthoff and Göker, 2019 (link)). The genome sequences of strain K1 were submitted to NCBI and assigned an accession number (CP093546 ).
A total of 5,378,358,199-bp paired end reads were generated. These reads were assembled using Unicycler v0.4.7 (
7
Genomic DNA Sequencing and Assembly
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Genomic DNA extracts of BMU 00512 and BMU 04928 were sequenced with 1 μg DNA per sample as input material with an Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., using paired-end and mate-paired libraries. Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, U.S.A.) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Quality control of the reads was performed using FastQC v0.11.81 and PCR adapter reads and low-quality sequences were removed by BBMap2. High-quality reads were de novo assembled using SOAP (16 (link), 17 (link)), SPAdes v3.10.0 (18 (link)) and ABySS (19 (link)) with default parameters for Illumina paired-end reads. The completeness of the genome assemblies was accessed by quantifying the presence of the Core Eukaryotic Genes (CEGs) via CEGMA (20 (link)).
8
Whole Genome Sequencing and Assembly of P. polymyxa SF05
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Genomic DNA was extracted with the SDS method [21 (link)]. The harvested DNA was detected by the agarose gel electrophoresis and quantified by Qubit® 2.0 (Invitrogen, Thermo Fisher Scientific, Hillsboro, OR, USA). The whole genome of P. polymyxa strain SF05 was sequenced using the Nanopore PromethION platform and Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Unicycler was used to combine PE150 data and Nanopore data which were used to assemble then compare the readings to the assembled sequence, count the distribution of sequencing depth, and distinguish whether the assembled sequence was a chromosomal sequence or a plasmid sequence according to sequence length and alignment, and check whether it was a circular genome. The genes of the genome were predicted by the Pan-genome analysis pipeline (PAGP) [22 (link)].
9
RNA-seq analysis of BMDM and PM
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The integrity of RNA purified from indicated BMDM or PM samples was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). All samples showed an RNA integrity number > 8. RNA sequencing libraries were generated using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB), and sequenced on an Illumina Novaseq PE150 platform. Sequencing was performed at Genewiz Co. Ltd (Suzhou, China). The filtered reads were mapped to the mouse genome reference sequence (GRCm38/mm10 Ensembl release 81) using HISAT2. Gene expression was quantified as fragments per kilobase of coding sequence per million reads (FPKM) algorithm. Genes were ranked by log2 of fold change (log2FC), and −log10 of false discovery rate (−log10 (FDR)). For the gene ontology analysis, top upregulated genes were uploaded to DAVID Bioinformatics Resources 6.8 to perform gene ontology (GO) analysis. The top enriched items (p < 0.05) were shown in the figure, as described in the legend of Figure 4.
10
Genomic Analysis of P4R5 Yeast
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The genomic DNA of P4R5 was extracted and purified using the TIANamp yeast DNA kit (TIANGEN, Beijing, China). Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s protocol. The whole genome of P4R5 was sequenced using Illumina NovaSeq PE150 and assembled with SOAP de novo software at the Beijing Novogene Bioinformatics Technology Co., Ltd. The coding genes were predicted using the Augustus 2.7 program, and the gene function was predicted based on the Non-Redundant Protein Database [45 (link)] and the Kyoto Encyclopedia of Genes and Genomes database. The genome information of P4R5 has been deposited to GenBank under the accession number ID: JAHERV000000000.
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