WGS was conducted to investigate the genomic differences between CC17/III GBS isolates with different RFLP patterns and CRISPR1 locus. WGS was performed using both PacBioTM SMRT (Pacific Biosciences, Menlo Park, CA, USA) [41 (link)] and MiSeqTM (Illumina, San Diego, CA, USA) [42 (link)] sequencing technologies. The sequencing library was prepared using a TruSeq DNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) for the Illumina MiSeq system. Genomic libraries were generated using Nextera XT kits (Illumina, San Diego, CA, USA). All the sequencing processes were performed using a DNA-sequencing kit 4.02v2 (QIAGEN, Dusseldorf, Germany) and SMRT cell 8 Pac (QIAGEN, Dusseldorf, Germany). The circlator tool (v1.4.0) (Cambridge, UK) was used to correct and linearize the genome, and QUAST (v4.5) (San Diego, CA, USA) was applied to evaluate the assembled genome quality.
Pacbio smrt
Manufactured by Pacific Biosciences
Sourced in United States
The PacBio SMRT (Single Molecule, Real-Time) is a DNA sequencing platform that utilizes a unique technology to sequence individual DNA molecules in real-time. The system employs proprietary SMRT cells and a specialized polymerase enzyme to capture the process of DNA synthesis, allowing for the detection of DNA base incorporations as they occur.
Automatically generated - may contain errors
Lab products found in correlation
Truseq dna lt sample prep kit, by Illumina (3 mentions)
Nextera xt kit, by Illumina (3 mentions)
Dna sequencing kit 4.02v2, by Qiagen (2 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Miseq system, by Illumina (1 mentions)
Hiseq 2000, by Pacific Biosciences (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
9 protocols using pacbio smrt
1
Comparative Genomic Analysis of GBS
2
Genomic Characterization of Invasive GBS Strain N5
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An invasive III/ST-17 GBS strain N5 was arbitrarily chosen as the reference strain for genomic comparisons. The genome of N5 was sequenced by WGS, which was performed using both PacBioTM SMRT (Pacific Biosciences, Menlo Park, CA, USA) [23 (link)] and MiSeqTM (Illumina, San Diego, CA, USA) [24 (link)] sequencing technologies. The sequencing library was prepared using a TruSeq DNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) for the Illumina MiSeq system. Genomic libraries were generated using Nextera XT kits (Illumina, San Diego, CA, USA). The genome assembly was completely concordant with full-length perfectly aligning Illumina short reads. All the sequencing processes were performed using a DNA sequencing kit 4.02v2 (QIAGEN, Hilden, Germany) and SMRT cell 8 Pac (PacBio, Menlo Park, CA, USA). The Circlator tool (v1.4.0) was used to correct and linearize the genome, and QUAST (v4.5) was applied to evaluate the assembled genome quality. Other GBS strains including A28, N48, N96, P103 and P65 were used to generate contigs by the MiSeqTM sequencing method. All genome sequences were blasted against the NCBI genome database to search for possible plasmid sequences. After the de novo assembled genome was generated, Prokka (v1,12) [25 (link)] was used for genome annotation and identification of rRNA-encoding and tRNA-encoding regions.
3
Genomic DNA Extraction and Sequencing
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Total genomic DNA was extracted from the leaves of each clone using the DNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions for plant tissue. Sequencing of the ten clones was carried out using both 10X Genomics’ GemCode technology (https://www.10xgenomics.com/ ) and PacBio SMRT (Pacific Biosciences, Menlo Park, CA, USA) sequencing technology.
4
Complete Genome Sequencing of GBS Strains
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Three isolates were selected from the type Ib ST12 (termed N92) and type III ST-17 (termed N48, N96, and N5) GBS strains. We used the lysozyme-sodium dodecyl sulfate-proteinase K method to extract the DNA. WGS was performed using both PacBio SMRT (Pacific Biosciences, Menlo Park, CA, USA) (26 (link)) and MiSeq (Illumina, San Diego, CA, USA) (45 (link)) sequencing technologies. The sequencing library was prepared using a TruSeq DNA LT sample prep kit (Illumina, San Diego, CA, USA) for the Illumina MiSeq system. Genomic libraries were generated using Nextera XT kits (Illumina, San Diego, CA, USA). We used SPAdes (version 3.9.0) to assemble the sequence. All genome sequences were subjected to BLAST analysis using the NCBI genome database to identify possible plasmid sequences. After the de novo-assembled genome was generated, Prokka (version 1,12) (46 (link)) was used for genome annotation and the identification of rRNA-encoding and tRNA-encoding regions.
5
Phage DNA Extraction and Sequencing
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In this study, the saturated phenol–chloroform extraction method was used to extract phage DNA [17 (link)], and the phage genome was sequenced by PacBio SMRT (Pacific Biosciences, Menlo Park, CA, USA) platform in Anshan Biotechnology Co., Ltd. (Tianjin, China). Finally, the phage genome was assembled using Flye 2.8.3 [18 (link)].
6
Accurate Long-Read Error Correction with Hercules
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Hercules corrects errors (insertions, deletions, and substitutions) present in long read sequencing platforms such as PacBio SMRT (26 (link)) and Oxford Nanopore Technologies (8 (link)), using reads from a more accurate orthogonal sequencing platform, such as Illumina (27 (link)). We refer to reads from the former as ‘long reads’ and the latter as ‘short reads’ in the remainder of the paper. The algorithm starts with preprocessing the data and obtains the short-to-long read alignment. Then, for each long read, Hercules constructs a pHMM template using the error profile of the underlying platform as priors. It then uses the Forward–Backward algorithm to learn the posterior transition/emission probabilities, and finally, uses the Viterbi algorithm to decode the pHMM to output the corrected long read (Figure 1 ).
7
Fecal Microbiome Profiling via 16S rRNA Sequencing
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Fecal samples were collected from all 749 subjects who completed the questionnaire, and within 2 h, these samples were placed in a refrigerator at 4°C for temporary preservation. Within 24 h, the samples were stored at -80°C before analysis.
According to the instructions provided with the QIAamp DNA Stool Mini kit (QIAGEN, Hilden, Germany), total DNA were extracted from fecal samples. The V4 region of 16S rRNA gene was amplified by polymerase chain reaction (PCR) from the total isolated DNA samples. The amplification reaction conditions were as follows: initial activation step of 94°C for 2 min followed by 30 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 30 s and a final incubation at 72°C for 5 min. The V4 region of 16S rRNA was sequenced by second-generation sequencing (Illumina HiSeq 2000) in combination with third-generation sequencing (PacBio SMRT).
According to the instructions provided with the QIAamp DNA Stool Mini kit (QIAGEN, Hilden, Germany), total DNA were extracted from fecal samples. The V4 region of 16S rRNA gene was amplified by polymerase chain reaction (PCR) from the total isolated DNA samples. The amplification reaction conditions were as follows: initial activation step of 94°C for 2 min followed by 30 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 30 s and a final incubation at 72°C for 5 min. The V4 region of 16S rRNA was sequenced by second-generation sequencing (Illumina HiSeq 2000) in combination with third-generation sequencing (PacBio SMRT).
8
Rotifer Reference Genome Sequencing
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We selected one rotifer clone (called: OHJ7i3n10) as DNA donor for the reference genome This clone ultimately derives from the natural population since it is a descendant of clone OHJ7, which was hatched from sediments of Obere Halbjochlacke. However, its immediate ancestors were passed through three generations of selfing (i.e., mating one male and female of the same clone). More details on the genealogy of this lineage and its biological characteristics can be found in [7 (link)]. Using long-read sequencing technology (PacBio SMRT® on the Sequel-platform), we obtained 16.3 Gbp from two SMRTcells, which corresponds to a 57-fold coverage assuming a haploid genome size of 284 Mbp. Additionally, we obtained 35.5 Gbp of Iso-Seq transcriptome data and 12 Gbp of short-read Illumina data of OHJ7i3n10. All sequencing and library preps related to the reference genome were performed by the Next Generation Sequencing Facility at Vienna BioCenter Core Facilities (VBCF), a member of the Vienna BioCenter (VBC), Austria.
9
Rotifer Genome Sequencing Protocol
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We selected one rotifer clone (called: OHJ7i3n10) as DNA-donor for the reference genome. This clone ultimately derives from an ancestor of the natural OHJ population. However, its immediate ancestors were passed through three generations of selfing (i.e., mating one male and female of the same clone). More details on the genealogy of this lineage and its biological characteristics can be found in (Stelzer et al. 2019) . According to this study, OHJ7i3n10 has a 2C-genome size of 568 Mbp, and thus contains approximately 40% excess genomic sequences, compared to the smallest genome size of the OHJ-population (~410 Mbp).
Our B. asplanchnoidis reference genome is based on long-read sequencing technology (PacBio SMRT® on the Sequel-platform). In total, we obtained 16.3 Gbp from two SMRTcells, which confers to 57-fold coverage assuming a haploid genome size of 284 Mbp. Additionally, we obtained 35.5 Gbp of Iso-Seq transcriptome data and 12 Gbp of short-read Illumina data of OHJ7i3n10. All sequencing and library preps related to the reference genome were performed by the Next Generation Sequencing Facility at Vienna BioCenter Core Facilities (VBCF), member of the Vienna BioCenter (VBC), Austria.
Our B. asplanchnoidis reference genome is based on long-read sequencing technology (PacBio SMRT® on the Sequel-platform). In total, we obtained 16.3 Gbp from two SMRTcells, which confers to 57-fold coverage assuming a haploid genome size of 284 Mbp. Additionally, we obtained 35.5 Gbp of Iso-Seq transcriptome data and 12 Gbp of short-read Illumina data of OHJ7i3n10. All sequencing and library preps related to the reference genome were performed by the Next Generation Sequencing Facility at Vienna BioCenter Core Facilities (VBCF), member of the Vienna BioCenter (VBC), Austria.
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