Pacbio sequel 1

Manufactured by Pacific Biosciences

Sourced in Canada, United States

The PacBio Sequel I is a DNA sequencing platform that utilizes single-molecule, real-time (SMRT) technology. It is designed to generate long read lengths and high-quality genomic data.

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PacBio Sequel (25 citations) Sequel I (5 citations)

8 protocols using pacbio sequel 1

Sequencing High-Quality Passionfruit Genome

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Fresh developing fruits of “Rojo Pasión” were frozen in liquid nitrogen immediately after being sampled in Murcia, Spain. After being shipped to the Max Planck Institute for Plant Breeding Research (MPIPZ, Cologne, Germany), DNA was extracted from the mesocarp and exocarp of the fruits using the Plant DNA Kit of Macherey-Nagel™ to create a PacBio sequencing library. Meanwhile, fresh leaves were sampled from the parental cultivars (“Currot” and “Orange Red”) at the experimental field of CEBAS-CSIC in Murcia, Spain, and Illumina short-read libraries were prepared after DNA extraction using the Plant DNA Kit of Macherey-Nagel™.
All libraries were sequenced with the respective sequencing machines (Illumina HiSeq 3000 and PacBio Sequel I) at Max Planck Genome-centre Cologne (MP-GC), which led to 19.9-Gb long reads for “Rojo Pasión” (PacBio; Additional file 1: Fig. S2) and 15.7- and 16.2-Gb short reads for the parental cultivars (Illumina). Note that the parental WGS data were only used for haplotype validation and for sorting the individual chromosome assemblies to two sets of eight chromosomes to match the inheritance of the chromosomes.

Campoy J.A., Sun H., Goel M., Jiao W.B., Folz-Donahue K., Wang N., Rubio M., Liu C., Kukat C., Ruiz D., Huettel B, & Schneeberger K. (2020). Gamete binning: chromosome-level and haplotype-resolved genome assembly enabled by high-throughput single-cell sequencing of gamete genomes. Genome Biology, 21, 306.

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Large DNA Sequencing with PacBio Sequel I

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High-molecular-weight DNA (no shearing required) was purified with 0.6× AMPure PB beads (Beckman Coulter) and eluted in Elution Buffer (EB) buffer. Quantification was done on a Qubit (Thermo Fisher Scientific) by using the ‘Broad Range Kit’. A sequencing library was prepared by using the SMRTbell express template prep kit (PacBio) according to the ‘Procedure & Checklist - Preparing > 15 kb Libraries Using SMRTBell Template Preparation Kit’ (PacBio). Library fragments in the 15–50-kb range were size-selected on a Blue Pippin (Sage Science) by using a 0.75% dye-free agarose gel cassette according to the ‘0.75% DF Marker S1 high-pass 15–20 kb protocol’. Library quality was checked on a Fragment Analyzer (Agilent) by using the ‘High Sensitivity Large Fragment 50 kb Kit’. Final yield was measured on a Qubit by using the ‘High Sensitivity Kit’. The library was sequenced on a PacBio Sequel I (PacBio) by using the V2.1 Binding kit, Chemistry kit and three SMRT cells. The loading type was ‘diffusion’ with an on-plate concentration of 6 pM. There was no pre-extension, and data were collected as a 10-h movie.

Amorim M.J., Gansemans Y., Gomes S.I., Van Nieuwerburgh F, & Scott-Fordsmand J.J. (2021). Annelid genomes: Enchytraeus crypticus, a soil model for the innate (and primed) immune system. Lab Animal, 50(10), 285-294.

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PacBio Iso-seq Protocol Benchmarking

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PAIso-seq1 and PAIso-seq2 The PAIso-seq1 and PAIso-seq2 libraries were constructed following the PAIso-seq1 and PAIso-seq2 protocols starting with purified total RNA as described previously 27, 32 . The libraries were sequenced using PacBio Sequel I or Sequel II instruments at Annoroad.

Liu Y., Zhang Y., Nie H., Z L., Wang J., & Lu F. (2021). Re-polyadenylation occurs predominantly on maternal mRNA degradation intermediates during mammalian oocyte-to-embryo transition.

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PacBio Metagenomic DNA Sequencing

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Bacterial pellets were resuspended in 200 µL of 1× PBS and transferred to a 2-mL bead beating tube (Matrix E; MP Biomedicals). Proteinase K (20 µL; Qiagen) was added, and the cells homogenized (SPEX 1600 MiniG; 1 min; 1,500 Hz Fisher Scientific). DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer’s instructions (Qiagen). The DNA was quantified using the 1× dsDNA HS kit (ThermoFisher Scientific on a Qubit). DNA was prepped with the SMRTbell Template Prep Kit 2.0 (Pacific Biosciences) to make PacBio SMRTbell libraries with barcodes sourced from the Barcoded Overhang Adaptor Kit 8A and 8B (Pacific Biosciences). The sequencing primers were annealed and bound to Polymerase 3.0 using the Sequel Binding Kit 3.0 (Pacific Biosciences). The bound complex was purified and sequenced on a PacBio Sequel I using an SMRT Cell M1 v3 tray (Pacific Biosciences). The spike-in controls for each PacBio Sequel I run were from the Internal Control Kit 3.0 (Pacific Biosciences).

Cramer N.A., Socarras K.M., Earl J., Ehrlich G.D, & Marconi R.T. (2024). Borreliella burgdorferi factor H-binding proteins are not required for serum resistance and infection in mammals. Infection and Immunity, 92(3), e00529-23.

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E. coli Genome Sequencing Using PacBio SMRT

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To enable an accurate SNP analysis, a reference chromosome of an E. coli isolate from our collection (ampC_0069) was constructed using PacBio SMRT sequencing. For sequencing, genomic DNA (gDNA) was extracted using a bacterial gDNA isolation kit (Norgen Biotek). A single E. coli isolate was subjected to DNA shearing using Covaris g-TUBEs for 30 s at 11 000 r.p.m. Each DNA sample was separated into two aliquots. Size selection was performed using a 0.75 % agarose cassette and marker S1 on the BluePippin system (Sage Science) to obtain either 4–8 kb or 4–12 kb DNA fragments. This size selection was chosen to maintain all DNA fragments, including these originating from plasmids (data not used in this study). Library preparation was performed using the PacBio SMRTbell template prep kit 1.0 (Pacific Biosciences). For cost-effectiveness, samples were barcoded and pooled with other samples that are not relevant for this study. Sequencing was conducted using the PacBio Sequel I (Pacific Biosciences) on a Sequel SMRT Cell 1M v2 (Pacific Biosciences) with a movie time of 10 h (and 186 min pre-extension time). Subreads per sample were obtained by extracting the bam files using SMRT Link version 5.1.0.26412 (Pacific Biosciences).

Coolen J.P., den Drijver E.P., Verweij J.J., Schildkraut J.A., Neveling K., Melchers W.J., Kolwijck E., Wertheim H.F., Kluytmans J.A, & Huynen M.A. (2021). Genome-wide analysis in Escherichia coli unravels a high level of genetic homoplasy associated with cefotaxime resistance. Microbial Genomics, 7(4), 000556.

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Hybrid genome sequencing of BP625

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The complete BP625 genome was sequenced using both short- and long-read sequencing methods. Genomic DNA was extracted using the Genomic-tip 100/G and DNA buffer set (Qiagen, Hilden, Germany). For short-read sequencing, a genomic library was prepared using the TruSeq DNA PCR-Free Kit (Illumina, San Diego, CA), followed by sequencing on the Illumina NovaSeq 6000 platform, generating 150-bp paired-end reads. Conversely, the long-read library was constructed using the SMRTbell Template Prep kit (Pacific Biosciences, Menlo Park, CA), and single-molecule real-time sequencing was performed using PacBio Sequel I (Pacific Biosciences, Menlo Park, CA). The Illumina platform yielded 6,411,695 reads, whereas the PacBio platform generated 176,974 subreads. All library preparation and sequencing steps were performed by Macrogen Co. (Tokyo, Japan). Before the downstream analysis, the raw read data were filtered using fastp version 0.20.1 for short reads (length of 20 bp; sequencing quality score of 20) [20 (link)] and Filtlong version 0.2.0 (https://github.com/rrwick/Filtlong) for long reads (length of 1,000 bp; 10% of poor-quality reads discarded).

Koide K., Uchitani Y., Yamaguchi T., Otsuka N., Goto M., Kenri T, & Kamachi K. (2024). Whole-genome comparison of two same-genotype macrolide-resistant Bordetella pertussis isolates collected in Japan. PLOS ONE, 19(2), e0298147.

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PacBio SMRT Sequencing of Bacterial Genomic DNA

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For PacBio SMRT sequencing, genomic DNA (gDNA) was extracted using the Bacterial gDNA Isolation Kit (Norgen Biotek Corp., CAN, ON, Thorold). A single E. coli isolate was subjected for DNA shearing using Covaris g-TUBEs (Covaris Inc, US, MA, Woburn) for 30 seconds on 11,000 RPM (g). Each DNA sample was separated into two aliquots. Size selection was performed using a 0.75% agarose cassette and marker S1 on the BluePippin (Sage Science Inc, US, MA, Beverly) to obtain either 4-8 kb or 4-12 kb DNA fragments. This size selection was chosen to maintain all DNA fragments including these originating from plasmids (data not used in this study). Library preparation was performed using the SMRTbell Template prep kit 1.0 (Pacific Biosciences, US, CA, Menlo Park). For cost-effectiveness, samples were barcoded and pooled with other samples that are not relevant for this study. Sequencing was conducted using the PacBio Sequel I (Pacific Biosciences, US, CA, Menlo Park) on a Sequel SMRT Cell 1M v2 (Pacific Biosciences, US, CA, Menlo Park) with a movie time of 10 h (and 186 min pre-extension time). Subreads per sample were obtained by extracting the bam files using SMRT Link version 5.1.0.26412 (Pacific Biosciences, US, CA, Menlo Park).

Coolen J.P.M., Drijver E.d., Verweij J.J., Schildkraut J.A., Neveling K., Melchers W.J.G., Kolwijck E., Wertheim H., Kluytmans J., & Huynen M.A. (2020). Genome-wide analysis in Escherichia coli unravels an unprecedented level of genetic homoplasy associated with cefotaxime resistance.

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Full-length Transcriptome Profiling using PacBio Sequencing

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Colo320 cDNA was prepared using the SMARTer cDNA synthesis kit (Clonetech) and converted into a SMRTbell library using the SMRTbell Template Prep Kit 2.0 (Pacific Biosciences) according to the manufacturer's instructions. The samples were sequenced on a PacBio Sequel I (Pacific Biosciences). Obtained data were analyzed using the Iso-Seq3 in the PacBio SMRT Analysis (v. 6.0) as described elsewhere (25) . Briefly, circular consensus sequences with both 5 0 and 3 0 primers were regarded as full-length reads, and they were pooled for isoform-level clustering analysis. The Arrow algorithm in the SMRT Link software called high-quality sequences (predicted consensus accuracy ≥ 99%), which were then mapped to GRCh38.p13 using minimap2 (v. 2.1). The full-length isoforms were ultimately annotated and characterized using a SQANTI2 pipeline (26) . Library preparation and sequencing were performed by Macrogen (Korea).

Kikuchi Y., Tokita S., Hirama T., Kochin V., Nakatsugawa M., Shinkawa T., Hirohashi Y., Tsukahara T., Hata F., Takemasa I., Sato N., Kanaseki T, & Torigoe T. (2021). CD8⁺ T-cell Immune Surveillance against a Tumor Antigen Encoded by the Oncogenic Long Noncoding RNA PVT1. Cancer immunology research, 9(11).

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