For each reaction, ~1 µg of either input cDNA or captured cDNA was converted into a SMRTbell library using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences) and sequenced on either a PacBio RSII or Sequel I (Pacific Biosciences).
Sequel 1
Manufactured by Pacific Biosciences
Sourced in United States
The Sequel I is a single-molecule real-time (SMRT) sequencing system developed by Pacific Biosciences. It is designed to perform high-throughput DNA and RNA sequencing. The Sequel I utilizes PacBio's proprietary SMRT technology to generate long read lengths and high-quality genomic data.
Automatically generated - may contain errors
5 protocols using sequel 1
1
PacBio RSII/Sequel I cDNA Sequencing
2
Whole Genome Sequencing of E. coli 550
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The E. coli strain designated as 550 was subjected to whole genome DNA extraction using the NucleoSpin Microbial DNA kit (Macherey-Nagel, Duren, Germany). The obtained DNA was sheared using Megaruptor 2 using the Hydropore-long (Diagenode). Library preparation of the sheared DNA was performed in accordance with the manufacturer’s recommendation for microbial multiplexing for the Express kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). No size selection was performed during library preparation. The constructed library was sequenced using long-reads sequencing technology on Sequel I (Pacific Biosciences, Menlo Park, CA, USA).
3
Long-read Sequencing of E. coli Strains
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The E. coli strain designated as C7 and C9-3 were subjected to DNA extraction using the Macherey-Nagel™ NucleoSpin™ Tissue kit (Carlo Erba Reagents, Milan, Italy). The extracted DNA was fragmented with Megaruptor 2 using the Hydropore-long (Diagenode, Belgium). Libraries' preparation of the fragmented DNA was accomplished according to the manufacturer's recommendation for microbial multiplexing with the Express kit 2.0. No size selection was performed during library preparation. Constructed libraries were sequenced using long-read sequencing technology on Sequel I (Pacific Biosciences, Menlo Park, California, USA).
4
Phylogenetic Analysis of Bacterial Genomes
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The genomes of bacteria from which phages were isolated were sequenced with Sequel I technology (Pacific Biosciences, Menlo Park, USA) (SI file 1 text ). After genome assembly the quality was checked and 16S rRNA operons were retrieved using the MiGA online platform [64 (link)]. Additionally, the 16S rRNA gene from all the other bacterial strains was amplified and sequenced using the Sanger technology (see SI file 1 text ).
For phylogenetic analysis, a neighbor joining tree with Jukes-Cantor correction and a RAxML tree (version 8, [65 (link)]) were calculated using ARB [66 (link)]. The reference data set Ref132 was used, with the termini filter and Capnocytophaga as outgroup [67 (link)]. Afterwards, a consensus tree was calculated.
For phylogenetic analysis, a neighbor joining tree with Jukes-Cantor correction and a RAxML tree (version 8, [65 (link)]) were calculated using ARB [66 (link)]. The reference data set Ref132 was used, with the termini filter and Capnocytophaga as outgroup [67 (link)]. Afterwards, a consensus tree was calculated.
5
Long-read sequencing of bacterial genomes
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Bacteria were cultured at 28°C with shaking (225 rpm) for 24 h in nutrient broth (BD Biosciences, San Jose, CA, U.S.A.), and genomic DNA was isolated using the MasterPure gram-positive DNA purification kit (Lucigen, Middleton, WI, U.S.A.), according to manufacturer instructions. Ten-kilobase genomic libraries were prepared and size-selected as previously described (Booher et al. 2015) (link). The libraries were multiplexed on a SMRT cell (10, 24, or 48 libraries per cell) on a SEQUEL I or SEQUEL II machine (Pacific Biosciences, Menlo Park, CA, U.S.A.) at the Icahn School of Medicine at Mt. Sinai (New York). The genomes were assembled using the HGAP assembler version 4.0 or the PacBio Microbial Assembler and were annotated via the National Center for Biotechnology Information (NCBI) with the Prokaryotic Genome Annotation Pipeline (Chin et al. 2013; Li et al. 2021) (link).
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