Computational protocol: Cultivation-Independent and Cultivation-Dependent Analysis of Microbes in the Shallow-Sea Hydrothermal System Off Kueishantao Island, Taiwan: Unmasking Heterotrophic Bacterial Diversity and Functional Capacity

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Protocol publication

[…] Whole genome sequencing of strain P5 was accomplished using a hybrid approach, combining Illumina short read data with PacBio long read data (). For PacBio sequencing, 5 μg of sample DNA was sheared to 10 Kb by a Covaris® g-TUBE® (Covaris, United States). A PacBio® SMRTbellTM Template Prep Kit (PacBio, United States) was used to construct the library. The sequencing primers were annealed using a PacBio DNA/Polymerase Kit (PacBio, United States) and polymerase combined with the SMRTbell templates. We obtained long read data from PacBio RS II PacBio RS II (PacBio, United States). The raw Illumina data were filtered by the FASTX-Toolkit to remove the adapters, N bases, and low-quality reads. The clean data were assembled using Velvet v1.2.03 with default parameters. The PacBio long reads were assembled by RS HGAP assembly 3. The complete genome was finally gap closed by Sanger sequencing. The final assembled genomes of P5 were automatically annotated and analyzed through the IMG/ER. The comparison and visualization of multiple genomes was conducted with BRIG (). […]

Pipeline specifications

Software tools FASTX-Toolkit, BRIG
Applications WGS analysis, Genome data visualization
Organisms Bacteria, Escherichia coli
Chemicals Carbon, Sulfur