Fungal Community Profiling using PacBio CCS

A DNA library was prepared using the DNA Template Prep kit 2.0 (Pacific Biosciences) according to the manufacturer's instructions. Sequencing was performed with the PacBio RS II system using the DNA Sequencing Kit C2 (Pacific Biosciences) with P4 polymerase. Circular Consensus Sequence (CCS) constructed from more than eight full-pass subreads were produced using PacBio SMRT Analysis, and then primer sequences were removed using the FASTX-Toolkit (http://bbmap.sourceforge.net/). For fungal analyses, 2202 reads in average in 1 sample were generated. Sequences were clustered into OTUs, defined at 95% similarity using UCLUST version 1.2.22q (http://sco.h-its.org/exelixis/web/software/pear/). Representative sequences for each OTU were classified taxonomically using RDP Classifier version 2.2 with the ntF-ITS1 database.^{16 (link)} The Mann–Whitney U test and the Fisher’s probability test were used to compare the relative abundance and detection ratio for statistical analyses, respectively.

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Pareek S., Kurakawa T., Das B., Motooka D., Nakaya S., Rongsen-Chandola T., Goyal N., Kayama H., Dodd D., Okumura R., Maeda Y., Fujimoto K., Nii T., Ogawa T., Iida T., Bhandari N., Kida T., Nakamura S., Nair G.B, & Takeda K. (2019). Comparison of Japanese and Indian intestinal microbiota shows diet-dependent interaction between bacteria and fungi. NPJ Biofilms and Microbiomes, 5, 37.

Publication 2019

DNA Template Prep kit 2.0 DNA Sequencing Kit C2

A dna Consensus sequence Library Pear Primer Smrt

Corresponding Organization :

Other organizations : Osaka University, Translational Health Science and Technology Institute, Shimadzu (Japan), Society for Applied Studies, Stanford University, Japan Agency for Medical Research and Development

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Variable analysis

independent variables

DNA library preparation using the DNA Template Prep kit 2.0 (Pacific Biosciences)
Sequencing with the PacBio RS II system using the DNA Sequencing Kit C2 (Pacific Biosciences) with P4 polymerase
Primer sequence removal using the FASTX-Toolkit

dependent variables

Circular Consensus Sequence (CCS) constructed from more than eight full-pass subreads
Fungal read counts (average of 2202 reads per sample)

control variables

Manufacturer's instructions for DNA library preparation
UCLUST version 1.2.22q for OTU clustering at 95% similarity
RDP Classifier version 2.2 with the ntF-ITS1 database for taxonomic classification

controls

No positive or negative controls were explicitly mentioned.

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