# Full-Length 16S Sequencing Report

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#### Zymo Research Corporation

# 1 Results

Click on the links below to view the results from each analysis:

00…AllSamples.Bac16Sv19PB
NegativeControls.Bac16Sv19PB
PositiveControls.Bac16Sv19PB

# 2 Raw Data

Click on the link below to download the raw FASTQ files for the samples in this project:

https://zymo-microbiomics-service.s3.amazonaws.com/epiquest/epiquest_zr24059/EEAPKUPLYOBDFFZJ/rawdata/250912.FL16S/zr24059_rawdata_links.tsv

# 3 Materials and Methods

The samples were processed and analyzed with the ZymoBIOMICS® Targeted Sequencing Service (Zymo Research, Irvine, CA).

DNA Extraction: One of two different DNA extraction kits was used depending on the sample type and sample volume. In most cases, the ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research, Irvine, CA) was used to extract DNA using an automated platform. In some cases, ZymoBIOMICS® DNA Miniprep Kit (Zymo Research, Irvine, CA) was used for low biomass samples, such as skin swabs, as it permits for a lower elution volume, resulting in more concentrated DNA samples.

Targeted Library Preparation: The library of whole 16S sequencing was prepared by following the full-length 16S amplification protocol from PacBio (https://www.pacb.com/wp-content/uploads/Procedure-checklist-Amplification-of-bacterial-full-length-16S-rRNA-gene-with-barcoded-primers.pdf). In brief, the whole 16S gene was amplified using the 27f (AGRGTTYGATYMTGGCTCAG) and 1492r(RGYTACCTTGTTACGACTT) primers with barcodes and adapters. 2ng DNA was used as the PCR tempalte for each sample. The PCR was run with 25 cycles following the conditions mentioned in the protocol. After that, the PCR product of each reaction underwent clean up with Select-a-Size DNA Clean & Concentrator MagBead Kit (Zymo Research, Irvine, CA) keeping fragments >300bp. The library of each reaction is then quantified using NanoDrop and pooled together with equal DNA mass. The pooled library was prepared for PacBio sequencing using the SMRTbell® prep kit 3.0.

Control Samples: The ZymoBIOMICS® Microbial Community Standard (Zymo Research, Irvine, CA) was used as a positive control for each batch of DNA extraction. The ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was also used as a positive control for the library preparation process. Negative controls (i.e. blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process.

Sequencing: The library was sequenced on one 8M SMRT cell on the Sequel IIe system.

Bioinformatics Analysis: Unique amplicon sequences variants were inferred from raw reads using the DADA2 pipeline (Callahan et al, 2016). Potential sequencing errors and chimeric sequences were also removed with the Dada2 pipeline. Taxonomy assignment was performed using Uclust from Qiime v.1.9.1 with the Zymo Research Database, a 16S database that is internally designed and curated, as reference. Composition visualization, alpha-diversity, and beta-diversity analyses were performed with Qiime v.1.9.1 (Caporaso et al., 2010). If applicable, taxonomy that have significant abundance among different groups were identified by LEfSe (Segata et al., 2011) using default settings. Other analyses such as heatmaps, Taxa2ASV Deomposer, and PCoA plots were performed with internal scripts.

# 4 References

Callahan B.J., McMurdie P.J., Rosen M.J., Han A.W., Johnson A.J., Holmes S.P., (2016) DADA2: High resolution sample inference from Illumina amplicon data. Nat Methods 13(7):581-3.

Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K. et al. (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335-336.

Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett, W.S., and Huttenhower, C. (2011) Metagenomic biomarker discovery and explanation. Genome Biol 12: R60.