Zymobiomics 96 magbead dna kit

DNA was extracted from 100 mg of cecal contents using the ZymoBIOMICS 96 Magbead DNA Kit from Zymo Research. The sequencing library preparation was performed by following the protocol published by Illumina (document number 15044223, Rev. B). Briefly, the variable regions V3-V4 of the bacterial 16S rRNA gene were targeted by primers designed by Klindworth et al. [38 (link)]. In a two-step protocol, the first round of PCR amplified the target and added Illumina sequencing adapters. Nextera XT set A barcodes were added during the second round of PCR. KAPA HiFi DNA polymerase was used for all amplification reactions. Sequencing was performed on an Illumina MiSeq, using the v3 600-cycle reagent kit to produce 2 × 300 bp paired-end reads. A DNA extraction negative control and mock community DNA (ATCC MSA-1002) were sequenced along with the sample libraries for quality control purposes.

Following the manufacturer’s protocols, tissue samples (including fish stomach and mussel tissue) were extracted with the Zymo Research ZymoBIOMICS 96 MagBead DNA Kit (San Diego, CA). Random samples of DNA extracts were analyzed on an Agilent 2100 Bioanalyzer using a high-sensitivity assay kit. Fragments in the target amplicon range were apparent (albeit not known to be of mussel origin). All samples were stored at − 20 °C until PCR was performed. DNA from both the T. cylindrica and L. complanata samples were analyzed for DNA quality.

Fecal microbiota was analyzed by 16S rRNA V4 sequencing methodology. In brief, total fecal DNA was extracted using ZymoBIOMICS™ 96 MagBead DNA kit (Zymo Research, Irvine, CA) with automated epMotion (Eppendorf, Hamburg, Germany) robotic system. Mixed template amplicon library was prepared according to the protocol from Earth Microbiome Project (http://www.earthmicro biome.org/emp-standard-protocols/) form extracted fecal total DNA using the primer sets (515 F and barcoded 806 R)^{60 (link)}. The PCR master mix, primer, and samples were plated in triplicate using automated epMotion robotic system (Eppendorf, Hamburg, Germany). The PCR composition and the reaction cycle for the amplicon library preparation has been previously described^{31 (link)}. Amplicon DNA was multiplexed and sequenced using the Illumina MiSEQ platform with 2 × 250 bp paired-end sequencing. Obtained sequence data were de-multiplexed and analyzed using the open-source software QIIME2-DADA2 pipeline²⁵ . Taxonomy was assigned using the SILVA 132 reference database^{61 (link)} customized for QIIME2 for 16S  V4 (515 F/806 R) region of sequences at the threshold of 99% pairwise identity. A detailed analysis has been described in the supplemental methodology.

Fecal material was collected from Pancreatic cancer model mice cohort and healthy mice cohort at Mayo Clinic Jacksonville, FL. Genomic DNA was isolated from the cancer and control fecal material according to the manufacturer’s instructions using the ZymoBIOMICS^®-96 MagBead DNA Kit (Zymo Research, Irvine, CA). The DNA was run on a 1% agarose gel to confirm the successful extraction and concentration measured using a Nanodrop instrument (VWR, USA).

The samples used in this study were analyzed using the ZymoBIOMICS^® service performed by Zymo Research (Irvine, CA, United States). DNA was extracted from samples collected and processed with the ZymoBIOMICS™ Service - Targeted Metagenomic Sequencing (Zymo Research, Irvine, CA, United States). The ZymoBIOMICS^®-96 MagBead DNA Kit (Zymo Research, Irvine, CA, United States) was used to extract DNA.