DOI: 10.5281/zenodo.17716312
title: WildR F7 murine gut microbiome metagenome-assembled genomes

description: Due to limitations in current experimental approaches, the physiological role of microbial interactions in a mammalian gut microbiome remains poorly understood. Thus, to explore the consequences of bacterial antagonism in this environment, we characterized a wild murine gut microbiome, WildR. This microbiome was initially obtained from wild mice and reconstituted in germ-free laboratory mice in 2017 (Rosshart, et al. 2017 Cell. PMID: 29056339). To identify the speices present in this community, which has since been propogated for multiple generations, we performed long and short-read metagenomic sequencing and assembled metagenome-assembled genomes using a hybrid assembly approach of the current F7 generation. These MAGs facilitated identification of the community composition along with systems involved in microbial interactions. 

MAG sequences are uploaded as fasta files or with annotations from Prokka as genbank files. 



Fasta files containing contigs for each genome: WildR-MAG-fasta.tar.gz (0.25 GB)

Prokka-annotated MAGs: WildR-MAG-prokka-genbank.tar.gz (0.45 GB)


Assembly metrics and taxonomic annotations for each MAG are also uploaded. 



Assembly metrics: Table1-WildR-MAG-metrics.xlsx (50 KB)

Taxonomic annotation: Table1-WildR-MAG-metrics.tsv (230 KB)
description: Metagenomic sequencing of the WildR community 

For shotgun sequencing on the Illumina platform, genomic DNA was extracted from two aliquots of the propagated, cryopreserved WildR stock (~250 mg of pelleted cells) and fecal pellets (~50 mg) from two of the six mice inoculated with the Taconic WildR material using the MoBio PowerFecal DNA Isolation kit (Catalog #12830-50, MoBio Laboratories, California, USA). Sequencing libraries were prepared using an in-house protocol (Salipante et al. 2014) and sequenced on the Illumina NextSeq using the NextSeq 500/550 Highoutput kit v2.5 300 cycles (Cat # 20024908). Samples were processed in technical duplicates to correct for PCR errors.

 For long-read sequencing (Oxford Nanopore MinION), high-molecular weight (HMW) gDNA was extracted from ~500 µL of the WildR cryopreserved stock using the Qiagen Genomic-tip 20/G (Cat#13323) following the protocol for bacterial DNA extraction, except 5 µL of Qiagen lytic enzyme (catalog #158928) and 10 µL of MetaPolyzme (Millipore Sigma MAC4L-5MG resuspended in 750 µL of water) were added instead of lysosome for more efficient enzymatic lysis of diverse bacteria in the community (Maghini et al. 2021). HMW DNA was then cleaned and concentrated with an isopropanol precipitation as described and eluted in 100 µL of water as previously described (Maghini et al. 2021). DNA was incubated at 55 ˚C for 1 hr before placing at 4 ˚C for 3 days to increase resuspension of HMW gDNA fragments. Quality and quantity of HMW gDNA were assessed by NanoDrop UV-Vis spectroscopy and Qubit dsDNA HS fluorimetry assay (Cat# Q33231), respectively, and the presence of fragments >10 kb was confirmed by agarose gel electrophoresis. Libraries for sequencing using the Oxford Nanopore MinION were prepared using the Oxford Nanopore SQK-LSK110 kit and 902 ng of WildR HMW gDNA as the starting material. The sequencing library was loaded onto a R9.4.1 SpotON flow cell and run for 74 hrs on the Mk1C until ~50 pores remained. The run resulted in 22 Gb of sequencing data from 8.6 million reads with an N50 of 5.3 kb. Raw reads have been deposited with links to BioProject PRJNA1367654 in the NCBI database. 

 

Metagenome assembly

WildR metagenome-assembled genomes (MAGs) were generated from Illumina and MinION reads using the MUFFIN 1.0.6 pipeline ((Van Damme et al. 2021); https://github.com/RVanDamme/MUFFIN). Briefly, short and long reads were filtered for high-quality before performing hybrid assembly with MetaFlye (version 2.7; https://github.com/mikolmogorov/Flye). Instead of the default polishing step using Pilon, we modified the MUFFIN pipeline to reduce error by using Polypolish for short read polishing of our assemblies ((Wick and Holt 2022); https://github.com/rrwick/Polypolish). Binning was performed with Concot (version 1.1.0) (Alneberg et al. 2014), Metabat2 (version 2.13) (Kang et al. 2019), and Maxbin2 (version 2.2.7, https://sourceforge.net/projects/maxbin2/), and refined by metawrap (version 1.2.2) (Uritskiy et al. 2018). Hybrid reassembly was then performed with Unicycler (version 0.4.7) (Wick et al. 2017). Bin quality was determined using CheckM (version 1.0.13) (Parks et al. 2015) and taxonomic classification was performed with GTDB-tk (version 2.1.0) (Chaumeil et al. 2019) . Finally, all genomes were annotated with Prokka (version 1.12) (Seemann 2014).