Miseq 2500

The DNA of samples was extracted using the MOBIO PowerSoil^® DNA Isolation Kit. The genomic DNA was amplified by PCR using the 16S rRNA gene V4 region primers (515F and 806R), barcode-specific primers, and Ta KaRa Premix Taq^® Version 2.0 [32 (link)]. The PCR conditions were as follows: denaturation at 94°C for 5 min, followed by 30 cycles (94 °C 30 s, 52 °C for 30 s, 72 °C for 45 s), and finally, an extension at 72°C for 10 min. The amplified product was verified by 1.0% agarose gel electrophoresis. The PCR (polymerase chain reaction) amplification product was further purified using a purification kit, and the PCR mixed product was recovered using an EZNA Gel Extraction Kit (Omega, Norcross, Georgia, USA). Finally, the Illumina MiSeq2500 (Guangzhou Magigene Biotechnology Co. Ltd., Guangzhou, China) platform was used for sequencing and microbial community analysis. The bacterial sequencing data were uploaded into the Sequence Read Archive (SRA) of NCBI (National Center for Biotechnology Information), and can be accessed through accession number PRJNA642756.

Microbial genomic DNA from colonic digesta was extracted and amplified using specific primers with barcodes (16S V3 + V4). Amplicon libraries were sequenced on the Illumina MiSeq 2500 platform (Illumina, San Diego, CA, USA) for paired-end reads. Clustering of the highest quality sequences with a 97% match resulted in the production of operational taxonomic units (OTUs) by using Uparse V.7.0 in QIIME V.1.8 (http://qiime.org/, accessed on 23 December 2020). The 16S rRNA Silva database was employed to assign taxonomy to OTUs, based on the ribosomal database project (RDP). OUT-based functional predictions were conducted using FAPROTAX. The correlation between femur and tibia bone density and the colonic microbial profile was conducted using Spearman’s correlation analysis. Visualizations were constructed using R (Version 2.15.3).

Genomic DNA of ZGZA7-10 strain was extracted using a Maxwell^® DNA Cell kit in a Maxwell^® 16 Research System instrument (Maxwell, Promega, Madison, WI, USA). Sequencing was performed on an Illumina MiSeq 2500 (Illumina, San Diego, CA, USA) at IGA Technology Services (IGA Technology Services Srl, Udine, Italy) using a paired-end approach, as described in Banić et al. [31 (link)]. Contigs were classified as belonging to E. faecium ZGZA7-10 when obtaining the best BLASTn v2.2.27 hit (45) in the NCBI nt database, where the whole genome sequence was submitted. The genome of ZGZA7-10 was uploaded to the web annotation service Rapid Annotations using Subsystems Technology (RAST; http://rast.nmpdr.org/rast.cgi accessed on: 25 November 2021) for automated annotation of sequenced genes, followed by manual scanning. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JAJAGE000000000 (BioProject PRJNA388578, Biosample SAMN22155556). This version of the project (01) consists of sequences JAJAGE010000001-JAJAGE010000158. A circular map of the ZGZA7-10 genome was created using the PATRIC.

Total bacterial DNA was extracted from fecal samples using the DNA extraction kit (TIANGEN, China). For analysis of the taxonomic composition of gut microbiota, the hypervariable regions V3-V4 region of 16S rRNA genes was pooled and sequenced at the Illumina MiSeq 2500 platform (Illumina MiSeq, USA). Reads were trimmed and classified using QIIME (V 1.8.0).^{46 (link)} After quality filtering and chimera removal, these sequences were assigned to operational taxonomic units (OTUs) with ≥ 97% similarity using UPARSE^{47 (link)} and RDP classifier^{48 (link)} was used to classify OTUs at a given taxonomic rank. The gut microbial signature was identified using selbal,^{49 (link)} a greedy stepwise algorithm for balance selection, and unsupervised RandomForest classification analysis.

Tumors and adjacent full-thickness colonic tissues (n = 21 pairs) were collected in an Eppendorf microcentrifuge tube containing RNAlater and homogenized with a pestle. Chloroform was added, and the samples were centrifuged at 12,000× g for 15 min. The aqueous layer was subjected to further purification using an RNAEasy Mini Kit (Qiagen, Germantown, MD, USA), and RNA integrity values were assessed using a Bioanalyzer (Agilent, Santa Clara, CA, USA). Samples were further selected for transcripts that were poly-adenylated, and cDNA libraries were sequenced on a MiSeq2500 (Illumina, San Diego, CA, USA) sequencer as described previously [30 (link),31 (link),32 (link)]. Paired-end reads were generated at a read length of 100 nucleotides. Read quality was assessed with the program FastQC (v11.8, Babraham Bioinformatics, Cambridge, UK), all Phred scores were above 30, and GC bias was not detected in the samples. An average of 32 million reads per sample were aligned to the human genome (build hg38) using STAR version 2.7.3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) with default parameters [33 (link)]. Aligned reads were counted using HTSeq [34 (link)]. Samples contained an average of 88% uniquely mapped reads. Quality assessment of the RNA-seq libraries is presented in Supplementary Figure S2.

A stratified salt crust ~ 14 cm thick was sampled from the surface of the Qi Jiao Jing (QJJ) Lake in Xinjiang Province of China (91.5881°E, 43.3806°N), in September 2019. The crust was characterized by distinct colored layers due to photosynthetic pigments and different mineral phases, and the bottom layer was at the interface with the underlying water (Additional file 2: Fig. S6). The salt crust was dissected into eight distinctive layers based on differences in color (QJJ1-8), and one sample from the underlying salt water was also collected (QJJ9). All nine samples were placed into 15 ml sterile tubes and stored in liquid nitrogen during shipment to the lab. DNA was extracted within 48 h as described previously [45 (link)]. The standard primer 515F-806R for the V4 region of the 16S rRNA gene was used for DNA amplification [46 (link), 47 (link)]. High-throughput sequencing was performed on an Illumina MiSeq 2500 platform to generate 250 bp paired-end reads. Separately, a library with an insert size of ~ 400 bp was constructed from the total genomic DNA and was sequenced using the Illumina HiSeq 2500 instrument, generating ~ 36 Gbp (2 × 150 bp) raw data for each sample.