Fecal microbiota transplantation in treating methylmercury-poisoned rats: Reconstruction of gut microbiome and modulation of gut-brain axis related metabolites
Yanfei Zhang1#, Hongxin Xie2#, Xiaoying Lin1*, Xue Tian2, Piaoxue Ao2, Liwei Cui3, Yan Xin2, Ming Yin2, Nianqiu Shi1*, Bai Li2, Yuxi Gao2,3, Lihai Shang4, Yu-Feng Li2,3*
1 Jilin Medical University, Jilin 132013, Jilin, China
2 CAS-HKU Joint Laboratory of Metallomics on Health and Environment, & Beijing Metallomics Facility, & National Consortium for Excellence in Metallomics, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China
3 University of Chinese Academy of Sciences, Beijing 100049, China
4 State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, Guiyang 550002, China
*Corresponding authors: 
Yu-Feng Li, E-mail: liyf@ihep.ac.cn, Tel: +86-10-88233908; Xiaoying Lin, E-mail: linxytime@163.com, Tel: +86-15110115663. Nianqiu Shi, E-mail: shinianqiu2009@163.com
# These authors contribute equally to this work. 

2.1 Materials and methods
2.1.1 16SrRNA 
 Sequencing
（1）Extraction of genome DNA 
Total genome DNA from samples was extracted using CTAB/SDS method. DNA concentration and purity was monitored on 1% agarosegels. According to the concentration, DNA was diluted to 1ng/μl using sterile water. 
（2）Amplicon Generation 
Primer：16S V3-V4: 341F-806R, 18S V9: 1380F-1510R, ITS1: ITS1F- ITS2R. 16S /18S rRNA genes were amplified used the specific primer with the barcode. All PCR reactions were carried out in 30μL reactions with 15μL of Phusion®High-Fidelity PCR Master Mix (New England Biolabs); 0.2μM of forward and reverse primers, and about 10 ng template DNA.Thermal cycling consisted of initial denaturation at 98 ℃ for 1 min, followed by 30 cycles of denaturation at 98℃ for 10 s, annealing at 50 ℃ for 30 s, and elongation at 72 ℃ for 60 s. Finally 72 ℃ for 5 min. 
（3）PCR Products quantification and qualification 
Mix same volume of 1X loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. Samples with bright main strip between 400-450bp were chosen for further experiments. 
（4）PCR Products Mixing and Purification 
PCR products was mixed in equidensity ratios. Then, mixture PCR products was purified with AxyPrepDNA Gel Extraction Kit (AXYGEN). 
（5）Library preparation and sequencing 
Sequencing libraries were generated using NEB Next®Ultra™DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina Miseq/HiSeq2500 platform and 250bp/300bp paired-end reads were generated.
Data analysis 
（1）Paired-end reads assemblies 
Paired-end reads from the original DNA fragments were merged using FLASH, a very fast and accurate analysis tool, which was designed to merge paired-end reads when at least some of the reads overlap the read generated from the opposite end of the same DNA fragment. Paired-end reads was assigned to each sample according to the unique barcodes. 
（2）OTU cluster and Species annotation 
Sequences analysis were performed by UPARSE software package using the UPARSE-OTU and UPARSE-OTUref algorithms. In-house Perl scripts were used to analyze alpha (within samples) and beta (among samples) diversity. Sequences with ≥97% similarity were assigned to the same OTUs. We pick a representative sequences for each OTU and use the RDP classifier to annotate taxonomic information for each representative sequence. In order to compute Alpha Diversity, we rarify the OTU table and calculate three metrics: Chao1 estimates the species abundance; Observed Species estimates the amount of unique OTUs found in each sample, and Shannon index. Rarefaction curves were generated based on these three metrics. 
（3）Phylogenics distance and community distribution 
Graphical representation of the relative abundance of bacterial diversity from phylum to species can be visualized using Krona chart. Cluster analysis was preceded by principal component analysis (PCA), which was applied to reduce the dimension of the original variables using the QIIME software package. QIIME calculates both weighted and unweighted unifrac distance, which are phylogenetic measures of beta diversity. We used unweighted unifrac distance for Principal Coordinate Analysis (PCoA) and Unweighted Pair Group Method with Arithmetic mean (UPGMA) Clustering. PCoA helps to get principal coordinates and visualize them from complex, multidimensional data. It takes a transformation from a distance matrix to a new set of orthogonal axes. By which the maximum variation factor is demonstrated by first principal coordinate, and the second maximum one by the second principal coordinate, and so on. UPGMA Clustering is a type of hierarchical clustering method using average linkage and can be used to interpret the distance matrix.
2.1.2 Metabolomics proﬁling
Chemicals
Ammonium acetate (NH4AC) was purchased from Sigma Aldrich, Acetonitrile was purchased from Merck, ammonium hydroxide (NH4OH) and methanol were purchased from Fisher.
Sample Collection and Preparation
The mice feces were quickly frozen in liquid nitrogen immediately after dissection. Then the tissue were cut on dry ice (~80 mg) into an Eppendorf tube (2 mL). The tissue samples with 200 μL of H2O and five ceramic beads were homogenized using the homogenizer. 800 μL methanol/acetonitrile (1:1, v/v) were added to homogenized solution for metabolite extraction. The mixture was centrifuged for 15 min (14000 g, 4 °C). The supernatant was dried in a vacuum centrifuge. For LC-MS analysis, the samples were re-dissolved in 100 μL acetonitrile/water (1:1, v/v) solvent.
LC-MS/MS Analysis
Analysis was performed using an UHPLC (1290 Infinity LC, Agilent Technologies) coupled to a quadrupole time-of-flight (AB Sciex TripleTOF 6600) in Shanghai Applied Protein Technology Co., Ltd. For HILIC separation, samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH 1.7 µm column (waters, Ireland). In both ESI positive and negative modes, the mobile phase contained A=25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B= acetonitrile. The gradient was 85% B for 1 min and was linearly reduced to 65% in 11 min, and then was reduced to 40% in 0.1 min and kept for 4 min, and then increased to 85% in 0.1 min, with a 5 min re-equilibration period employed.
For RPLC separation, a 2.1 mm × 100 mm ACQUIY UPLC HSS T3 1.8 µm column (Waters, Ireland) was used. In ESI positive mode, the mobile phase contained A= water with 0.1% formic acid and B= acetonitrile with 0.1% formic acid; and in ESI negative mode, the mobile phase contained A=0.5 mM ammonium fluoride in water and B= acetonitrile. The gradient was 1%B for 1.5 min and was linearly increased to 99% in 11.5 min and kept for 3.5 min. Then it was reduced to 1% in 0.1 min and a 3.4 min of re-equilibration period was employed. The gradients were at a flow rate of 0.3mL/min, and the column temperatures were kept constant at 25℃. A 2 µL aliquot of each sample was injected.
The ESI source conditions were set as follows: Ion Source Gas1 (Gas1) as 60, Ion Source Gas2 (Gas2) as 60, curtain gas (CUR) as 30, source temperature: 600℃, IonSpray Voltage Floating (ISVF) ± 5500 V. In MS only acquisition, the instrument was set to acquire over the m/z range 60-1000 Da, and the accumulation time for TOF MS scan was set at 0.20 s/spectra. In auto MS/MS acquisition, the instrument was set to acquire over the m/z range 25-1000 Da, and the accumulation time for product ion scan was set at 0.05 s/spectra. The product ion scan is acquired using information 
dependent acquisition (IDA) with high sensitivity mode selected. The parameters were set as follows: the collision energy (CE) was fixed at 35 V with ± 15 eV; declustering potential (DP), 60 V (+) and −60 V (−); exclude isotopes within 4 Da, candidate ions to monitor per cycle: 10.
Data processing
The raw MS data (wiff.scan files) were converted to MzXML files using ProteoWizard MSConvert before importing into freely available XCMS software. For peak picking, the following parameters were used: centWave m/z = 25 ppm, peakwidth = c (10, 60), prefilter = c (10, 100). For peak grouping, bw = 5, mzwid = 0.025, minfrac = 0.5 were used. CAMERA (Collection of Algorithms of MEtabolite pRofile Annotation) was sued for annotation of isotopes and adducts. In the extracted 
ion features, only the variables having more than 50% of the nonzero measurement values in at least one group were kept. Compound identification of metabolites was performed by comparing of accuracy m/z value (<25 ppm), and MS/MS spectra with an in-house database established with available authentic standards.

3. Results
3.1 Effect on pathological injury of neurotoxic rat organs.

Figure S1 H&E staining to detect the pathology of spleen (400X), liver (400X) , kidney (800X), Lung(200X), Heart(400X) in the Control, MeHg and MeHg+FT group. (n=5)
3.2 Perturbance to gut microbiota 14 d after oral exposure FMT to MeHg poisoning rats
Table S2 Relative abundance of typical gut bacterial in the measurements for control, MeHg poisoning rats, and MeHg+FT rats. Note: Relative abundance of >0.1% is significant.

3.3 Metabolome changes 14 d after MeHg poisoning rats

3.4 Metabolome changes 14 d after oral exposure to FMT to MeHg poisoning rats