#METABOLOMICS WORKBENCH FMT_AID_20250328_005539 DATATRACK_ID:5801 STUDY_ID:ST003865 ANALYSIS_ID:AN006352 PROJECT_ID:PR002421
VERSION             	1
CREATED_ON             	April 4, 2025, 11:06 am
#PROJECT
PR:PROJECT_TITLE                 	Restoration of mucosal and faecal microbiome and metabolome is associated with
PR:PROJECT_TITLE                 	response to faecal microbiota transplantation and anti-inflammatory diet in
PR:PROJECT_TITLE                 	active ulcerative colitis
PR:PROJECT_SUMMARY               	Background Faecal microbiota transplantation with anti-inflammatory diet
PR:PROJECT_SUMMARY               	(FMT-AID) supports clinical response and deep remission in patients with active
PR:PROJECT_SUMMARY               	ulcerative colitis (UC). The present study aims to assess FMT-associated
PR:PROJECT_SUMMARY               	bacterial, fungal and metabolomic shifts in mucosal and faecal niches in active
PR:PROJECT_SUMMARY               	UC, and to correlate these changes with clinical and endoscopic outcomes of FMT.
PR:PROJECT_SUMMARY               	Study also determines if baseline microbial signatures in UC can determine FMT
PR:PROJECT_SUMMARY               	engraftment and clinical outcomes. We performed a prospective cohort study of
PR:PROJECT_SUMMARY               	patients with UC, recruited as part of the FMT-AID trial, and randomised to
PR:PROJECT_SUMMARY               	receive either 8-week FMT-AID or standard medical therapy (SMT). Non-IBD
PR:PROJECT_SUMMARY               	controls were also recruited in the study. Faecal and mucosal microbiome and
PR:PROJECT_SUMMARY               	mycobiome were characterised in 104 paired pre- and post-intervention samples
PR:PROJECT_SUMMARY               	(n=52, faecal; n=52, mucosal) from 26 patients (16 in FMT-AID arm + 10 in SMT
PR:PROJECT_SUMMARY               	arm) In addition, 48 non-IBD control samples (n=21, faecal; n=27, mucosal) were
PR:PROJECT_SUMMARY               	collected at single time point from 27 subjects. Clinical response (reduction in
PR:PROJECT_SUMMARY               	SCCAI≥3) was the primary outcome. A subset of samples (n=71; 40 paired pre-and
PR:PROJECT_SUMMARY               	post-FMT samples and 31 non-IBD control samples) were used for untargeted
PR:PROJECT_SUMMARY               	metabolomics. Results FMT-AID remodelled mucosal (β-diversity-adonis R2=2.13,
PR:PROJECT_SUMMARY               	p=0.02) and faecal (R2=2.76, p=0.003) bacterial communities and enhanced faecal
PR:PROJECT_SUMMARY               	bacterial diversity (p<0.001). FMT-AID enriched the gut with beneficial
PR:PROJECT_SUMMARY               	bacterial taxa (mucosal-Odoribacter and faecal- Slackia, Agathobaculum and
PR:PROJECT_SUMMARY               	Aldercreutzia) and reduced pathobiont abundances (mucosal- Enterococcus,
PR:PROJECT_SUMMARY               	Veillonella, Malassezia; faecal- Enterococcus, Candida tropicalis). Compared to
PR:PROJECT_SUMMARY               	clinical responders to FMT-AID, the non-responders had a distinct gut microbiome
PR:PROJECT_SUMMARY               	signature, which associated with elevated disease activity (Megasphaera and
PR:PROJECT_SUMMARY               	UCEIS (R=0.77, FDR-q=0.02), Malassezia slooffiae and SCCAI (R=0.81; FDR-q=0.01),
PR:PROJECT_SUMMARY               	UCEIS (R=0.62; FDR-q=0.12) and FCP (R=0.88; FDR-q=0.003)]. FMT-AID also restored
PR:PROJECT_SUMMARY               	mucosal and faecal metabolome with ‘health-associated’ metabolites. Higher
PR:PROJECT_SUMMARY               	pre-intervention abundances of beneficial taxa in mucosa and faeces associated
PR:PROJECT_SUMMARY               	with positive outcomes of FMT-AID. Pre-FMT faecal bacteriome also correlated
PR:PROJECT_SUMMARY               	with post-FMT engraftment of beneficial bacteria. Conclusions Enrichment of
PR:PROJECT_SUMMARY               	specific beneficial bacterial, fungal and metabolomic signatures in faecal and
PR:PROJECT_SUMMARY               	mucosal niches was associated with positive clinical, biochemical and endoscopic
PR:PROJECT_SUMMARY               	outcomes following FMT-AID in patients with UC. Pre-FMT bacteriome was
PR:PROJECT_SUMMARY               	associated with post-FMT engraftment of beneficial bacteria and clinical
PR:PROJECT_SUMMARY               	response.
PR:INSTITUTE                     	All India Institute of Medical Sciences
PR:DEPARTMENT                    	Gastroenterology
PR:LABORATORY                    	IBD Research Group
PR:LAST_NAME                     	Bajaj
PR:FIRST_NAME                    	Aditya
PR:ADDRESS                       	AIIMS Campus, Ansari Nagar East, Delhi, Delhi, 110092, India
PR:EMAIL                         	adityabajaj93@gmail.com
PR:PHONE                         	+91-9718405090
PR:FUNDING_SOURCE                	The work has been funded by the Indian Council of Medical Research: Centre for
PR:FUNDING_SOURCE                	Advanced Research and Excellence in Intestinal Diseases (grant number:
PR:FUNDING_SOURCE                	55/4/11/CARE-ID/2018-NCD-II); Science and Engineering Research Board (SERB):
PR:FUNDING_SOURCE                	Core Research Grant (CRG/2019/005292); and Scheme for Promotion of Academic and
PR:FUNDING_SOURCE                	Research Collaboration (SPARC P1492).
PR:PUBLICATIONS                  	Restoration of Mucosal and Fecal Microbiome, Mycobiome and Metabolome is
PR:PUBLICATIONS                  	Associated with Response to Fecal Microbiota Transplantation and
PR:PUBLICATIONS                  	Anti-Inflammatory Diet in Active Ulcerative Colitis. Available at SSRN:
PR:PUBLICATIONS                  	https://ssrn.com/abstract=4953224 or http://dx.doi.org/10.2139/ssrn.4953224
PR:CONTRIBUTORS                  	Markandey, Manasvini and Bajaj, Aditya and Kshetrapal, Pallavi and Virmani,
PR:CONTRIBUTORS                  	Shubi and Singh, Mukesh and Verma, Mahak and Thirunavukkarasu, Ramasamy and
PR:CONTRIBUTORS                  	Vuyyuru, Sudheer Kumar and Kante, Bhaskar and Kumar, Peeyush and Makharia,
PR:CONTRIBUTORS                  	Govind and Das, Bhabatosh and Kumar, Dhiraj and Kedia, Saurabh and Ahuja,
PR:CONTRIBUTORS                  	Vineet,
#STUDY
ST:STUDY_TITLE                   	Assessment of the effects of Faecal microbiota transplantation with
ST:STUDY_TITLE                   	anti-inflammatory diet (FMT-AID) on faecal metabolome in patients with
ST:STUDY_TITLE                   	ulcerative colitis.
ST:STUDY_SUMMARY                 	The study aims to understand FMT-AID driven recuperation of faecal metabolome in
ST:STUDY_SUMMARY                 	patients with UC.
ST:INSTITUTE                     	All India Institute of Medical Sciences
ST:DEPARTMENT                    	Gastroenterology
ST:LABORATORY                    	IBD Research Group
ST:LAST_NAME                     	Bajaj
ST:FIRST_NAME                    	Aditya
ST:ADDRESS                       	AIIMS Campus, Ansari Nagar East, Delhi, Delhi, 110092, India
ST:EMAIL                         	adityabajaj93@gmail.com
ST:PHONE                         	+91-9718405090
ST:NUM_GROUPS                    	3 (Non-IBD Controls, Patients with UC before FMT and Patients with UC after FMT)
ST:TOTAL_SUBJECTS                	41 (13 Non-IBD controls + 28 patients with UC)
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:AGE_OR_AGE_RANGE              	Non-IBD controls, 36.9 ± 12.64; UC group, 33.28 ± 11.13
SU:GENDER                        	Male and female
SU:HUMAN_ETHNICITY               	Indian
SU:HUMAN_LIFESTYLE_FACTORS       	BMI (kg/m2), Non-IBD controls, 21.17 ± 3.1; UC group, 21.17 ± 4.6
SU:HUMAN_MEDICATIONS             	UC group, Prior 5-ASA course (%) 100; Prior steroid course (%) 6.3; Prior
SU:HUMAN_MEDICATIONS             	Azathioprine therapy (%) 37.5; Prior biological use (%) 0; Prior methotrexate
SU:HUMAN_MEDICATIONS             	use (%) 0; Topical 5-ASA use (%) 100; Topical steroid enema use (%) 75
SU:HUMAN_SMOKING_STATUS          	Smokers (%), Non-IBD controls, 9.5% ; UC group, 11.42%
SU:HUMAN_ALCOHOL_DRUG_USE        	Alcohol consumption (%), Non-IBD controls, 0% ; UC group, 2.8%
SU:HUMAN_INCLUSION_CRITERIA      	Age, 20-60 years; Clinical disease activity, Mild-moderate UC (SCCAI 3-9);
SU:HUMAN_INCLUSION_CRITERIA      	Endoscopic disease activity, UCEIS >1; Disease extent, Left-sided/pancolitis;
SU:HUMAN_INCLUSION_CRITERIA      	Permitted medications, 1.Oral 5-ASA (stable dose for >4 weeks)
SU:HUMAN_INCLUSION_CRITERIA      	2.Azathioprine/6-MP (stable dose for >3 months) 3.Topical 5-ASA or topical
SU:HUMAN_INCLUSION_CRITERIA      	steroids (stable dose for >2 weeks) 4.Oral steroids (<20 mg prednisolone with
SU:HUMAN_INCLUSION_CRITERIA      	taper of 5 mg/week) 5.Anti-TNF mAb (If used more than 6 months back); Others,
SU:HUMAN_INCLUSION_CRITERIA      	1.Patients who gave written informed consent, 2.Agreement to adhere to diet
SU:HUMAN_INCLUSION_CRITERIA      	schedule
SU:HUMAN_EXCLUSION_CRITERIA      	Disease activity, Patients with severe disease activity or acute severe colitis;
SU:HUMAN_EXCLUSION_CRITERIA      	Prohibited medications, 1.Topical steroids (if used within 2 weeks),
SU:HUMAN_EXCLUSION_CRITERIA      	2.Antibiotics and Probiotics (if used within 4 weeks of randomization); Others,
SU:HUMAN_EXCLUSION_CRITERIA      	1.History of bowel surgery, 2.Pregnancy/lactation, 3.Presence of co-morbid
SU:HUMAN_EXCLUSION_CRITERIA      	illnesses, 4.Concomitant gastrointestinal infection
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	01-C	01-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=01-C-F.raw
SUBJECT_SAMPLE_FACTORS           	02-C	02-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=02-C-F.raw
SUBJECT_SAMPLE_FACTORS           	03-C	03-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=03-C-F.raw
SUBJECT_SAMPLE_FACTORS           	04-C	04-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=04-C-F.raw
SUBJECT_SAMPLE_FACTORS           	05-C	05-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=05-C-F.raw
SUBJECT_SAMPLE_FACTORS           	06-C	06-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=06-C-F.raw
SUBJECT_SAMPLE_FACTORS           	07-C	07-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=07-C-F.raw
SUBJECT_SAMPLE_FACTORS           	08-C	08-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=08-C-F.raw
SUBJECT_SAMPLE_FACTORS           	10-C	10-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=10-C-F.raw
SUBJECT_SAMPLE_FACTORS           	12-C	12-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=12-C-F.raw
SUBJECT_SAMPLE_FACTORS           	13-C	13-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=13-C-F.raw
SUBJECT_SAMPLE_FACTORS           	14-C	14-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=14-C-F.raw
SUBJECT_SAMPLE_FACTORS           	16-C	16-C-F	Sample source:Faecal | Factor:Control	RAW_FILE_NAME(Raw file name)=16-C-F.raw
SUBJECT_SAMPLE_FACTORS           	01-UC	01-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=01-A-F.raw
SUBJECT_SAMPLE_FACTORS           	01-UC	01-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=01-B-F.raw
SUBJECT_SAMPLE_FACTORS           	02-UC	02-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=02-A-F.raw
SUBJECT_SAMPLE_FACTORS           	02-UC	02-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=02-B-F.raw
SUBJECT_SAMPLE_FACTORS           	03-UC	03-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=03-A-F.raw
SUBJECT_SAMPLE_FACTORS           	03-UC	03-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=03-B-F.raw
SUBJECT_SAMPLE_FACTORS           	04-UC	04-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=04-A-F.raw
SUBJECT_SAMPLE_FACTORS           	05-UC	05-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=05-A-F.raw
SUBJECT_SAMPLE_FACTORS           	05-UC	05-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=05-B-F.raw
SUBJECT_SAMPLE_FACTORS           	06-UC	06-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=06-A-F.raw
SUBJECT_SAMPLE_FACTORS           	06-UC	06-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=06-B-F.raw
SUBJECT_SAMPLE_FACTORS           	07-UC	07-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=07-A-F.raw
SUBJECT_SAMPLE_FACTORS           	08-UC	08-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=08-A-F.raw
SUBJECT_SAMPLE_FACTORS           	08-UC	08-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=08-B-F.raw
SUBJECT_SAMPLE_FACTORS           	09-UC	09-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=09-A-F.raw
SUBJECT_SAMPLE_FACTORS           	09-UC	09-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=09-B-F.raw
SUBJECT_SAMPLE_FACTORS           	10-UC	10-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=10-A-F.raw
SUBJECT_SAMPLE_FACTORS           	11-UC	11-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=11-A-F.raw
SUBJECT_SAMPLE_FACTORS           	11-UC	11-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=11-B-F.raw
SUBJECT_SAMPLE_FACTORS           	12-UC	12-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=12-A-F.raw
SUBJECT_SAMPLE_FACTORS           	12-UC	12-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=12-B-F.raw
SUBJECT_SAMPLE_FACTORS           	13-UC	13-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=13-A-F.raw
SUBJECT_SAMPLE_FACTORS           	13-UC	13-B-F	Sample source:Faecal | Factor:Post-FMT	RAW_FILE_NAME(Raw file name)=13-B-F.raw
SUBJECT_SAMPLE_FACTORS           	14-UC	14-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=14-A-F.raw
SUBJECT_SAMPLE_FACTORS           	15-UC	15-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=15-A-F.raw
SUBJECT_SAMPLE_FACTORS           	16-UC	16-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=16-A-F.raw
SUBJECT_SAMPLE_FACTORS           	17-UC	17-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=17-A-F.raw
SUBJECT_SAMPLE_FACTORS           	18-UC	18-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=18-A-F.raw
SUBJECT_SAMPLE_FACTORS           	19-UC	19-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=19-A-F.raw
SUBJECT_SAMPLE_FACTORS           	20-UC	20-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=20-A-F.raw
SUBJECT_SAMPLE_FACTORS           	21-UC	21-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=21-A-F.raw
SUBJECT_SAMPLE_FACTORS           	22-UC	22-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=22-A-F.raw
SUBJECT_SAMPLE_FACTORS           	23-UC	23-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=23-A-F.raw
SUBJECT_SAMPLE_FACTORS           	24-UC	24-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=24-A-F.raw
SUBJECT_SAMPLE_FACTORS           	25-UC	25-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=25-A-F.raw
SUBJECT_SAMPLE_FACTORS           	26-UC	26-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=26-A-F.raw
SUBJECT_SAMPLE_FACTORS           	27-UC	27-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=27-A-F.raw
SUBJECT_SAMPLE_FACTORS           	28-UC	28-A-F	Sample source:Faecal | Factor:Pre-FMT	RAW_FILE_NAME(Raw file name)=28-A-F.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Faecal samples were collected at a single time point from non-IBD controls and
CO:COLLECTION_SUMMARY            	twice from patients with UC, once before the commencement of FMT-AID (Pre-FMT)
CO:COLLECTION_SUMMARY            	and once after 7 weekly FMT sessions (Post-FMT). Faecal samples were collected
CO:COLLECTION_SUMMARY            	in sterile stool vials, homogenised manually and distributed in 50mg aliquots in
CO:COLLECTION_SUMMARY            	sterile 2ml cryovials. The aliquots were flash-frozen in liquid nitrogen and
CO:COLLECTION_SUMMARY            	stored at -80°C for metabolite extraction.
CO:SAMPLE_TYPE                   	Feces
CO:COLLECTION_LOCATION           	IBD Clinic, All India Institute of Medical Sciences, New Delhi
CO:COLLECTION_FREQUENCY          	Faecal samples were collected at a single time point from non-IBD controls and
CO:COLLECTION_FREQUENCY          	twice from patients with UC, once before the commencement of FMT-AID (Pre-FMT)
CO:COLLECTION_FREQUENCY          	and once after 7 weekly FMT sessions (Post-FMT).
CO:VOLUMEORAMOUNT_COLLECTED      	Stored two aliquots of 50mg each.
CO:STORAGE_CONDITIONS            	-80℃
CO:COLLECTION_VIALS              	Sterile stool vials
CO:STORAGE_VIALS                 	Cryovials
CO:COLLECTION_TUBE_TEMP          	4°C
CO:ADDITIVES                     	None
#TREATMENT
TR:TREATMENT_SUMMARY             	FMT-AID arm received seven consecutive weekly, multi-donor pooled faecal
TR:TREATMENT_SUMMARY             	microbiota transplantations (FMT), administered colonoscopically with an
TR:TREATMENT_SUMMARY             	anti-inflammatory diet (AID). The AID was enriched in components supporting the
TR:TREATMENT_SUMMARY             	expansion of T-regulatory cells, healthy gut microbiota and improvement of gut
TR:TREATMENT_SUMMARY             	barrier integrity. AID suggested avoidance of gluten-based grains, meat, refined
TR:TREATMENT_SUMMARY             	sugars, food additives and dairy products and promoted the consumption of fresh
TR:TREATMENT_SUMMARY             	fruits and vegetables (especially cruciferous vegetables as a source of AhR
TR:TREATMENT_SUMMARY             	ligands), fermented foods and polyphenols.
TR:TREATMENT                     	Faecal microbiota transplantation with anti-inflammatory diet
TR:TREATMENT_COMPOUND            	Pooled, multi-donor faecal suspension
TR:TREATMENT_ROUTE               	Colonoscopic infusion of FMT
TR:TREATMENT_DOSE                	50 g of stool homogenised with 200-250 ml of 0.9% normal saline till liquid
TR:TREATMENT_DOSE                	consistency.
TR:TREATMENT_DOSEVOLUME          	200-250 ml of faecal suspension
TR:TREATMENT_VEHICLE             	0.9% normal saline
TR:HUMAN_FASTING                 	Not required
TR:HUMAN_ENDP_CLINICAL_SIGNS     	The primary outcome measure for the present study is clinical response
TR:HUMAN_ENDP_CLINICAL_SIGNS     	(reduction in SCCAI score by 3 or more points), and secondary outcomes are
TR:HUMAN_ENDP_CLINICAL_SIGNS     	clinical remission (SCCAI <2), endoscopic remission (UCEIS <1), and endoscopic
TR:HUMAN_ENDP_CLINICAL_SIGNS     	response (decline in UCEIS by 2 or more points).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	For faecal metabolome extraction, 100uL of 50% methanol was added to each
SP:SAMPLEPREP_SUMMARY            	Precellys homogenization tube containing ~50mg stool sample, followed by
SP:SAMPLEPREP_SUMMARY            	homogenization in the Precellys 24 tissue homogenizer. Homogenized samples were
SP:SAMPLEPREP_SUMMARY            	subjected to 90-second heat shock treatment at 37°C. 150uL of 100% methanol was
SP:SAMPLEPREP_SUMMARY            	then added per sample, followed by a 10 min incubation on ice. Samples were
SP:SAMPLEPREP_SUMMARY            	centrifuged at 1400rpm for 15 min at 4 ̊C, and 200uL of supernatant from each
SP:SAMPLEPREP_SUMMARY            	sample was mixed with 200uL of methyl-tert-butyl-ether (MTBE). This mixture was
SP:SAMPLEPREP_SUMMARY            	vortexed for 1 hour at RT, followed by centrifugation at 14000 rpm for 15 min at
SP:SAMPLEPREP_SUMMARY            	4 ̊C. 80uL from each of the aqueous and organic phases were pooled together in
SP:SAMPLEPREP_SUMMARY            	a fresh tube from each sample and vacuum dried overnight in a Speed Vac.
SP:SAMPLEPREP_SUMMARY            	Metabolite extracts were reconstituted in 15% acetonitrile for reverse phase
SP:SAMPLEPREP_SUMMARY            	chromatography and run on the Orbitrap Fusion Tibrid Mass Spectrometer
SP:SAMPLEPREP_SUMMARY            	(Thermofisher Scientific) for an untargeted LCMS-based metabolomic screening.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	fecal-metabolite-extraction-protocol.pdf
SP:PROCESSING_STORAGE_CONDITIONS 	-20℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000
CH:COLUMN_NAME                   	Waters ACQUITY UPLC CSH C18 (100 x 1mm,1.7um)
CH:SOLVENT_A                     	0.1% Formic acid in water
CH:SOLVENT_B                     	0.1% Formic acid in methanol
CH:FLOW_GRADIENT                 	Formic acid in water: Formic acid in methanol: 0.3ml per min rate for 5 minutes
CH:FLOW_RATE                     	0.3 ml/min
CH:COLUMN_TEMPERATURE            	40°C
CH:RETENTION_TIME                	0.01 to 14.0 min
CH:SAMPLE_INJECTION              	5 µL
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Fusion Tribrid Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Metabolite extracts were reconstituted in 15% acetonitrile for reverse phase
MS:MS_COMMENTS                   	chromatography and run on the Orbitrap Fusion Tibrid Mass Spectrometer
MS:MS_COMMENTS                   	(Thermofisher Scientific) for an untargeted LCMS-based metabolomic screening.
MS:MS_COMMENTS                   	Raw m/z ratios (peaks) were filtered through ProgenesisQI and StatTarget.
MS:MS_COMMENTS                   	Shortlisted peaks were normalized using Pareto-scaling and log transformation
MS:MS_COMMENTS                   	using MetaboAnalyst 5.0. Significant, differentially abundant peaks in our two
MS:MS_COMMENTS                   	comparison groups – controls vs. pre-FMT and pre-FMT vs. post-FMT, were
MS:MS_COMMENTS                   	identified as having a VIP score>1, Fold change>2 and significant T-score (FDR
MS:MS_COMMENTS                   	corrected). Putative identities were manually assigned, using 4 different
MS:MS_COMMENTS                   	databases – LipidMaps, METLIN, KEGG and HMDB, with a ppm error of <1 and
MS:MS_COMMENTS                   	sorted into functional categories based on the pathways they participate in the
MS:MS_COMMENTS                   	host or microbial metabolism.
MS:CAPILLARY_VOLTAGE             	3500V
MS:MS_RESULTS_FILE               	ST003865_AN006352_Results.txt	UNITS:Peak intensity	Has m/z:Yes	Has RT:Yes	RT units:Seconds
#END