Multiomics of faecal samples collected from individuals in families with multiple cases of type 1 diabetes mellitus (T1DM) over 3 or 4 months. Metagenomic and metatranscriptomic sequencing and metaproteomics were carried out, as well as whole human genome sequencing. Phenotypic data is available.
Sample Processing Protocol
DNA and proteins were extracted from flash-frozen, unthawed faecal samples after pre-treatment with 1.5 ml RNAlater ICE (LifeTechnologies), using differential centrifugation as described in Roume (Roume, H., Heintz-Buschart, A., Muller, E. E. L. & Wilmes, P. Sequential isolation of metabolites, RNA, DNA, and proteins from the same unique sample. Meth. Enzymol. 531, 219–236 (2013)) and the All-In-One kit (Norgen Biotek). An aliquot of 15 µg isolated protein fraction was reduced (DTT) and alkylated (Iodoacetamide) and the proteins precipitated using the Clean-up Kit (GE Healthcare) according to the manufacturer’s instructions. The protein pellets were re-suspended at a concentration of 500 µg/ml in 50 mM ammonium bicarbonate to perform a complete trypsin digestion. 3.5 µg digested protein was purified on a Ziptip C18 (Millipore), dried and re-suspended in 100 mM ammonium formiate (pH 10) at 333 ng/µl. 3 µg digested protein were injected on a Nano 2D UPLC – Orbitrap MS system (2D-nanoAcquity UPLC (Waters) and Q-Exactive (Thermo)). MPDSMIX (Waters) was spiked in at 150 fmoles of ADH digest per injection. A 2D LC method with three steps of 180 min was run. The three steps were run on a column at high pH with increasing percentage of acetonitrile and the eluted peptides were diluted and loaded on a low pH column, where each step consisted of a gradient of 5 min from 99 % of A (A = 0.1 % formic acid in water; B = acetonitrile) to 93 % of A followed by a gradient of 135 min from 93 % of A to 65 % of A. Mass spectrometry was done using a TopN-MSMS method (N = 12), with MS parameters as follows: mass range from 400 to 1750 m/z, resolution 70,000, AGC target 106 or maximum injection time 200 ms. MS2 parameters for spectrum acquisition were: isolation window 1.6 m/z, collision energy (NSE) 25, resolution 17,500, AGC target 105 or maximum injection time 50 ms.
Data Processing Protocol
Sample specific SEARCH databases were constructed from individual-specific pseudo-phased human proteins including all homo- and heterozygous variants annotated using ANNOVAR76, as well as microbial protein predictions derived from the combined metagenomic and metatranscriptomic assemblies after inclusion of variants. The database SEARCHes were performed using Proteome Discoverer (v 1.4; Thermo Scientific) and Sequest HT on the protein predictions. Peptides were grouped by mass and sequences, PSM with at least a medium confidence interval and a delta Cn better than 0.15 were considered, strict maximum parsimony principle was applied and protein grouping was enabled. A minimal number of one unique peptide was required for protein identification.
Corresponding dataset(s) in other omics resources
PRJNA289586 (BioProject, ENA, EMBL-EBI)
Heintz-Buschart A, May P, Laczny CC, Lebrun LA, Bellora C, Krishna A, Wampach L, Schneider JG, Hogan A, de Beaufort C, Wilmes P. Integrated multi-omics of the human gut microbiome in a case study of familial type 1 diabetes. Nat Microbiol. 2016 Oct 10;2:16180 PubMed: 27723761