ABI2 protein, human

The eMERGE network consists of seven adult sites and two pediatric sites, each with DNA databanks linked to EHR. Each site in the network has a set of at least 3000 samples that have been genotyped on one or more genotyping platforms (Gottesman et al., 2013 (link)). Table 1 provides a summary of the number of samples from each site and the genotyping platforms used. Previous studies have shown that the quality of input genotype data does not affect imputation quality in a significant manner (Southam et al., 2011 (link)), but nevertheless we selected the genomic data sets for the current imputation study that had all undergone the pre-processing recommended by the eMERGE CC to eliminate samples and SNPs with call rates less than 99–95% depending on the coverage of genotyping for each platform (Zuvich et al., 2011 (link)). Minor allele frequency (MAF) threshold of 5% was also applied. This ensures that only high quality data were considered for imputation and downstream analyses.
Several eMERGE sites genotyped duplicate samples on multiple different genotyping platforms, for quality control purposes. A total of 56,890 samples were submitted to the eMERGE CC for imputation, out of which 53,200 samples were unique. All of these samples were genotyped and deposited to CC at different times, so imputation was performed as the datasets arrived. This resulted in imputing some datasets with fewer than 100 samples. When the dataset was less than 100 samples, we included the 1000 Genomes dataset with the study data during phasing. We imputed all samples; however, for the purpose of merging the data, we only merged high quality datasets (defined by having masked analysis concordance rate greater than 80%; described in more detail below). We included only one sample from pairs of duplicates; specifically the sample genotyped on the higher density genotyping platform. Our final merged dataset contains 51,035 samples. Samples that had low quality due to either of the following two reasons were not included:

Samples not genotyped on dense, genome-wide genotyping platform (e.g., the MetaboChip).

Sample size of the dataset on the specific platform for phasing was fewer than 100 (as recommended in SHAPEIT2 guidelines).

A small number of samples were also genotyped for two SNPs (rs1799945 and rs1800562) using commercially available 5′-nuclease assays (TaqMan® Assay; Life Technologies). Genotyping reactions were carried out in 10 μl volumes in an ABI 7500 Fast Real-Time PCR System (Life Technologies). The genotypes were called using ABI 7500 software version 2.0.4 (Life Technologies). These data were used to evaluate the concordance of imputed genotypes with TaqMan generated genotypes.

The protein profiles used were either retrieved from existing databases (PFAM^{42 (link)}, COG^{43 (link)}) or built from scratch when no adequate profiles existed (see below for details on the building of HMM profiles and Supplementary Data 2).
New protein profiles for the proteins involved in anti-phage systems were built using a homogeneous procedure. We collected a set of sequences from the protein family that were representative of the diversity of the bacterial taxonomy. Homologous proteins were aligned using MAFFT v7.475^{44 (link)} (default options, mode auto) and then used to produce protein profiles with Hmmbuild (default options) from the HMMer suite v3.3⁴⁵ . To ensure a better detection we curated each profile manually by assigning a GA score (used with the hmmsearch option–cut_ga) (Supplementary Figs. 1–2). GA score defines the threshold above which a hit is considered significant. This threshold was determined manually by inspecting the distribution of the scores. All accession numbers for proteins used to build custom HMM profiles are available in Supplementary Data 2.
Protein scrapping was done using different methods depending on the available information about the system in the literature (Details in Supplementary Data 1). For systems from^{1 (link)}, dGTPase⁸ , dCTPdeaminase⁸ , BREX^{3 (link)}, part of Cyclic-oligonucleotide-based anti-phage signaling systems (CBASS)^{17 (link)}, all the reverse transcriptases of retrons, BstA¹⁰ , viperins^{7 (link)} and DISARM² , we used a subset (between 20 and 100 proteins) of the proteins available in the supplementary data of each publication. We then tested if the HMM allows for detection of all known occurrences of such proteins. If a lot of proteins were undetected, we added proteins reported in the supplementary materials but not detected through our HMM to the list of sequences for the alignment and subsequent HMM generation.
For AbiEii, AbiH,Abi2, Stk2, Pif, Lit, PrrC, RexAB, part of CBASS, part brxA, and brxB from BREX, PARIS (AAA15 and AAA21), we used PFAM available at (http://pfam.xfam.org/) or the sequence available on COG (https://www.ncbi.nlm.nih.gov/research/cog-project/). For part of BREX, DndABCDEFGH^{46 (link)}, we searched for proteins with this name available on NCBI and curated manually such list. For systems when only one sequence was provided such as Gao’s systems^{4 (link)}, Rousset’s systems¹² , Dnd type SspBCDE, part of retrons, the protein sequence was BLASTed. Between 20 and 50 sequences with high coverage were selected. For retrons other than reverse transcriptase, we used the IMG genome neighborhood feature to get adjacent protein of the reverse transcriptase and repeated the BLAST process. For CAS systems, HMM protein profiles were downloaded from^{22 (link),47 (link)}. All hmm profiles used are available at https://github.com/mdmparis/defense-finder-models.

The total RNA of HCN cells and their differentiated cells, or Angptl2^+/+ and Angptl2^−/− mouse brain tissues were extracted and used for real-time RT-PCR. The reactions were performed as previously described [70 (link)]. Briefly, 10 μL reactions with 2 × ABI SYBR^® Green PCR master mix, primers and cDNA were used for the evaluation of indicated gene expression levels. The experiments were conducted in triplicate with Applied Biosystems 7900HT. The mRNA level was normalized to the level of β-actin RNA transcripts. The primer sequences for related genes were showed in Additional files 2. The unedited agarose gel figures were showed in Additional file 3.

Gene expression analyses from buffy coat cells, ilium, and colon biopsies were performed by RT-qPCR. Briefly, peripheral whole blood from CON (n = 8) and BEO (n = 8) groups was collected on days 30 and 60 and centrifuged at 800 g for 10 min at room temperature for buffy coat isolation. The white blood cells and platelets (buffy coat) formed a layer on red blood cells that were carefully removed with a micropipette. According to the fabricant instructions, red blood cells were then lysate by Ammonium-Chloride-Potassium Lysing Buffer (ACK; Thermoscientific, Waltham, USA), and only the white layer of cells was frozen at RNA protect reagent (Qiagen, Hilden, Germany) until RNA extraction. The ileum and colon biopsies were obtained from the animal’s necropsy and kept on RNAprotect reagent (Qigen, Hilden, Germany) until analysis. The RNA extraction from buffy coat and organ samples was performed with RNeasy Mini kit (Qiagen, Hilden, Germany). The obtained total RNA was quantified by the Nanodrop 1000 Spectrophotometer (Thermo Scientific, Waltham, USA), and the cDNA synthesis was performed by SuperScript III First-Strand kit (Thermo Scientific, Waltham, USA), all according to the manufacturer's instructions^{33 (link)}.
The RT-qPCR assays occurred in 7500 Fast Real-Time PCR System (Thermo Scientific, Waltham, USA), using the PowerUp SYBR Green Master Mix (Thermo Scientific, Waltham, USA) to verify the expression of interleukin 6 (IL-6) and interleukin 10 (IL-10) genes. It was used β-actin, GAPDH, and Ubiquitin as reference genes based on expression stability calculated with RefFinder online software. Each sample calculated the average of Ct values from targets and reference genes using ABI Real-Time PCR 7500 software v.2.3 (Thermo Scientific, Waltham, USA).

Quantitative real-time PCR was performed to quantify the bacterial biomass in each gut sample. Amplification reactions were carried out in an ABI StepOne^TM Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) in a 10 μL mixture containing 1X BlasTaq^TM qPCR MasterMix (Applied Biological Materials Inc., Richmond, BC, Canada), 400 nM of each primer (341F and 515R [28 (link)]), and 1 μL of template DNA. The amplification conditions consisted of incubation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 60 s, and extension at 72 °C for 45 s. All samples were carried out in triplicate in optical 96-well plates along with the negative control and standard curve. Standard curve was created for absolute quantification of 16S rRNA gene using a plasmid containing the target gene fragment from Pseudomonas sp. DSM1650 10-fold diluted from 3.60 × 10⁷ to 3.60 × 10³ gene copy numbers μL⁻¹. Fluorescent light outputs were collected during each elongation step and analyzed with the ABI StepOne Real-Time PCR system SDS software v 2.3 (Applied Biosystems). Melting curves were generated after amplification by increasing the temperature of 0.5 °C every 30 s from 65 to 95 °C, in order to verify the absence of primer dimers or artifacts.