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A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease

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A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease Supplementary Materials: A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease S1. Library preparation and NGS sequencing Qualified DNA samples were used to prepare sequence library with an insert size of 180 bp. First of all, purified DNA samples were sheared into smaller fragments by Nebulization technique. Then the overhangs resulting from fragmentation were converted into blunt ends by using T4 DNA polymerase, Klenow Fragment and T4 Polynucleotide Kinase. After adding an ‘A’ base to the 3' end of the blunt phosphorylated DNA fragments, adapters were ligated to the ends of the DNA fragments. After that, too short fragments were removed using Ampure beads. The qualified library was used for sequencing with 2G raw data output per sample and a length of 100bp per read. After raw data generation, low quality reads were removed. Data filtration was done by BGI custom scripts, and listed as follows: 1. removing reads with 3 N; 2. removing reads contaminated by adapter; 3. removing reads with a certain proportion of low quality bases (40% as default, parameter setting at 36 bp); 4. removing duplication contamination. Finally, the clean data obtained were used for subsequent bioinformatic analysis. The library preparation procedures, as well as the sequencing pipeline described here were implemented and performed by the Beijing Genomics Institute (BGI, Shenzhen, Guangdong, China). Additional details about the bioinformatics pipeline used in this work including information about raw data control were reported in the main text (Material and Methods section) and in S2 Table. S2. File to be uploaded to iPath site (http://pathways.embl.de/) Supplementary Figure Legends and Tables Figure S1. Metabolic and regulatory pathway distribution as obtained from HUMAnN analysis. Only pathways reporting a coverage values higher than 80% were reported. Figure S2. Metabolic map drawn with iPath. Equally distributed pathways were reported in green, whereas pathways with a higher abundance in normal/mild and severe groups were reported in blue and red. In particular, pathways reporting higher values in patients belonging to the normal/mild group were reported in blue and pathways more represented in patients belonging to the severe group were reported in red. An interactive version of this plot can be generated using Supporting Information S2 directly from iPath site (http://pathways.embl.de/). Figure S3. Regulatory distribution derived from the metabolic map as reported in S2 Fig. Different colors refer to different distributions as reported in S2 Fig legend. An interactive version of this plot can be generated using S2 Supporting Information directly from iPath site (http://pathways.embl.de/). Figure S4. Contig length distribution. Boxplots were drawn reporting the interquartile range (IQR) between the 25th and the 75th percentile (first and third quartiles), whereas the inner line represents the median. Whiskers represent the lowest and highest values within 1.5 times IQR from the first and third quartiles Outliers were not reported. Figure S5. Distribution patterns of transporter proteins as reported by TCDB. On the left part of the panel, the top 30 transporter categories were enlarged for easy reading. “m” and “s” correspond to “normal/mild” and “severe” groups. Figure S6. Virulence factor distribution for each patient. Only factors displaying a significant (Student’s t-test p-value ≤ 0.05) diverging distribution in the two groups of patients were reported. Figure S7. Distribution patterns of virulence factors classified with MvirDB. The top 20 sub- categories were reported for each main-category. Figure S8. Distribution of antibiotic resistance genes for each category detected at least in one sample. The percentage of resistance genes found has been reported for each patient. Figure S9. Top-20 taxa detected with both NGS and culture Table S1. Culture-based diagnostic microbiology of sputum from patients with CF. Table S2. Number of metagenomic sequences obtained and processed for each patient. Table S3. Mantel test P values and R statistics obtained comparing all biotic features. In the right part of the table, the values of the R statistics were reported whereas the P values were reported in the left part. T=taxonomic; K=KEGG pathways; E=gene frequencies (eggNOG database); V=virulence factors (MvirDB database); A=antibiotic resistance genes (Resfam database). Table S4. Variable loadings on the PCs across samples. The top-50% variables were reported in bold. Lipoic acid metabolism (Ko:00785) Scale Mismatch repair (Ko:03430) DNA replication (Ko:03030) Thiamine metabolism (Ko:00730) 0.09 Nucleotide excision repair (Ko:03420) One carbon pool by folate (Ko:00670) Pyruvate metabolism (Ko:00620) Sulfur relay system (Ko:04122) 0.06 beta−Alanine metabolism (Ko:00410) Protein export (Ko:03060) D−Alanine metabolism (Ko:00473) 0.03 Homologous recombination (Ko:03440) Folate biosynthesis (Ko:00790) Peptidoglycan biosynthesis (Ko:00550) Biotin metabolism (Ko:00780) Cysteine and methionine metabolism (Ko:00270) Valine, leucine and isoleucine biosynthesis (Ko:00290) Histidine metabolism (Ko:00340) Citrate cycle (TCA cycle) (Ko:00020) D−Glutamine and D−glutamate metabolism (Ko:00471) Selenocompound metabolism (Ko:00450) Fatty acid biosynthesis (Ko:00061) Nicotinate and nicotinamide metabolism (Ko:00760) Pentose phosphate pathway (Ko:00030) Riboflavin metabolism (Ko:00740) Ribosome (Ko:03010) Pyrimidine metabolism (Ko:00240) RNA degradation (Ko:03018) Alanine, aspartate and glutamate metabolism (Ko:00250) Terpenoid backbone biosynthesis (Ko:00900) C5−Branched dibasic acid metabolism (Ko:00660) Vitamin B6 metabolism (Ko:00750) Glycine, serine and threonine metabolism (Ko:00260) Bacterial chemotaxis (Ko:02030) Carbon fixation pathways in prokaryotes (Ko:00720) Bacterial secretion system (Ko:03070) Butanoate metabolism (Ko:00650) Synthesis and degradation of ketone bodies (Ko:00072) Glycosylphosphatidylinositol(GPI)−anchor biosynthesis (Ko:00563) Valine, leucine and isoleucine degradation (Ko:00280) Flagellar assembly (Ko:02040) Nitrotoluene degradation (Ko:00633) Other glycan degradation (Ko:00511) RNA polymerase (Ko:03020) Geraniol degradation (Ko:00281) Caprolactam degradation (Ko:00930) Basal transcription factors (Ko:03022) normal/mild severe Other types of Mucin type O-glycan biosynthesis O-glycan biosynthesis Lipopolysaccharide Butirosin and neomycin biosynthesis N-Glycan Various types of biosynthesis Glycan Biosynthesis biosynthesis N-glycan biosynthesis and Metabolism Glycosphingolipid biosynthesis - Glycosaminoglycan Streptomycin Nucleotide globoseries biosynthesis - biosynthesis chondroitin sulfate Metabolism Glycosphingolipid biosynthesis - lact and neolacto series Other glycan Puromycin degradation Biosynthesis of biosynthesis Thiamine [B] Glycosyltransferases Glycosaminoglycan ansamycins metabolism biosynthesis - heparan sulfate Purine metabolism Starch and sucrose metabolism Metabolism of Cofactors and Vitamins Glycosaminoglycan degradation Galactose Ascorbate and metabolism aldarate metabolism Glycosylphosphatidylinositol Caffeine (GPI)-anchor biosynthesis Folate beta-Alanine metabolism biosynthesis alpha-Linolenic acid metabolism Riboflavin Glycosphingolipid Glycosaminoglycan metabolism metabolism biosynthesis - biosynthesis - Pyrimidine metabolism [B] Proteoglycans ganglioseries keratan sulfate Polyketide sugar Pentose and glucuronate unit biosynthesis interconversions Vitamin B6 Biosynthesis of vancomycin Tyrosine metabolism metabolism group antibiotics Amino sugar and nucleotide sugar metabolism Pentose phosphate metabolism Biosynthesis of 12-, 14- and Fructose and 16-membered macrolides Phenylalanine , tyrosine and mannose metabolism tryptophan biosynthesis One carbon pool Glycerophospholipid by folate metabolism Phenylalanine Linoleic acid Arachidonic acid metabolism Isoquinoline alkaloid metabolism biosynthesis metabolism Photosynthesis - Biosynthesis of antenna proteins Novobiocin Biosynthesis of Phenylpropanoid unsaturated fatty acids Glycerolipid [B] Lipids metabolism biosynthesis Other Secondary Metabolites biosynthesis Amino Acid [B] Phytochemical Carbohydrate Acridone alkaloid compounds Lipid Metabolism biosynthesis Metabolism Ether lipid Metabolism metabolism Photosynthesis Sphingolipid Sulfur Taurine and [B] Lipids biosynthesis Cutin, suberine and metabolism metabolism hypotaurine Isoflavonoid Inositol phosphate metabolism biosynthesis proteins wax biosynthesis metabolism Indole alkaloid Glycolysis / [B] Photosynthesis Biosynthesis of biosynthesis Gluconeogenesis proteins siderophore group Fatty acid nonribosomal biosynthesis peptides Tryptophan Peptidoglycan Energy metabolism Flavonoid Carotenoid biosynthesis Metabolism Ubiquinone and biosynthesis biosynthesis other terpenoid-quinone Fatty acid biosynthesis metabolism Biosynthesis of type II Phosphonate and Histidine metabolism polyketide products phosphinate metabolism Retinol metabolism D-Alanine Benzoxazinone Pyruvate metabolism biosynthesis metabolism Glycine, serine and Valine, leucine and threonine metabolism isoleucine biosynthesis Pantothenate and CoA biosynthesis Flavone and flavonol Anthocyanin Synthesis and Porphyrin and biosynthesis biosynthesis degradation of chlorophyll Biosynthesis of type II Ketone bodies metabolism Limonene and polyketide backbone Selenocompound pinene degradation Zeatin metabolism Cyanoamino acid biosynthesis metabolism Butanoate Fatty acid elongation Monoterpenoid metabolism in mitochondria
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