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Supporting Information Supporting Information McLean et al. “Candidate Phylum TM6 Genome Recovered from a Hospital Sink Biofilm Provides the First Genomic Insights into this Uncultivated Phylum “ SI Appendix McLean et al. PNAS Supporting Information a b High Fluor TM6 Background 1 Fig. S1. Biofilm sample biomass and Fluorescence Activated Cell Sorting (FACS) plot of a 2 biofilm sample. a) Sample biomass collected directly into buffer solution and from a biofilm in 3 a sink drain within a restroom adjacent to an emergency waiting room. b) Sorting gates set to 4 sort events after staining with SYBR Green DNA stain. The P1 gate includes high fluorescent 5 SYBR Green stained particles, and the background gate indicates that region in which unstained 6 sample events were located. The low fluorescent P2 region was chosen as a sort gate to target a 7 total of 100 events in each of 32 wells of a 384 well plate. 1 Fig. S2. Custom integrated Agilent Technologies BioCel 1200 liquid handling automated 2 platform for high throughput single cell genomics. The BioCel platform allows processing of 3 more than 5,000 single cells per week through a multi-stage protocol that includes multiple 4 displacement amplification (MDA) of DNA, MDA dilution and 16S PCR, MDA and PCR hit 5 picking, Picogreen (Life Technologies) DNA quantitation, 16S Syto 9 (Life Technologies) melt 6 curve assay, 16S Taqman qPCR, and SAP/Exonuclease I (Affymetrix) PCR treatment. All liquid 7 handling is performed on the BioCel with the BioRAPTR (Beckman Coulter) and Bravo 8 (Agilent) performing non-contact dispensing and liquid transfer steps, respectively. The MDA 9 isothermal reaction and PCR are performed offline on GeneAmp PCR system 9700 10 thermocyclers (Applied Biosystems), while TaqMan or melt curve analysis are performed in- 11 line on the ABI 7900HT (Applied Biosystems). The platform includes barcode tracking of 384- 12 well plates, and is integrated with a JCVI Laboratory Information Management System (LIMS). leum 7 more 2 more 15% Chryseobacterium Flavobacteriaceae g 0.9% Bacteroides Sphingobacteriales 45% Bacteroidales MDA1 Bacteria 21% TM6 1,041,076bp Bacteroidetes Bacteroidetes/Chlorobi group MDA2 27% TM6 1,074,690bp Viruses Proteobacteria 38% TM6 MDA3 Clostridia 998,785bp Enterobacteriaceae 0.8% Siphoviridae 0.7% Bacillales 0.8% Clostridiales 0.7% Escherichia coli 14 more 6 more Chitinophaga pinensis 4% Sphingobacteriaceae 8% Sphingobacterium 2% Pedobacter saltans 0.9% 1 Fig. S3. Identification of TM6 contigs in the assembled metagenome. Taxonomic 2 classification of assembled contigs for three independent wells representing the mini- 3 metagenomes from 100 event sorts using MGTAXA software. A 273 kb contig containing the 4 TM6 16S rRNA gene was used as a training sequence to generate a predictive model of 5 nucleotide patterns for this genome. All assembled contigs were run through this pipeline and 6 the percentage of contigs sharing similar profiles were identified and classified as belonging to 7 TM6 (green). The total contig size representing TM6 are shown for each of the three 8 independent amplified genomes. 1 Fig. S4. Comparison of assembled TM6 genomes from MDA1 and MDA3 datasets with the 2 concatenated MDA2 TM6 contigs (contigs were ordered by length and then concatenated). 3 a) Contigs for each MDA were aligned with Progressive Mauve against the concatenated MDA2 4 contigs. b) Similarity dotplots between the concatenated MDA2 TM6 contigs and TM6 contigs 5 from MDA1 and MDA3. Fig. S5. Read coverage and single nucleotide polymorphisms across the concatenated MDA2 TM6 contigs (contigs were ordered by length and then concatenated). Row 1) Reference TM6 MDA2 contigs; Row 2) GC content; Row 3) MDA2 contigs; Row 4) CDS; Row 5) RNA genes; Row 6-8) depth of mapped Illumina reads from each amplified sample; Rows 9- 11) SNPs at a cutoff of 10X coverage for each single cell amplification. 10000 2000 9000 1500 TM6SC1 1000 8000 500 y = 0.0009x + 45.77 R² = 0.8564 7000 0 0 1000000 2000000 6000 5000 Predicted CDS Predicted y = 0.0009x + 149.94 R² = 0.9721 4000 3000 2000 1000 TM6SC1 0 0 2,000,000 4,000,000 6,000,000 8,000,000 10,000,000 12,000,000 Genome Size (bp) 1 Fig. S6. Relationships between genome size of finished bacterial genomes and the number 2 of predicted coding DNA sequences CDS. (Inset) Small bacterial genomes that have less than 3 2000 predicted CDS. The TM6SC1 genome is marked in red terium 48 ium sea bacterium cterium 6 bacterium RNA uncultured candidate division TM6 bac TM6 division candidate uncultured RNA erium um HM187093 uncultured bacterium uncultured HM187093 DQ814727 uncultured bacterium uncultured DQ814727 EF203192 uncultured bacterium DQ815137 uncultured bacter uncultured DQ815137 HQ003609 uncultured bacterium AB630784 uncultured bacterium uncultured AB630784 HM186396 uncultured bacterium AB630932 uncultured ba uncultured AB630932 HQ330601 uncultured bacterium FJ405888 Verrucomicrobia bacterium YJF2 bacterium Verrucomicrobia FJ405888 HM187321 uncultured bacterium GU172187 uncultured bacterium uncultured GU172187 JF833548 uncultured proteobacterium ured bacterium ured AB630785 uncultured bacterium uncultured AB630785 EF076236 uncultured bacterium EU038006 uncultured candidate division TM6 bacterium TM6 division candidate uncultured EU038006 AM997772 uncultured deep AB630789 uncultured bacterium uncultured AB630789 EU925837 uncultured bacterium EU181509 uncultured bacterium uncultured EU181509 AM936568 uncultured candidate division TM6 bacterium JF344237 uncultured bacterium uncultured JF344237 terium acterium JN538639 uncultured organism unclassified m HQ330533 uncultured bacterium uncultured HQ330533 JN500482 uncultured organism unclassified HM270042 uncultured bacterium uncultured HM270042 HQ330553 uncultured bacterium elta proteobacterium JN869188 uncultured bacterium uncultured JN869188 AY491598 uncultured bacterium GQ402788 uncultured bacterium uncultured GQ402788 JN886887 uncultured bacterium FJ543088 uncultured bact uncultured FJ543088 EU491475 uncultured bacterium JF428967 uncult JF428967 JN860315 uncultured Acidobacteria bacterium EU266789 small subunit ribosomal subunit small EU266789 EU491195 uncultured bacterium tured bacterium tured HQ588469 uncultured bacterium uncultured HQ588469 JF495290 uncultured bacterium AF507715 uncultured soil bacteri soil uncultured AF507715 DQ811946 uncultured candidate division TM cterium FJ516811 uncultured bacterium uncultured FJ516811 EU488115 uncultured b EU048675 uncultured bac uncultured EU048675 AJ704673 uncultured d EU385703 uncultured bacterium uncultured EU385703 EU592482 uncultured bacteriu HM598149 uncul HM598149 JF344625 uncultured bacterium GU363043 uncultured bacterium uncultured GU363043 EU488076 uncultured bacterium HM598234 uncultured bacterium uncultured HM598234 JF344150 uncultured bacterium JN886884 uncultured bacterium uncultured JN886884 HQ153869 uncultured ba ism unclassified ism EU181505 uncultured bacterium uncultured EU181505 DQ787720 uncultured bacterium EU373910 uncultured delta proteobacterium delta uncultured EU373910 EU386002 uncultured bacterium HM780294 uncultured bacterium uncultured HM780294 AB177131 uncultured bacterium AB630787 uncultured bacterium uncultured AB630787 JN532782 uncultured organism unclassified ism unclassified ism HM186162 uncultured bacterium uncultured HM186162 DQ413113 uncultured bacterium JN531129 uncultured organ uncultured JN531129 X97099 uncultured bacterium * JN531183 uncultured organism unclassified organism uncultured JN531183 AB630668 uncultured bacterium rium JN530766 uncultured organism unclassified organism uncultured JN530766 rium GQ402806 uncultured bacterium JN538095 uncultured organ uncultured JN538095 AY661981 small subunit ribosomal RNA uncultured bacterium JN454351 uncultured organism unclassified organism uncultured JN454351 GU368367 uncultured bacterium ate division TM6 bacte TM6 division ate FJ439879 uncultured bacterium uncultured FJ439879 TM6 JCVI HM598261 uncultured bacte uncultured HM598261 AM162488 uncultured bacterium HQ462496 uncultured candidate division TM6 bacterium TM6 division candidate uncultured HQ462496 JF800694 uncultured bacterium DQ070831 uncultured candid uncultured DQ070831 EU135363 uncultured bacterium EU735020 uncultured bacterium uncultured EU735020 EU135362 uncultured bacterium EU236370 uncultured bacterium uncultured EU236370 EF516771 uncultured bacterium JN977250 uncultured bacterium uncultured JN977250 FJ542964 uncultured bacterium AY212595 uncultured bacterium uncultured AY212595 AB619710 uncultured bacterium sea bacterium sea JN430793 uncultured organism unclassified organism uncultured JN430793 GQ402765 uncultured bacterium EU385862 uncultured bacterium uncultured EU385862 AB630665 uncultured bacterium AM997480 uncultured deep uncultured AM997480 DQ129127 uncultured soil bacterium FJ478536 uncultured bacterium uncultured FJ478536 JQ408066 uncultured bacterium HQ190363 uncultured bacterium uncultured HQ190363 AB630783 uncultured bacterium EF018807 uncultured bacterium uncultured EF018807 JF428914 uncultured bacterium FJ542881 uncultured bacterium uncultured FJ542881 AB630666 uncultured bacterium HM127881 uncultured bacterium uncultured HM127881 GU179759 uncultured candidate division TM6 bacterium FJ437950 uncultured bacterium uncultured FJ437950 EU335393 uncultured bacterium um AB630930 uncultured bacterium uncultured AB630930 FJ542880 uncultured bacterium JN540217 uncultured Nautiliales bacteri Nautiliales uncultured JN540217 GU208307 uncultured prokaryote unclassified FJ543045 uncultured bacterium uncultured
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