Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection

SUPPLEMENTARY INFORMATION Articles https://doi.org/10.1038/s41587-019-0156-5 In the format provided by the authors and unedited. Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection Themoula Charalampous 1,8, Gemma L. Kay 1,2,8, Hollian Richardson 1,8, Alp Aydin 2, Rossella Baldan 1,3, Christopher Jeanes 4, Duncan Rae 4, Sara Grundy 4, Daniel J. Turner 5, John Wain 1,2, Richard M. Leggett 6, David M. Livermore 1,7 and Justin O Grady 1,2 * 1 Bob Champion Research and Educational Building, University of East Anglia, Norwich Research Park, Norwich, UK. 2 Quadram Institute Bioscience, Norwich Research Park, Norwich, UK. 3 CIDR, King s College London, St Thomas Hospital, London, UK. 4 Microbiology Department, Norwich and Norfolk University Hospital, Norwich, UK. 5 Oxford Nanopore Technologies, Gosling Building, Oxford Science Park, Oxford, UK. 6 Earlham Institute, Norwich Research Park, Norwich, UK. 7 AMRHAI, Public Health England, London, UK. 8 These authors contributed equally: Themoula Charalampous, Gemma L. Kay, Hollian Richardson. *e-mail: justin.ogrady@quadram.ac.uk Nature Biotechnology www.nature.com/naturebiotechnology

Supplementary Information (A) TTGTTGCTTCGTTCAGTCGGGTGGGTGTTTATGGGACCGTAAGCCTACCGTGACGTTACCAGTAGGAATGAATCCTTCGTT TTTCGTGCGCCGCTTCACTCTATAACTTGTTCCACAGCCATTTTTTGAAGATCTGGTGGACCATATTTCTTTAATAGGCGTCT AACTTGAACTTCTCATTTTAGCTTTAGCACTGTCTCGTTTGAACCAGTCAACACTCATGTTATCATAACTGTTTTTAGTTAACT CATGTGCAATGGCTCTTAACTCTTTATCGCCAAAGCTTCTTTAGCTGTTCATGTGAAGCTCGATACTACATACAATATACGC AACAATGCGCTTGTTTCGATTAGGTTTAGGCATTGAATCATAAAGTGCAATATATACTTAACACGTTCTTTAATATTTGAATTA AGACCTAGTAAATATTGTGCCATTATTTATGTGCTGAGATTTTAAAATAGAACATTTTAATATCCCGATTCTACGCAGTATAT GTTCATGGCGATTCAAAATTTTTTAAAACGTTTCTATCACATACCTTTTCACAGTCATTGTCCATCATTGTTTTTACTTATATAT AGTGCAGGTTAAACCAGAATATCATTTTAAAAACAACATATAAAGCTGGTTGAATATTAAGTCATGACTTTTCGCATGTGATA GTTCATGTAGAATAATATATTCAATTTCTTTGTCATTCATGGTTTCATTACGACAGTTGGTATTGGTACAATTGGGATTTCACT AAACCAAATACCATCGGATTATCAATGTTTTGAACTATAACTAATTGTTATATGCTTTTTTGTGCGTTACATCTTACTTTGACA TACTTTAAGTCGTTCATTAAGATATGACGATTCCAATGACAGACTTTAATAACATCCAATTTGTCGGAATGCCTTAATCATAT AAAAATAAGCACAACAAACTACCGAATACCATATCAAAAGAATCATATACGTTATATTTGAGGTCTCGGCAGTTAACATTAAT TTTCAAGTCTTTCGTAACAGATGATTGTTGACCATCTAACATATGACTAACCAGAAAGAAGTCGTGTCAGATACATTTCGATT CATCATATCTTTGAAAATGGAATTCGATATTTTGTAAAATGGGTATTAATGGAATTAACGTGGAGACGAGCACCTAATAACCA AATCTTATGTGACATAATATTTTGAGTATATTTTATATAGAACATTCTCACTAAAAAATTACATATCGTGAGCAATGAACTGAT TATACTTAACATTAAAAAGGTATTAACATATTCTACACCTCCATATCACAAAATTGTAACATTATTTTGACATAAATACTACATT TGTAATATACTACAAATGTGGTCTTATATAAGAGTATATTGATGAAAAGAAGATAAAAATTGTTCACACTTATTTTTAATAGT TGTAGTTGTCGGGTTTGGTATATATTTTATGCTTCAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAA TTTCAAACAAGTTTATAAGATAACAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATAATGACTGAACGTCCGAT AAAAATATATATAATAGTTAGGCGTTAAAGATATAAACATTCAGGATCCGTAAAATAAAAAAGTATCTAAAATAAAAACA GTAGATGCTCAATATAAAATTAAAACAAACTGGTAGCATTGATCCGCAGCGTTCCGTTTAATTTTGTTAAAAGATGGTAT GTGGAAGTTAGGTGGGATCATAGCGTCATTATTCAGAATGCAGAAAGACCAAAGCATACATATTGAAAATTTAAAATCA GAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCAATACAGGAACAGCATATGAGATAGGCATCGTTCCAA AGAATGTATCTAAAAGATTATAAAACAATCGCTAAAGAACTAAGTATTTCTGAAGACTATATCAAACCAACAAATGGATC AAAATTGGGTACAAAGATGATACCTTCGTTCTTTTAAAACCGTTAAAAATGGATGAATATTTAAGTGATTTCGCAAAATTT CATCTTACAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGAAAAACGACTTCACATCTATTAGGTTATGTTGGTCCC ATTAACTCTGAAGAATTAAAACAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAAAAGGGACTCGAAAA ACTTTACGATAAAAAACCAACATGAAGATGGCTATCGTGTCACAATCGTTGACGATAATAGCAATACAATCGCACATAC ATTAATAGAGAAAAAGAAAAAGATGGCAAAGATATTCAATAACTATTGATGCTGAAGTTCAAAAGTGACTATAACAACA TGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAAACAGGTGAATTATTATTAGCACTTGTAAGCACACCTTCA TATGACGTCTATCCATTTTATGTATAGCATGAGTAACGAAAAGAATATAATATAAATTAACCGAAGATGAAAGAAAAGAA CCTCTGCTCCAACAAGTTCCAGATTACAACTTCACCGAGTTCAACTCAAAAATATTAACAGCAATGATTGGGTTAAATAA CAAAACATTAGACGATAAAACAAGTTATAAAATCGATGGTAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACG CGTTACAAGATATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTTTGC TAGAGTAGCACTCAGATTAGGCAGTGAAATTTGAAAAGGCATGAAAAACTAGGTGTTGGTGAAGATATACCAAGTGATT ATCCATTTTATAATGCTCAGAATTTCAAACAAAATTTAGATAATGAAATATTGATAGCTGATTCAGGTTACGGACAAGGT GAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGCATTAGAAAGAAATAATAGCAATATTAACGCACCTCA TGATAAAAGAACTTACGAAAAACAAAGTTTGGAAGAAAAATATTTCAATGAAGCGGCGCACGAAAACAGAGGATTCATTC CACGGTAACACGTCACC (B)

(C) ATGGTTCCGTGGTGTTTAACAGTCATGTAAATGACCTTGTCCAAGTTTGTAACCTTCGTTTTTCGTGCGCCGCTTCCAATCG CGTCCTTTGTTTACACGAATGTCTTTTGCTCTTAATATTAATTCCTGCTTCCTCATCTATAATCGCATGTGCTTTCATTTTGCT TTGTATGCGGGAAAACCCTGCGATTTTAGCAGGAAGAGTTGTAATTAAAATATAATATTCCTCCAATATTGTTTATTTTAAAT CCCTATTTTCTAACCTCAGTTATCATTTTGGCAAACTATAGCAATGTAATGAGTCTAAAATACCTGATAGTAAAACCAATG ATTTACACCGATTTTATCGGCAAAAGAATCCAAGAAGAATTATTAATTGTATGAAGTGACATGATACTTTAAATCAAGAA AATACACGTCCCTAAATATTCTAGACTTAATTTTCTCCTGAAAAAGAGCTGTTTACACCATATAAATATACCAGCCCAATG TGCACAACAAACTACGAATATTATAATCCCAGCTGGAGGAAGTATTCCGAAACGGCTAAACTGGTCCCCATAAAAAATG AACTTGTTATTGGTAATACATGCTTTTCAGAAGCCCCTAATCTTCAACCTAATAAGCCTCCTGCTGGCATCCCAAATACTG AGGAAATTTCCATGTAGCAAAGTAATATACGTTCCATTAAGAGTGTTCCATGCTTATTAAAGAAAATAATTGCATTGATTG GCATATAAACAAAGTATATAGTGTTCTAAAAGAGTATAAGGCAAACAAATCCCTTTGTTTTGTCTCCAGAACCACAACTC CTTCTTTCATCTCCCTTGTGAAATTTAGTTCAAACTTTGCATGATTACCCAGCTTGGTATACGGTACAATTTACCGTATAA ATCACAGTCTGGCACCCATACGTCGATGGCAATAATAGCATTTAAATCCCAAACGGAGGTATAAGAGTGCTGCAACTGC CGGACTATTTCATATAACATAGACTGCAGACTGACTATAGCTCGCATTTCGTTAATAATTTCTTCTGGTACTAAAATTGGT GTAACCGCATTGAGAGTGCTGGGGTGGCTGTTCAATGCTACGGATAACAATACTATCGCAAATCATCAGACAGGTAGCT CCCGTCAGCAACCGAAACACTGCACTGCTTCTGCGATATTAAATCGGCACCAATCATTCCTCTTCTTCCTATCGTGATCC ACTAACTTTTCAATGGCGGTCAAATCGCATGGGGTAAAAACCTACTAATGAAGCCATAGACAGGACAAAAATGCAGATC CTGTTTTTCTGTAGGTAAAAATAATCATAATTTACGGGATGGCACTGGTCATGATACTGCTTTGCCCACTTATGTGCATAG GTACTTCGTTTCCAATTGTTTGTATTTCCATTTATATTTATCTCACCATATTCTTTATGAATTTCTAATGGCCTGAATAGCATT CCAGGCAATAAAAATGCGAATGACCAAGTAAGCTTTTGGTCTTACCTTACGCTGTTAGGTATTCATATTAAAGACCTTGGAT TGAAATTAAAGGGTATGGTTAAATAACCAATGTCCTCACCGTAACTATGAATGCTCAATATCGTATAAATAAGCACAACAAAA AGCCTATCATCGGGTATAGATTCCTTTGTTGCCAGCCAAACGCATTAGAGGCTGTCATAGTTTCGATATCTATCTTTGTTTAT ATCAATTTATGAGTAACTTTAACAAGATGATATGTTCAATAAAGGATTATGGAATGGACTCATACTTTAATTCGATTAGTTTAA AGGATATGACAGAGTTTTCTTCATGAACCGTAACGCTCTAGTTATCTTCCTTAGTGAGCAAGCGATTAGCTTTCTAAAATCTT CTGTTTGTAGTTTAGAATTCATCAGGATTTTCTAACTCAACCTCAAATATTCATTTTACATTCCTATTTCATTTATTGATAAATT AGTTTGCAAAAAATTGGACAATTTTTGTCCTCTTAACCTTTAAATAAGTTTTTTTAATCGATTTAGGTTGCCTAAATTATTGCT TATTCGGTAAATGAAGTATTGCTTTCAACAGATTTCCTTCAACTACACTTCTTGATTCAAACAAGTTGTTTATTCTTCCACAAA CTCCTTGTCAATGGAATAAAACACATGCCACAGGTAAATGAATAGAACTGATGATTTCTAAACTATCGCTACCTCATTCCAG AATTTTCTCACTCTCTAGATGCCCACCCTGTAAAGGCAAAACTGCAATCAAACTCCATTATCCTTTCGTTCAAGGACCACAA GCTTTACAAACCGGGCGACCCACACTCAGCGGTTCGCTATCTCCGTTCTGTCACGCGTGCTAACTTGTCGTCACGGACGG CTATCGCATGGAGCGGAGTAAATGTAATCTTCGTCGTTTTGCTCCTTGACCGCCAAACTACCCGCCTCAGCATGCTAAATG CATCTCCTATATTAAAACGAAAGTAACTGAGTTGTTGTTTAAATTAAAATAGAATGAAACTATTTTATAGTTACCATTTTTCAA CACCAGCTGATATTCATCCGCCAAAAATTTACCCTTTAACTTACGGCAAACTTGTTGAGTCACATAGCACTTTTAACGTCCA ACTTCACCATTCTTCTTTAATCAACTCTTTTATTATTCAGCTTTTCTCAATCAAATGCCAACTAGGAATGGCCGTTGCTTTAAT ATCCATCGTACCATTCTCCATTCCCTGCGCTTGATTGCATTGCTGATATTTCTTCAGCTCCGTAGGAACGGGCCATGGAGC ACCAACTATGTTGTTGAAAGAATATAGTGAAGCGGCCGCACGAGAAAAACGAAGGTTACGCAAACCCTGGACAGGTGGG (D)

Supplementary Figure 1: The metagenomic pipeline with a turnaround time of approx. six hours (optimised) and approx. eight hours (pilot) from sample collection to sample result inferring antibiotic resistance gene/organism association using resistance gene flanking sequence. A: An example MinION read mapping to the meca gene (gene sequence in bold)* B: Identification of Staphylococcus aureus chromosome sequence using the meca gene 5 flanking sequence of the read*. C: An example of a MinION read mapping to the gene (gene sequence in bold)*. D: Identification of Streptococcus gordonii chromosome sequence using the gene 3 flanking sequence of the read*. *Three independent clinical samples were analysed (an example of a pathogen and a lung commensal are represented).

Supplementary Table 1a: Comparison of bacterial DNA extraction methods using qpcr. Sample Sample type Microbiology result O1 Sputum S. aureus O2 Sputum P. aeruginosa Condition 16S rrna gene V3-V4 fragment qpcr assay (Cq) Bacterial gain to standard depletion (DCq) Human qpcr assay (Cq) New DNA depletion compared to standard depletion (DCq) Standard depletion 18.22-27.34 - New depletion (enzyme 16.66 1.56 27.22 0.12 cocktail) (3 fold) New depletion (bead beating) 13.84 4.38 (21 fold) 28.4 1.04 (2 fold) Standard depletion 12.06-27.92 - New depletion (enzyme 12.65 0.59 27.46 0.46 cocktail) New depletion (bead beating) 12.18 0.12 27.95 0.03

Supplementary Table 1b: Comparison of the microbial community with altered PCR extension times for MinION sequencing. Sample Sample type Microbiology result PCR extension time (min) Average read length (Kb) Bacterial species from WIMP analysis ( 0.5% of classified reads) O1 Sputum S. aureus 4 2.6 S. aureus Streptococcus agalactiae Streptococcus anginosus group Veillonella parvula 6 3.2 S. aureus Streptococcus agalactiae Streptococcus anginosus group O2 Sputum P. aeruginosa 4 2.7 P. aeruginosa group Pseudomonas P. stutzeri group 6 2.4 P. aeruginosa group Pseudomonas Percentage of total classified reads (%) 76.1 7.1 2.3 0.9 0.5 78.7 6.1 1.4 0.9 87.1 1.1 1.0 0.7 88.2 1.0 0.6

Supplementary Table 2a: Limit of detection for Gram-positive and Gram-negative organisms using the optimised method in a high bacterial background. Sample Replicate Pathogen Approx. number of pathogen cells (CFU) DNA yield (ng/µl) Total raw read count Reads mapping to hg38 Nonhuman reads Classified from nonhuman reads Unclassified from non-human reads Total number of pathogen reads from total classified reads 1 27.0 66,528 581 (0.9%) 65,947 (99.1%) 50,922 15,015 12,658 (24.9%) SA 10 5 2 100,000 7.5 54,576 418 (0.8%) 54,158 (99.2%) 42,612 11,524 4,286 (10.1%) 3 20.6 36,285 221 (0.6%) 36,064 (99.4%) 29,303 6,749 3,882 (13.2%) 1 S. aureus 10.9 40,002 263 (0.7%) 39,739 (99.3%) 29,311 10,423 165** (0.6%) SA 10 4 2 10,000 34.6 36,416 312 (0.9%) 36,104 (99.1%) 27,233 8,860 389 (1.4%) 3 28.6 44,844 416 (0.9%) 44,428 (99.1%) 32,656 11,752 686 (2.1%) 1 26.6 37,222 259 (0.7%) 36,963 (99.3%) 28,559 8,398 1,346 (4.7%) EC 10 5 2 100,000 25.0 50,842 261 (0.5%) 50,581 (99.5%) 38,924 11,637 2,030 (5.2%) 3 25.6 45,167 208 (0.5%) 44,959 (99.5%) 34,140 10,798 1,092 (3.2%) 1 E. coli 7.8 28,035 119 (0.4%) 27,916 (99.6%) 22,324 5,586 90** (0.4%) EC 10 4 2 10,000 26.2 44,761 292 (0.7%) 44,469 (99.3%) 33,525 10,932 156** (0.5%) 3 36.6 46,920 331 (0.7%) 46,589 (99.3%) 34,319 12,244 149** (0.4%) *22Cq bacterial 16S rrna gene V3-V4 fragment qpcr assay **number of reads detected was below the ³1% of classified microbial reads and WIMP assignment q-score ³20 cut-off required for a sample to be considered positive.

Supplementary Table 2b: Limit of detection for Gram-positive and Gram-negative organisms using the optimised method in a low bacterial background*. Sample Replicate Pathogen Approx. number of pathogen cells (CFU) DNA yield (ng/µl) Total raw read count Reads mapping to hg38 Nonhuman reads Classified from nonhuman reads Unclassified from non-human reads Total number of pathogen reads from total classified reads** 1 5.0 38,213 589 (1.5%) 37,624 (98.5%) 36,271 1,343 23,514 (64.8%) SA 10 4 2 10,000 0.6 8,476 92 (1.1%) 8,384 (98.9%) 8,151 230 5,928 (72.7%) 3 1.5 14,300 581 (4.1%) 13,719 (95.9%) 12,642 1,074 3,129 (24.8%) 1 S. aureus 11.0 61,889 19,276 (31.1%) 42,613 (68.9%) 38,796 3,805 7,222 (18.6%) SA 10 3 2 1000 3.2 31,360 9,337 (29.8%) 22,023 (70.2%) 19,905 2,115 3,037 (15.3%) 3 1.8 25,346 2,531 (10.0%) 22,815 (90.0%) 20,6802 2,120 2,499 (12.1%) 1 1.9 29,322 879 (3.0%) 28,443 (97.0%) 26,452 1,983 10,609 (40.1%) EC 10 4 2 10,000 0.8 7,324 185 (2.5%) 7,139 (97.5%) 6,642 493 2,756 (41.5%) 3 0.9 4,058 93 (2.3%) 3,965 (97.7%) 3,637 327 1,072 (29.5%) 1 E. coli 0.8 2,645 88 (3.3%) 2,557 (96.7%) 2,271 285 110 (4.8%) EC 10 3 2 1,000 1.2 10,380 281 (2.7%) 10,099 (97.3%) 8,736 1,357 384 (4.4%) 3 1.4 15,016 543 (3.6%) 14,473 (96.4%) 12,663 1,805 555 (4.4%) *27Cq bacterial 16S rrna gene V3-V4 fragment qpcr assay **The number of reads detected for all samples was above the 1% of classified reads and WIMP assignment q-score 20 required for a sample to be considered positive.

Supplementary Table 3: Mock community qpcr results in triplicate for spiked NRF samples with and without the optimised method. qpcr assay Sample Triplicate 1 (Cq) Triplicate 2 (Cq) Triplicate 3 (Cq) Average (Cq) Bacterial loss (DCq) Human Undepleted 24.20 24.27 25.27 24.58 9.9 Depleted 33.97 34.52 34.96 34.48 (10 3 fold) E. coli Undepleted 23.47 23.53 23.99 23.66 0.12 Depleted 23.73 23.94 23.68 23.78 H. influenzae Undepleted 30.60 30.53 30.55 30.38 0.77 Depleted 31.55 30.66 31.25 31.15 K. pneumoniae Undepleted 29.96 29.78 30.29 30.01 0.05 Depleted 30.08 30.26 29.85 30.06 P. aeruginosa Undepleted 22.78 22.77 23.15 22.90 0.17 Depleted 23.07 23.14 22.99 23.07 S. aureus Undepleted 26.23 26.62 27.99 26.94 0.16 Depleted 26.19 27.35 26.80 26.78 S. maltophilia Undepleted 24.96 24.96 25.66 25.29 0.02 Depleted 25.56 24.93 25.45 25.31 S. pneumoniae Undepleted 25.66 25.70 26.68 26.01 2.52 Depleted 28.18 28.81 28.62 28.53 (5.7 fold)

Supplementary Table 4: Sequencing metadata for all respiratory samples processed with the optimised method. Sample number Number of raw reads from 2hrs Number of reads minus hg38 Human reads Classified reads Unclassified reads Metagenomics output S1 108610 108346 264 107971 364 E. coli 91178 S2 17516 17485 31 17303 182 K. pneumoniae 1692 S3 26641 26257 384 24673 1583 P. aeruginosa 15817 S4 7358 7348 10 6602 743 S. marcescens 3900 S5 19888 19865 23 19224 636 K. oxytoca 3254 K. pneumoniae 906 S6 16403 13295 3108 13271 24 S. aureus 11307 Process extraction negative 1 348 279 69 235 44 E. coli S. aureus S. pneumoniae S7 32730 21690 11040 17398 4289 P. aeruginosa H. influenzae 232 S8 49277 44120 5157 35023 9088 M. catarrhalis 7078 S. pneumoniae 504 S9 29111 28969 142 28527 437 E. coli 12974 S10 56005 51610 4395 49850 1743 H. influenzae 35348 S. pneumoniae 571 S11 43088 40944 2144 34012 6927 S. pneumoniae 693 S12 43267 38274 4993 35153 3116 M. catarrhalis H. influenzae 5573 414 Process extraction negative 2 76 63 13 57 6 S. aureus H. influenzae E. coli 12 8 5 S13 27592 27406 186 26979 425 S. marcescens 22758 S14 41154 40622 532 35413 5205 S. aureus 9465 Number of pathogen reads ( 1% of classified reads and WIMP assignment q- score 20) 10 6 3 1970

M. catarrhalis 8089 S15 37500 36537 963 30473 6058 S. aureus 6597 S. pneumoniae 390 S16 85298 85057 241 83750 1301 S. aureus 59392 S17 25499 23615 1884 19072 4541 - S16-undepleted 37352 11884 25468 11626 257 S. aureus 6203 Process extraction 27 18 9 17 1 - negative 3 S18 38902 38744 158 38092 650 H. influenzae 34396 S19 47994 42413 5581 33926 8485 - S20 46331 43070 3261 42143 920 H. influenzae 2827 S21 45214 45075 139 39288 5780 H. influenzae S. pneumoniae S22 36853 36194 659 27003 9183 - S23 33140 32933 207 29164 3766 H. influenzae 9635 20161 408 Process extraction negative 4 71 0 71 0 0 - S24 58752 57669 1083 51978 5686 H. influenzae 28443 S25 36621 35808 813 35716 89 H. influenzae 33307 S26 38138 36910 1228 33541 3367 M. catarrhalis 3831 S27 78311 78064 247 74697 3357 H. influenzae S. pyogenes S. aureus S28 35804 34699 1105 26717 7980 S. pneumoniae 464 S29 57865 9429 48436 9153 275 P. aeruginosa S. aureus 6838 1187 Process extraction negative 5 56 46 10 41 5 H. influenzae S. aureus E. coli S. pyogenes 13 5 2 2 S1-undepleted 51137 1121 50016 1101 17 E. coli 803 31884 19544 9969

S30 27371 27217 154 27154 60 P. aeruginosa 21577 S31 43823 13463 30360 9567 3895 H. influenzae 1644 S32 48271 47988 283 46882 1101 E. coli 35094 S33 32085 19181 12904 18722 459 - S34 40998 35522 5476 35031 489 - Process extraction 46 35 11 32 3 E. coli 10 negative 6 S35 47893 29765 18128 29663 96 E. coli 26316 S36 33155 32774 381 31170 1603 H. influenzae 23245 S37 60495 60083 412 59644 432 P. aeruginosa 47138 S38 23889 12947 10942 12833 113 S. aureus P. aeruginosa 1248 253 S39 60316 59972 344 57940 2029 M. catarrhalis H. influenzae 32133 14404 S40 48848 7444 41404 7340 104 S. aureus 6138 Process extraction negative 7 105 69 36 63 6 E. coli H. influenzae M. catarrhalis P. aeruginosa S. aureus S41 2320 2129 191 2040 89 S. aureus H. influenzae E. coli 12 9 4 3 3 744 343 35

Supplementary Table 5: Confirmatory qpcr analysis of additional pathogen detection. Discordant results compared to culture Sample qpcr gene target qpcr result (Cq) S32 22.6 Escherichia coli 1 S41 cyaa - S10 25.4 Haemophilus influenzae 1 S12 omp P6 - S21 32.0 S31 29.2 Klebsiella pneumoniae 1 S5 mdh - S12 23.8 Moraxella catarrhalis 1 S14 copb 22.0 S39 23.5 Pseudomonas aeruginosa* S9 oprl - Pseudomonas aeruginosa 1 S7 oprl 32.9 Staphylococcus aureus 1 S29 eap 32.7 S8 - S10 26.1 Streptococcus pneumoniae 1 S11 ply 32.2 S15 - S21 - S28 - Streptococcus pyogenes 1 S27 sdab 28.7 1 Detected by metagenomics and not culture *Detected by culture but not metagenomics

Supplementary Table 6: Species-specific gene analysis for H. influenzae and S. pneumoniae Pathobionts detected by metagenomics pipeline Haemophilus influenzae Streptococcus pneumoniae Sample Pathobiont specific gene Number of reads aligned to pathobiont specific gene S7 5 S10 85 S12 0 S18 85 S20 67 S21 siat 49 S23 21 S24 61 S25 90 S27 63 S31 5 S36 60 S39 43 S41 2 S8 0 S10 5 S11 19 S15 ply 0 S21 0 S28 0

Supplementary Table 7: Microbiology antibiogram and ARMA output for all optimised method samples. Sample Microbiology Antibiogram culture result S1 E. coli Amoxicillin R, Gentamicin S, Co-amoxiclav R, Co-trimoxazole R, Tazocin I, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Ceftriaxone S, Cefuroxime S, Amikacin S, Ertapenem S, Tigecycline S, Tobramycin S, Cefepime S S2 K. pneumoniae Amoxicillin R, Gentamicin S, Co-amoxiclav R, Co-trimoxazole S, Tazocin I, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Ceftriaxone S, Cefuroxime S, Amikacin S, Ertapenem S, Tigecycline S, Tobramycin S, Cefepime S S3 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Ceftazidime S, Meropenem S S4 S. marcescens Gentamicin S, Co-trimoxazole S, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Amikacin S, Tigecycline S, Tobramycin S, Levofloxacin S, Colistin R, Cefepime S, Minocycline S, Ticarcillin S S5 K. oxytoca Gentamicin S, Co-trimoxazole S, Tazocin I, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Amikacin S, Tigecycline S, Tobramycin S, Levofloxacin S, Colistin S, Cefepime S, Imipenem S, Minocycline S, Ticarcillin R S6 S. aureus Flucloxacillin S, Erythromycin/clarithromycin S, Clindamicin S, Fuscidic acid S, Tetracycline/doxycycline S, Mupirocin S S7 H. influenzae Amoxicillin R, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S8 M. catarrhalis Amoxicillin R, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S9 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Meropenem S, Ceftazidime S E. coli Co-amoxiclav R, Co-trimoxazole R, Tazocin S, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Ceftriaxone S, Cefuroxime S, Amikacin S, Ertapenem S, Tigecycline S, Tobramycin R, Cefepime S ARMA Output sul1 mpha dfra17 aada5 ACT-5 oqxb oqxa InuA OXA-50 catb7 APH(3 )- llb AAC(6 )- lc oqxb OXY-4-1 oqxb APH(3')- Ia vgac tet38 teto IsaC ermc sul1 aada2 AAC(3 )- lla mpha dfra12 AAC(3 )- llc S10 NSG ND

teto S11 NRF ND S12 NRF ND ErmB S13 S. marcescens Gentamicin S, Co-amoxiclav R, Co-trimoxazole S, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Ceftriaxone S, Cefuroxime R, Amikacin S, Ertapenem S, Tigecycline I, Tobramycin S, Cefepime S S14 S. aureus Flucloxacillin S, Erythromycin/clarithromycin S, Clindamicin S, Fuscidic acid S, Tetracycline/doxycycline S, Mupirocin S S15 S. aureus Flucloxacillin S, Erythromycin/clarithromycin S, Clindamicin S, Fuscidic acid S, Tetracycline/doxycycline S, Mupirocin S S16 S. aureus/mrsa Penicillin R, Flucloxacillin R, Oxacillin R, Erythromycin S, Clindamycin S, Trimethoprim R, Gentamicin R, Ciprofloxacin R, Fusidic acid R, Mupirocin S, Rifampicin S, Vancomycin S, Teicoplanin S, Tigecycline S, Linezolid S AAC(6 )- lc oqxb tet38 tetq tet38 tetw tet38 meca ErmB S17 NRF ND tetq ErmF CfxA3 S18 H. influenzae Amoxicillin R, Tetracycline/doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Co-trimoxazole S TEM-70 TEM-33 TEM-105 TEM-104 TEM-208 S19 NRF ND tetw ErmX

S20 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S teto S21 NRF ND tet32 S22 NRF ND tetq S23 H. influenzae Amoxicillin S, Tetracycline/doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Co-trimoxazole S S24 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S25 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S26 M. catarrhalis Amoxicillin R, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S27 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S teto tetd tet32 tetw teta teto ermt tet38 S. aureus Flucloxacillin S, Erythromycin R, Clindamycin R, Fuscidic acid S, Tetracycline S, Mupirocin S S28 NRF ND lsac S29 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Meropenem S, Ceftazidime S S30 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Meropenem S, Ceftazidime S OXA-50 catb7 tet38 OXA-50 catb7 S31 NRF ND S32 NSG ND

AAC(3)- lla AAC(3)- llc vgac TEM-1 S33 NRF ND S34 NSG ND lsaa ErmB S35 E. coli Amoxicillin R, Gentamicin S, Co-amoxiclav R, Co-trimoxazole S, Tazocin R, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime I, Ceftriaxone S, Cefuroxime S, Amikacin S, Ertapenem S, Tobramycin S S36 H. influenzae Amoxicillin R, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole R S37 P. aeruginosa Gentamicin S, Tazocin R, Ciprofloxacin S, Meropenem S, Ceftazidime R S38 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Meropenem S, Ceftazidime S TEM-11 TEM-2 TEM-187 ACT-5 TEM-67 ErmB Mel catb7 OXA-50 tet38 S. aureus Flucloxacillin S, Erythromycin S, Clindamycin S, Fuscidic acid S, Tetracycline S, Mupirocin S S39 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole R S40 S. aureus (MRSA) Penicillin R, Flucloxacillin R, Oxacillin R, Erythromycin S, Doxycycline S, Clindamycin S, Trimethoprim S, Gentamicin S, Ciprofloxacin R, Fuscidic acid S, Rifampicin S, Vancomycin S, Teicoplanin S, Tigecycline S, Linezolid S, Daptomycin S, Chloramphenicol S S41 S. aureus Flucloxacillin S, Erythromycin S, Clindamycin S, Fuscidic acid S, Tetracycline S, Mupirocin S meca tet38

H. influenzae Tetracycline S, Amoxicillin S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole R R = resistant S = sensitive

Supplementary Table 8: All microorganisms identified in all samples tested using the optimised method (above our thresholds). Sample Organism cultured by microbiology Organisms identified from metagenomic pipeline ( 1% of classified reads and WIMP assignment q-score 20) S1 Escherichia coli Escherichia coli S2 Klebsiella pneumoniae Klebsiella pneumoniae S3 Pseudomonas aeruginosa Pseudomonas aeruginosa S4 Serratia marcescens Serratia marcescens Pantoea stewartii Citrobacter freundii Salmonella enterica Serratia plymuthica S5 Klebsiella oxytoca Klebsiella oxytoca Citrobacter freundii Klebsiella sp. M5al Klebsiella pneumoniae Klebsiella michiganensis S6 Staphylococcus aureus Staphylococcus aureus S7 Haemophilus influenzae Pseudomonas aeruginosa Veillonella parvula Rothia mucilaginosa Streptococcus sanguinis Haemophilus influenzae Haemophilus parainfluenzae Neisseria sicca S8 Moraxella catarrhalis Moraxella catarrhalis Streptococcus mitis Veillonella parvula Streptococcus salivarius Rothia mucilaginosa Streptococcus pseudopneumoniae Streptococcus pneumoniae Neisseria sicca Streptococcus gordonii S9 Pseudomonas aeruginosa & Escherichia coli Escherichia coli Lactobacillus paracasei Lactobacillus casei Candida albicans S10 NSG Haemophilus influenzae Streptococcus pseudopneumoniae Streptococcus sp. oral taxon 431

Streptococcus pneumoniae Streptococcus mitis S11 NRF Streptococcus sp. A12 Streptococcus mitis Rothia mucilaginosa Bifidobacterium longum Streptococcus pseudopneumoniae Streptococcus pneumoniae Streptococcus sp. I-P16 Streptococcus sp. I-G2 Haemophilus parainfluenzae S12 NRF Haemophilus parainfluenzae Moraxella catarrhalis Streptococcus gordonii Neisseria sicca Haemophilus influenzae S13 Serratia marcescens Serratia marcescens S14 Staphylococcus aureus Staphylococcus aureus Moraxella catarrhalis Rothia mucilaginosa Streptococcus constellatus Fusobacterium nucleatum Fusobacterium periodonticum Streptococcus anginosus Streptococcus intermedius S15 Staphylococcus aureus Staphylococcus aureus Streptococcus mitis Citrobacter koseri Rothia mucilaginosa Streptococcus salivarius Haemophilus parainfluenzae Streptococcus pseudopneumoniae Streptococcus pneumoniae Prevotella aninogenica S16 MRSA Staphylococcus aureus Lactobacillus rhamnosus S17 NRF Streptococcus salivarius Prevotella aninogenica Streptococcus sp. oral taxon 431 Streptococcus sp. A12 Streptococcus sp. NPS 308 Streptococcus sp.fdaargos_192 S18 Haemophilus influenzae Haemophilus influenzae

S19 NRF Streptococcus salivarius Streptococcus mitis Streptococcus sp.fdaargos_192 Streptococcus sanguinis Rothia mucilaginosa Veillonella parvula Streptococcus sp. oral taxon 431 Streptococcus gordonii Streptococcus constellatus S20 Haemophilus influenzae Haemophilus influenzae Rothia mucilaginosa Streptococcus mitis S21 NRF Haemophilus influenzae Streptococcus mitis Haemophilus parainfluenzae Streptococcus sp. oral taxon 431 Streptococcus salivarius Streptococcus pneumoniae S22 NRF Prevotella aninogenica Streptococcus mitis Streptococcus sanguinis Rothia mucilaginosa Veillonella parvula Haemophilus parainfluenzae Streptococcus salivarius Streptococcus sp. A12 Streptococcus cristatus S23 Haemophilus influenzae Haemophilus influenzae Streptococcus salivarius Veillonella parvula Streptococcus pseudopneumoniae Streptococcus mitis Streptococcus sp.fdaargos_192 S24 Haemophilus influenzae Haemophilus influenzae Haemophilus parainfluenzae Veillonella parvula Streptococcus salivarius Streptococcus sp. oral taxon 431 S25 Haemophilus influenzae Haemophilus influenzae S26 Moraxella catarrhalis Streptococcus mutans Moraxella catarrhalis Veillonella parvula

S27 Haemophilus influenzae & Staphylococcus aureus Streptococcus salivarius Streptococcus gordonii Rothia dentocariosa Lactobacillus salivarius Haemophilus influenzae Streptococcus pyogenes Staphylococcus aureus Streptococcus salivarius S28 NRF Streptococcus mitis Streptococcus salivarius Veillonella parvula Streptococcus sp. A12 Streptococcus sanguinis Rothia mucilaginosa Streptococcus sp. oral taxon 431 Streptococcus pneumoniae Streptococcus pseudopneumoniae Prevotella aninogenica Streptococcus sp. I-G2 Streptococcus sp. I-P16 Haemophilus parainfluenzae S29 Pseudomonas aeruginosa Pseudomonas aeruginosa Staphylococcus aureus S30 Pseudomonas aeruginosa Pseudomonas aeruginosa S31 NRF Haemophilus influenzae Streptococcus anginosus Prevotella intermedia Tannerella forsythia Bifidobacterium longum Rothia mucilaginosa Streptococcus gordonii Veillonella parvula Campylobacter concisus S32 NSG Escherichia coli S33 NRF Streptococcus salivarius Clavispora lusitaniae Streptococcus sp.fdaargos_192 Rothia mucilaginosa Saccharomyces cerevisiae S34 NSG Candida albicans Enterococcus faecalis S35 Escherichia coli Escherichia coli S36 Haemophilus influenzae Haemophilus influenzae Veillonella parvula

Streptococcus salivarius Streptococcus mitis S37 Pseudomonas aeruginosa Pseudomonas aeruginosa S38 Staphylococcus aureus & Pseudomonas aeruginosa Staphylococcus aureus Pseudomonas aeruginosa S39 Haemophilus influenzae Moraxella catarrhalis Haemophilus influenzae S40 MRSA Staphylococcus aureus Streptococcus constellatus Streptococcus anginosus Streptococcus intermedius S41 Haemophilus influenzae & Staphylococcus aureus Staphylococcus aureus Haemophilus influenzae Neisseria sicca Escherichia coli

Supplementary Table 9: qpcr gene targets and primer sequences. Organism Gene target Forward primer (5-3 ) Reverse primer (5-3 ) Probe (5-3 ) Reference Human Bacteria RNA polymerase A 16S rrna gene V3-V4 fragment TGAAGCCGTGCGGAA GG TCGTCGGCAGCGTCA GATGTGTATAAGAGAC AGCCTACGGGNGGCW GCAG E. coli cyaa CGATAATCGCCAGATG GC H. influenzae omp P6 AGCGGCTTGTAGTTCC TCTAACA K. pneumoniae mdh CGGGCGTAGCGCGTA A M. catarrhalis copb GGTGAGTGCCGCTTTT ACAAC P. aeruginosa oprl AGCCTTCCTGGTCCCC TTAC S. aureus eap ACTGTAACTTTGGCAC TGG S. maltophilia 23S rrna GCCGAAAGCCCAAGG TTT S. pneumoniae ply GCTTATGGGCGCCAA GTCTA ACAAGAGAGCCAAGTGTCG GTCTCGTGGGCTCGGAGATGTGTA TAAGAGACAGGACTACHVGGGTAT CTAATCC CCTAAGTTGCAGGAGATGG CAACAGAGTATCCGCCAAAAGTT GATACCCGCATTCACATTAAACAG TGTATCGCCTGCCAAGACAA CCTAATGAACCCCAGTGTATAAGT TTG GCAGATACCTCATTACCTGC CGACTTTCGTCCTCGCCTTA CAAAGCTTCAAAAGCAGCCTCTA [6FAM]TACCACGTCAT CTCCTTTGATGGCTCC TAT[BHQ1] Sybr Green Master Mix [6FAM]TAGAGCGCCTT CGGTGTCGGT[BHQ1] [6FAM]CGATGCTGCAG GCAATGGTGCT[BHQ1] [6FAM]CCCGGCATGGA TCGTTCCGA [BHQ1] [6FAM]TGCTTTTGCAG CTGTTAGCCAGCCTAA G[BHQ1] [6FAM]TGAACTGACGG TCGCCAACGGTT[BHQ 1] [6FAM]ATCGCAACGAC TGGCGCTA[BHQ1] Sybr Green Master Mix [6FAM]CTCAAGTTGGA AACCACGAGTAAGAGT GATGAA[BHQ1] Designed in house 56 Designed in house 15 15 15 15 Designed in house 15 15

S. pyogenes sdab GGRACACGTACCCAA AATGTAGGA TCTTGAGCTCTTTGTTCGGTRTAG [6FAM]CGTGACCAAAA AGGCGGCATGC[BHQ1 ] 15