Complete Genomes of E. coli with Diverse K antigens

Complete Genomes of E. coli with Diverse K antigens | Research Square Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Data Note Complete Genomes of E. coli with Diverse K antigens Carine Roese Mores, Christopher M. Field, Hans-Joachim Ruscheweyh, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9087730/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Escherichia coli is a diverse species with commensal and pathogenic lifestyles. Capsular polysaccharides (K antigens) are prominent surface structures, important virulence factors and targets for immune recognition. The complete genomes of 68 strains with serologically distinct capsule types were sequenced using a combination of PacBio/Oxford Nanopore and Illumina technologies. General Microbiology ANNOUNCEMENT Escherichia coli is a common and generally benign colonizer of the human gut. However, some strains can cause serious infections and pose a significant health risk ( 1 , 2 ). The capsular polysaccharide (CPS), a major surface antigen (K antigen), stands as an important virulence factor of pathogenic strains. Despite this, our understanding of CPS epidemiology in E. coli infections is limited due to the labor-intensive and costly nature of in vitro serotyping ( 3 ). Clinical and diagnostic laboratories have increasingly adopted whole genome sequencing (WGS) to phenotype bacterial isolates in silico . However, the scarcity of links between CPS serotyping and genome data limits the inference of E. coli capsule types based on WGS. Here, we sequenced the genomes of 68 E. coli strains from a K-antigen reference strain collection. Cryostocked strains were streaked on LB agar plates and incubated at 37°C for 24 h. Single colonies were transferred to LB, incubated at 37°C for 24 h and genomic DNA was extracted from cell pellets using NucleoBond AXG 20 columns with the buffer set III (Macherey-Nagel, Düren, Germany). Concentration, purity, and fragment size were measured using a Qubit fluorometer, a Nanodrop spectrophotometer and a Femto Pulse system, respectively (Thermo Fisher Scientific, USA, Agilent Technologies, USA). For Illumina sequencing, libraries were generated with the Nextera XT kit and sequenced (2 x 150 bp) on the NovaSeq 6000 system. Raw reads were quality-controlled to remove adapter and PhiX sequences, and to trim low-quality bases using bbmap v38.87 ( 4 ) with the "trimq"option of 14 and "minlength" of 45. For PacBio sequencing, DNA was sheared to an average size of 7–10 kb using a Megaruptor 3 device (Diagenode) and was not size selected. Libraries were constructed using the SMRTbell Prep Kit 3.0 and sequenced on the Sequel IIe system using the Sequencing Kit 2.0 (one SMRT Cell 8 M, HiFi mode, 15 h). QC was performed during the run using the Control SW Version 11.0.0.144466. Oxford Nanopore Technology (ONT) libraries were prepared using the rapid barcoding kit (SQK-RBK114) and sequenced using a MinION FLO-MIN114 flow cell (MinION Mk1C device, 72 h). Guppy v6.4.6 ( 5 ) was used for basecalling, demultiplexing and quality-controlling the ONT reads. Assemblies were performed with Flye v2.9 ( 6 ) with the "pacbio-hifi" and "nano-raw" options for PacBio (“Q20+”) and ONT (“pass”) reads, respectively. Quality-controlled Illumina reads were used to polish all assemblies using Pilon v1.23 ( 7 ). All tools were run with default parameters unless otherwise specified. The genomes exhibited a size range from 4.5 to 5.2 Mb (Table 1) and were annotated with the NCBI PGAP ( 8 ). Visualization of assembly graphs and verification of completeness was performed using Bandage ( 9 ). Overall, the reported genomes provide unprecedented access to study the molecular basis underlying the diversity of K antigens in E. coli . Declarations ACKNOWLEDGMENTS This work was supported by funding from the Basel Research Centre for Child Health (BRCCH) to E.S. and S.S., the Swiss National Science Foundation through the project grants 40B2-0_180953 and 310030_185128 to E.S. and the NCCR Microbiomes (51NF40_180575) to E.S. and S.S, and core funding from ETH Zürich. ONT basecalling was performed on the ETH Euler cluster. We thank Gregor Rot for bioinformatics support and acknowledge the IT Service and HPC facilities of ETH Zürich. Data availability. The genomes and sequencing data reported in this study have been deposited at the European Nucleotide Archive under the project PRJEB83217 with accession numbers listed in Table 1. All scripts used to generate the reported assemblies are available on GitHub ( https://github.com/carinemores/ecoliCPS ). References Bonten M, Johnson JR, van den Biggelaar AHJ, Georgalis L, Geurtsen J, De Ibarra P, Gravenstein S, Verstraeten T, Hermans P, Poolman JT Epidemiology of Escherichia coli Bacteremia: A Systematic Literature Review https://doi.org/10.1093/cid/ciaa210 MacKinnon MC, McEwen SA, Pearl DL, Lyytikäinen O, Jacobsson G, Collignon P, Gregson DB, Valiquette L, Laupland KB (2021) Mortality in Escherichia coli bloodstream infections: a multinational population-based cohort study. BMC Infect Dis 21:1–10 Fratamico PM, DebRoy C, Liu Y, Needleman DS, Baranzoni GM, Feng P (2016) Advances in Molecular Serotyping and Subtyping of Escherichia coli. Front Microbiol 7:644 Bushnell B (2014) BBMap: A Fast, Accurate, Splice-Aware Aligner. United States. https://www.osti.gov/servlets/purl/1241166 Wick RR, Judd LM, Holt KE Performance of neural network basecalling tools for Oxford Nanopore sequencing https://doi.org/10.1186/s13059-019-1727-y Kolmogorov M, Yuan J, Lin Y, Pevzner PA Assembly of long, error-prone reads using repeat graphs https://doi.org/10.1038/s41587-019-0072-8 Walker BJ, Abeel T¤, Shea T, Priest M, Abouelliel A (2014) Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement. PLoS ONE 9:112963 Tatusova T, Dicuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J (2016) NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 44:6614–6624 Wick RR, Schultz MB, Zobel J, Holt KE (2015) Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 31(20):3350–3352. 10.1093/bioinformatics/btv383 Table 1 Table 1. Genome assembly statistics and accession numbers Capsular serotype Source No. of short reads Long read technology No. of long reads Long read N50 (bp) Genome length (bp) GC content (%) Genome coverage ECEs GenBank accession no. Short read accession no. Long read accession no. K1 SSI 85361 8'843'528 PacBio 37'200 9'485 4'961'657 50.71 61 1 GCA_977947085 ERR14050185 ERR14041770 K2a CCUG 27 11'751'454 ONT 57'349 25'061 5'125'785 50.54 49 1 GCA_977947715 ERR14050194 ERR14041773 K2ab SSI 85362 9'401'764 PacBio 38'443 9'226 4'700'954 50.53 68 1 GCA_977947245 ERR14050196 ERR14041788 K3 SSI 85363 11'370'150 PacBio 34'889 9'383 5'141'176 50.41 57 2 GCA_977947575 ERR14050206 ERR14041791 K4 SSI 85364 14'322'598 PacBio 36'172 9'928 4'763'144 50.71 66 1 GCA_977947105 ERR14050210 ERR14041794 K5 SSI 85365 10'278'192 ONT 88'384 17'421 5'227'725 50.75 41 3 GCA_977947595 ERR14050213 ERR14041797 K6 SSI 81964 8'440'706 ONT 30'422 21'162 5'167'108 50.42 31 1 GCA_977947035 ERR14050216 ERR14041799 K7 SSI 81965 11'578'402 PacBio 41'401 9'538 4'736'535 50.63 76 1 GCA_977947585 ERR14050218 ERR14041800 K8 SSI 85366 10'442'820 PacBio 45'287 9'376 4'750'763 50.92 81 0 GCA_977947505 ERR14050223 ERR14041802 K9 SSI 85367 7'893'366 ONT 92'184 20'616 4'913'262 50.78 50 0 GCA_977946355 ERR14050225 ERR14041805 K10 SSI 85368 9'330'748 PacBio 30'281 9'653 4'883'361 50.79 53 2 GCA_977947665 ERR14050229 ERR14041806 K11 SSI 85369 9'423'246 ONT 88'383 19'801 4'573'209 50.74 53 4 GCA_977947875 ERR14050233 ERR14041809 K12 SSI 81966 3'639'474 ONT 96'876 15'401 5'286'215 50.45 40 2 GCA_977946375 ERR14050238 ERR14041813 K13 SSI 81967 11'463'720 PacBio 26'200 9'413 5'078'894 50.46 42 0 GCA_977947965 ERR14050242 ERR14041814 K14 SSI 85370 9'456'546 PacBio 35'501 9'568 4'581'132 50.71 68 0 GCA_977946545 ERR14050246 ERR14041815 K16 SSI 85371 8'768'754 PacBio 37'824 9'719 5'007'734 50.49 62 2 GCA_977947945 ERR14050249 ERR14041818 K17 SSI 85372 8'458'344 PacBio 37'922 9'152 4'600'220 50.8 69 1 GCA_977947915 ERR14050252 ERR14041819 K18a SSI 81968 8'888'756 PacBio 35'780 9'325 5'068'856 50.71 57 2 GCA_977946865 ERR14050254 ERR14041821 K18ab SSI 85373 486'230 ONT 27'461 15'050 5'069'166 50.72 35 2 GCA_977946875 ERR14050255 ERR14041824 K19 SSI 85374 8'343'316 PacBio 41'642 9'692 4'880'777 50.7 75 0 GCA_977947905 ERR14050258 ERR14041828 K20 SSI 85375 10'728'454 PacBio 38'318 9'860 4'991'724 50.86 70 0 GCA_977947155 ERR14050261 ERR14041829 K22 CCUG 11329 7'852'506 PacBio 38'024 8'664 5'085'183 50.72 56 2 GCA_977947955 ERR14050263 ERR14041831 K23 SSI 81970 8'639'116 ONT 95'956 21'074 5'125'131 50.42 92 0 GCA_977946625 ERR14050266 ERR14041833 K24 SSI 81971 11'535'816 ONT 58'864 34'261 5'105'026 50.46 59 0 GCA_977946345 ERR14050269 ERR14041836 K26 SSI 81972 9'819'104 PacBio 36'939 10'507 4'555'035 50.91 73 1 GCA_977946425 ERR14050272 ERR14041838 K27 SSI 81973 10'544'150 PacBio 31'751 11'071 4'646'608 50.93 68 0 GCA_977946285 ERR14050275 ERR14041842 K28 SSI 81974 7'338'250 PacBio 31'365 10'747 4'654'761 51.05 65 0 GCA_977946325 ERR14050278 ERR14041843 K29 SSI 81975 3'251'212 ONT 23'279 37'588 4'828'683 50.79 39 2 GCA_977946365 ERR14050279 ERR14041845 K30 SSI 81976 9'417'840 PacBio 37'295 9'715 4'808'187 50.87 64 2 GCA_977946635 ERR14050282 ERR14041847 K31 SSI 81977 10'874'260 PacBio 32'419 11'105 4'521'692 51.04 71 1 GCA_977947365 ERR14050286 ERR14041849 K32 SSI 81978 7'818'130 PacBio 31'234 10'974 4'943'816 50.84 62 1 GCA_977946405 ERR14050288 ERR14041851 K33 SSI 81979 8'403'202 PacBio 30'205 11'803 4'597'013 50.98 67 1 GCA_977946605 ERR14050291 ERR14041853 K34 SSI 81980 5'957'044 PacBio 29'718 11'398 4'529'990 50.91 67 0 GCA_977946575 ERR14050294 ERR14041857 K35 SSI 81981 6'432'850 PacBio 30'641 9'039 4'610'049 50.91 52 1 GCA_977946905 ERR14050296 ERR14041858 K36 SSI 81982 6'876'172 PacBio 42'137 9'185 4'897'268 50.83 72 0 GCA_977946735 ERR14050324 ERR14041861 K37 SSI 81983 8'387'472 PacBio 39'222 8'648 4'597'789 50.97 68 0 GCA_977946935 ERR14050363 ERR14041863 K38 SSI 81984 9'360'838 PacBio 29'217 8'653 4'842'863 51.08 47 0 GCA_977946685 ERR14050365 ERR14041864 K39 SSI 81985 10'294'304 PacBio 37'282 9'274 4'682'936 50.84 67 1 GCA_977946655 ERR14050368 ERR14041865 K40 SSI 81986 10'043'060 PacBio 33'985 9'822 4'542'529 50.81 66 1 GCA_977946775 ERR14051037 ERR14041867 K41 SSI 81987 8'383'774 PacBio 37'163 9'549 4'797'978 50.89 67 1 GCA_977946705 ERR14051039 ERR14041869 K42 SSI 81988 7'951'674 PacBio 37'978 9'526 4'791'211 50.89 67 1 GCA_977946795 ERR14051042 ERR14041870 K43 SSI 81989 4'254'874 PacBio 37'266 9'959 4'827'059 50.83 69 1 GCA_977946825 ERR14051044 ERR14041872 K44 SSI 81990 8'643'794 PacBio 28'320 11'510 4'938'150 50.73 56 2 GCA_977947275 ERR14051047 ERR14041874 K45 SSI 81991 10'978'198 PacBio 32'373 10'729 5'010'117 50.62 62 1 GCA_977947375 ERR14051121 ERR14041875 K46 SSI 81992 9'280'636 PacBio 32'459 10'445 4'805'835 50.8 63 0 GCA_977946895 ERR14051122 ERR14041877 K47 SSI 81993 10'409'706 PacBio 33'154 11'599 4'883'445 50.75 69 1 GCA_977947135 ERR14051123 ERR14041879 K48 SSI 81994 10'165'850 PacBio 23'045 10'255 4'960'040 50.74 41 1 GCA_977946915 ERR14051160 ERR14041881 K49 SSI 81995 9'169'518 PacBio 31'890 10'362 4'725'875 50.8 63 0 GCA_977946475 ERR14051161 ERR14041884 K50 SSI 81996 10'168'774 PacBio 31'925 10'689 5'081'262 50.66 59 1 GCA_977947205 ERR14051162 ERR14041886 K51 SSI 81997 8'834'586 PacBio 33'349 10'048 5'214'915 50.6 56 1 GCA_977947185 ERR14051202 ERR14041887 K52 SSI 81998 8'566'464 ONT 17'920 22'161 5'093'197 50.81 28 1 GCA_977947195 ERR14051208 ERR14041889 K53 SSI 81999 5'866'276 PacBio 35'373 9'496 5'110'598 50.55 60 0 GCA_977946495 ERR14051209 ERR14041894 K54 SSI 82000 11'910'714 PacBio 34'639 9'683 4'597'013 50.89 62 3 GCA_977946555 ERR14051210 ERR14041895 K55 SSI 82001 10'047'660 PacBio 37'216 9'746 4'661'405 51.02 70 1 GCA_977947805 ERR14051217 ERR14041897 K83 SSI 82003 9'893'698 PacBio 32'658 9'627 4'957'187 50.78 57 0 GCA_977946385 ERR14051218 ERR14041899 K84 SSI 82004 10'079'880 PacBio 35'163 10'084 4'949'990 50.74 62 3 GCA_977947815 ERR14051219 ERR14041901 K87 SSI 82005 8'400'154 PacBio 39'286 10'039 4'943'518 50.79 69 4 GCA_977946395 ERR14051220 ERR14041903 K92 SSI 82006 10'654'126 PacBio 33'033 10'439 5'156'189 50.58 58 2 GCA_977947695 ERR14051221 ERR14041906 K93 SSI 85376 7'285'978 PacBio 25'459 9'987 4'906'586 50.55 43 2 GCA_977946505 ERR14051222 ERR14041909 K94 SSI 85377 9'715'314 PacBio 35'202 10'259 4'886'020 50.71 66 0 GCA_977947735 ERR14051223 ERR14041911 K95 SSI 85378 13'358'338 PacBio 33'676 11'076 5'220'776 50.3 65 0 GCA_977947145 ERR14051224 ERR14041913 K96 SSI 85379 6'724'984 PacBio 28'338 11'097 5'063'914 50.63 54 1 GCA_977947645 ERR14051225 ERR14041914 K97 SSI 85380 6'641'816 ONT 30'822 26'522 5'298'499 50.74 30 5 GCA_977946295 ERR14051226 ERR14041915 K98 SSI 85381 13'814'536 PacBio 33'350 10'737 4'741'888 50.88 69 0 GCA_977947765 ERR14051229 ERR14041918 K100 SSI 82007 10'314'626 PacBio 33'594 9'707 5'098'252 50.62 57 1 GCA_977946755 ERR14051230 ERR14041920 K101 SSI 82008 10'089'768 PacBio 37'418 8'992 4'764'323 50.75 62 2 GCA_977946665 ERR14051231 ERR14041921 K102 SSI 82009 10'424'902 PacBio 37'305 9'394 4'726'166 50.73 64 3 GCA_977947395 ERR14051246 ERR14041925 K103 SSI 82010 10'278'752 PacBio 26'680 9'068 4'798'391 50.78 45 0 GCA_977946695 ERR14051248 ERR14041926 Additional Declarations The authors declare no competing interests. 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