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Sequencing Info

Standards Run Information
Project Name ASM NGS
Experimenter C. Mena
Experiment Name (project_run#_YYYY_MM_DD) 2024_07_03_cm_2
Run Start Date/Time 7/3/24
Run Length (hrs): 48 hours
Total Library Concentration (ng/ul) 193 ng
Positive Control (5ng BC10 PBK) (yes/no) (If yes label it on BC10) no
Sequencing Kit (RAB204, PBK004, RBK004, ect.) RBK114
Sequencing Kit Batch Number NA
Flowcell ID FAY83186
QC Total Pores 1570
Run Start Total Pore 1459
Device Number (111633, 111632, 110540, 111583, ect) MN43813
Standard Settings Correct settings if different from below
Basecalling ON (super accurate)
Basecalling Config: Super accuracy
Barcoding ON
Barcoding Options: Trim Barcodes OFF
Barcoding Options: Barcodes both ends OFF
Barcoding Options: Mid-read barcode filtering OFF
Barcoding Options: Override minimum barcoding score OFF (keep default at 60)
Filtering OFF
Filtering Options: Qscore 10
Output format: Compression (.vbz, .gzip) ON (default)
MinKNOW version 24.02.16
Nanopore Basecalling version (Guppy) super accuracy
Notes R.10 flow cell

Barcodes

The bacteria that were selected for whole genome sequencing and their respective barcodes can be found below.

Barcode Sample Name
barcode01 Paenibacillus sp.
barcode02 Bacillus cereus
barcode03 Klebsiella aerogenes
barcode04 Staphylococcus aureus
barcode05 Staphylococcus hominis
barcode06 Bacillus subtilis
barcode07 Staphylococcus epidermidis
barcode08 Klebsiella aerogenes

Run Performance

The nanopore flowcell performance can be determined using the following charts. They help to determine if the library chemistry and DNA template are going to provide high quality reads.

The nanopores through which DNA is passed, and signal collected, are arrayed as a 2-dimensional matrix. A heatmap can be plotted showing channel productivity against spatial position on the matrix. Such a plot enables the identification of spatial artifacts that could result from membrane damage through e.g. the introduction of an air-bubble. This heatmap representation of spatial activity shows only gross spatial aberations. Since each channel can address four different pores (Mux) the activity plot below shows the number of sequences produced per channel, not per pore.

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Translocation Speed

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The speed/time plot is used to observe any substantial changes in sequencing speed. A marked slow-down in sequencing speed can indicate challenges within the sequencing chemistry that could have been caused by the method of DNA isolation or an abundance of small DNA fragments.

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Quality vs. Length

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The density plot of mean sequence quality plotted against log10 sequence length is a used to show patterns within the broader sequence collection. The density plot shown in the figure below has been de-speckled by omitting the rarer sequence bins containing only 1 read or fewer have been omitted. This is mainly aesthetic and masks some speckle around the periphery of the main density map.

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Assembly Information

ID Length of Assembly No. of Contigs Genome N50 GC%
Paenibacillus sp. 6,613,973 1 6,613,973 47.16
B. cereus 5,292,514 2 5,216,597 35.40
K. aerogenes 5,422,585 3 5,325,448 54.85
S. aureus 2,791,163 1 2,791,163 32.88
S. hominis 2,316,782 2 2,304,307 31.54
B. subtilis 4,197,947 2 4,159,298 43.60
S. epidermidis 2,518,551 3 2,428,929 32.08
K. aerogenes 5,423,897 3 5,325,436 54.85

The statistics from the whole genome assemblies were calculated using seqkit stats version 2.8.0.

ANI & Assembly Quality

ID ANI ANI Best Match Completeness Contamination
Paenibacillus sp. 98.15 NZ_CP106740.1 Paenibacillus sp. CC-CFT742 chromosome, complete genome 100.00 0.00
B. cereus 100.00 NZ_CP018933.1 Bacillus cereus strain ISSFR-9F chromosome, complete genome 100.00 0.04
K. aerogenes 99.99 NZ_CP014029.2 Klebsiella aerogenes strain FDAARGOS_152 chromosome, complete genome 100.00 0.81
S. aureus 99.98 NZ_CP047815.1 Staphylococcus aureus strain UP_1484 chromosome, complete genome 100.00 0.19
S. hominis 99.02 NZ_CP080457.1 Staphylococcus hominis subsp. hominis strain WiKim0113 chromosome, complete genome 99.99 0.07
B. subtilis 99.87 NZ_CP072845.1 Bacillus subtilis strain XP chromosome, complete genome 100.00 0.06
S. epidermidis 99.45 NZ_CP090912.1 Staphylococcus epidermidis strain 44 chromosome, complete genome 99.99 0.05
K. aerogenes 99.99 NZ_CP024885.1 Klebsiella aerogenes strain AR_0009 chromosome, complete genome 100.00 0.84

To determine the identification of the bacteria using the whole genome sequence, average nucleotide identity was calculated using skani version 0.2.1 and used references downloaded from NCBI with ncbi-genome-download and assemblies that are considered complete. The completeness and contamination was determined using CheckM2 version 1.0.2.

Genome Annotation

Nanopore whole genome sequencing assemblies were annotated with Bakta version 1.9.4 annotation pipeline.

ID Coding Density tRNAs rRNAs CDSs pseudogenes hypotheticals
Paenibacillus sp. 87.2 110 39 5,944 13 392
B. cereus 85.9 104 42 5,326 26 98
K. aerogenes 89.5 88 25 5,047 10 142
S. aureus 85.4 61 19 2,568 7 43
S. hominis 88.1 63 19 2,205 6 92
B. subtilis 89.0 88 30 4,435 25 228
S. epidermidis 84.2 61 19 2,323 11 80
K. aerogenes 89.5 88 25 5,047 10 143

Genome Map

Circular genome maps were created using bakta_plot via Circos. The plots are created as a part of the standard workflow and are saved as png and svg files. The two default plot types, features and cog, were created following assembly. Both plot types share two innermost GC content and GC skew rings.

Features

All features are plotted on two rings representing the forward and reverse strand from outer to inner, respectively using the following feature colors:
CDS
tRNA/tmRNA
rRNA
ncRNA
ncRNA-region
CRISPR
GAP
Misc

COGs

All protein-coding genes (CDS) are colored according to assigned COG functional categories. To better distinguish non-coding genes, these are plotted on the additional 3rd ring.

1 RNA processing and modification
2 Chromatin structure and dynamics
3 Energy production and conversion
4 Cell cycle control, cell division, chromosome partitioning
5 Amino acid transport and metabolism
6 Nucleotide transport and metabolism
7 Carbohydrate transport and metabolism
8 Coenzyme transport and metabolism
9 Lipid transport and metabolism
10 Translation, ribosomal structure and biogenesis
11 Transcription
12 Replication, recombination and repair
13 Cell wall/membrane/envelope biogenesis
14 Cell motility
15 Posttranslational modification, protein turnover, chaperones
16 Inorganic ion transport and metabolism
17 Secondary metabolites biosynthesis, transport and catabolism
18 General function prediction only
19 Function unknown
20 Signal transduction mechanisms
21 Intracellular trafficking, secretion, and vesicular transport
22 Defense mechanisms
23 Extracellular structures
24 Mobilome: prophages, transposons
25 Nuclear structure
26 Cytoskeleton

Resistance Genes

Antimicrobial resistance and disinfection resistance gene determination was performed using ResFinder version 4.5.0 and the flags, -l 0.1 -t 0.1 –acquired –point –nanopore –ignore_missing_species –disinfectant. AMR genes were identified with a minimum identity of 80%.

Antimicrobial

ID Resistance gene Phenotype Identity Coverage
B. cereus fosB1 Fosfomycin 87.89 99.04
K. aerogenes fosA5 Fosfomycin 89.05 100.00
K. aerogenes OqxB Chloramphenicol, Nalidixic acid, Ciprofloxacin, Trimethoprim 87.97 99.24
K. aerogenes OqxA Chloramphenicol, Nalidixic acid, Ciprofloxacin, Trimethoprim 84.01 100.00
S. aureus blaZ Amoxicillin, Ampicillin, Penicillin, Piperacillin 100.00 100.00
S. hominis aph(3’)-III Kanamycin, Amikacin, Neomycin, Butirosin, Isepamicin, Lividomycin, Paromomycin, Ribostamycin 100.00 100.00
S. hominis ant(6)-Ia Streptomycin 99.84 70.52
S. hominis blaZ Amoxicillin, Ampicillin, Penicillin, Piperacillin 99.76 100.00
B. subtilis aadK Streptomycin 98.83 100.00
B. subtilis mph(K) Spiramycin, Telithromycin 99.46 100.00
B. subtilis tet(L) Doxycycline, Tetracycline, Doxycycline, Tetracycline, Minocycline, Tigecycline 99.13 100.00
S. epidermidis fosB Fosfomycin 99.30 100.00

Disinfection

ID Disinfection Resistance gene Phenotype Identity Coverage
K. aerogenes OqxB Benzalkonium Chloride, Cetylpyridinium Chloride 87.97 99.24
K. aerogenes OqxA Benzalkonium Chloride, Cetylpyridinium Chloride 84.01 100.00
S. aureus qacD Benzalkonium Chloride, Ethidium Bromide, Chlorhexidine, Cetylpyridinium Chloride 85.40 33.63
S. hominis qacD Benzalkonium Chloride, Ethidium Bromide, Chlorhexidine, Cetylpyridinium Chloride 97.63 33.63
S. hominis qacD Benzalkonium Chloride, Ethidium Bromide, Chlorhexidine, Cetylpyridinium Chloride 95.53 23.45
S. hominis qacA Benzalkonium Chloride, Ethidium Bromide, Chlorhexidine, Cetylpyridinium Chloride 100.00 100.00
S. epidermidis qacD Benzalkonium Chloride, Ethidium Bromide, Chlorhexidine, Cetylpyridinium Chloride 96.84 23.70
S. epidermidis qacD Benzalkonium Chloride, Ethidium Bromide, Chlorhexidine, Cetylpyridinium Chloride 97.77 33.63
S. epidermidis qacD Benzalkonium Chloride, Ethidium Bromide, Chlorhexidine, Cetylpyridinium Chloride 97.19 33.68
K. aerogenes 2 OqxB Benzalkonium Chloride, Cetylpyridinium Chloride 87.97 99.24
K. aerogenes 2 OqxA Benzalkonium Chloride, Cetylpyridinium Chloride 84.01 100.00

Plasmid Reconstruction

Plasmids are mobile genetic elements (MGEs), which allow for rapid evolution and adaption of bacteria to new niches through horizontal transmission of novel traits to different genetic backgrounds. MOB-suite version 3.1.9 was used to carry out the typing and reconstruction of plasmid sequences from the WGS assemblies.

ID Plasmid Size GC % Plasmid Name Accession Mobility prediction
B. cereus 75,917 0.32 AC347 CP000047 conjugative
K. aerogenes 57,190 0.52 AA002 U27345 conjugative
K. aerogenes 39,947 0.43 AB876 000687__KX348146_00033 non-mobilizable
S. hominis 12,475 0.28 AA852 000368__GQ900452_00020 mobilizable
S. epidermidis 62,882 0.28 AB820 000510__LN866850_00038,CP000737 mobilizable
S. epidermidis 62,882 0.28 AB820 000510__LN866850_00038,CP000737 mobilizable

R Packages

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## R version 4.4.1 (2024-06-14)
## Platform: aarch64-apple-darwin23.4.0
## Running under: macOS Sonoma 14.6.1
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## Matrix products: default
## BLAS:   /opt/homebrew/Cellar/openblas/0.3.28/lib/libopenblasp-r0.3.28.dylib 
## LAPACK: /opt/homebrew/Cellar/r/4.4.1/lib/R/lib/libRlapack.dylib;  LAPACK version 3.12.0
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## time zone: America/Chicago
## tzcode source: internal
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## attached base packages:
## [1] grid      stats     graphics  grDevices utils     datasets  methods  
## [8] base     
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## other attached packages:
##  [1] gridExtra_2.3      png_0.1-8          xlsx_0.6.5         scales_1.3.0      
##  [5] RColorBrewer_1.1-3 ggpubr_0.6.0       janitor_2.2.0      data.table_1.15.99
##  [9] lubridate_1.9.3    forcats_1.0.0      stringr_1.5.1      dplyr_1.1.4       
## [13] purrr_1.0.2        readr_2.1.5        tidyr_1.3.1        tibble_3.2.1      
## [17] tidyverse_2.0.0    ggplot2_3.5.1      kableExtra_1.4.0  
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## loaded via a namespace (and not attached):
##  [1] gtable_0.3.5      xfun_0.45         bslib_0.7.0       rstatix_0.7.2    
##  [5] rJava_1.0-11      tzdb_0.4.0        vctrs_0.6.5       tools_4.4.1      
##  [9] generics_0.1.3    fansi_1.0.6       highr_0.11        pkgconfig_2.0.3  
## [13] lifecycle_1.0.4   farver_2.1.2      compiler_4.4.1    munsell_0.5.1    
## [17] carData_3.0-5     snakecase_0.11.1  htmltools_0.5.8.1 sass_0.4.9       
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## [25] cachem_1.1.0      abind_1.4-5       mime_0.12         tidyselect_1.2.1 
## [29] digest_0.6.36     stringi_1.8.4     reshape2_1.4.4    labeling_0.4.3   
## [33] cowplot_1.1.3     fastmap_1.2.0     colorspace_2.1-0  cli_3.6.3        
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## [41] withr_3.0.0       backports_1.5.0   timechange_0.3.0  rmarkdown_2.27   
## [45] ggsignif_0.6.4    hms_1.1.3         evaluate_0.24.0   knitr_1.48       
## [49] viridisLite_0.4.2 rlang_1.1.4       Rcpp_1.0.13       glue_1.7.0       
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## [57] plyr_1.8.9        R6_2.5.1          systemfonts_1.1.0