TITLE: A step-by-step beginner’s protocol for whole genome sequencing of human bacterial pathogens
AUTHORS: Sanjay Gautam, Rajendra KC, Kelvin WC Leong, Micheál Mac Aogáin, Ronan F. O’Toole

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Bacterial whole genome sequencing (WGS) is becoming a widely-used technique in research, clinical diagnostic, and public health laboratories. It enables high resolution characterization of bacterial pathogens in terms of properties that include antibiotic resistance, molecular epidemiology, and virulence. The introduction of next-generation sequencing instrumentation has made WGS attainable in terms of costs. However, the lack of a beginner’s protocol for WGS still represents a barrier to its adoption in some settings. Here, we present detailed step-by-step methods for obtaining WGS data from a range of different bacteria (Gram-positive, Gram-negative, and acid-fast) using the Illumina platform. Modifications have been performed with respect to DNA extraction and library normalization to maximize the output from the laboratory consumables invested. The protocol represents a simplified and reproducible method for producing high quality sequencing data. The key advantages of this protocol include simplicity of the protocol for users with no prior genome sequencing experience and reproducibility of the protocol across a wide range of bacteria.</div><div class = "text-block"><div class = "justify" style = "text-align:center">  </div></div></div>

[STEPS]
?. [Extraction of bacterial genomic DNA]
Pellet the liquid culture media () by centrifuging at  for  in a sterile microfuge tube.
200 µl
CRITICAL STEP: All bacterial cultures should be treated as potentially pathogenic to the laboratory worker and colleagues. Therefore, the use of appropriate aseptic techniques, and the wearing of appropriate personal protective equipment are strongly recommended to maintain acceptable work health and safety standards and minimise exposure to harmful agents.
?. [Extraction of bacterial genomic DNA]
Resuspend the pellet in  phosphate-buffered saline (1x) until the absorbance at 600 nm (A600) is between 1.0 and 2.0.
600 µl
?. [Extraction of bacterial genomic DNA]
Lyse the cells by adding  lysozyme (50 mg/ml), vortex, and incubate at  for .
30 µl
37 °C
?. [Extraction of bacterial genomic DNA]
Follow the DNeasy® Blood and Tissue Kit Quick-start protocol to extract the DNA.
?. [Extraction of bacterial genomic DNA]
Elute the DNA in  volume.
100 µl
?. [Extraction of bacterial genomic DNA]
Treat it with  RNase (100 mg/ml) (Qiagen, Hilden, Germany) and incubate at  for .
2 µl
0 Room temperature
?. [Extraction of bacterial genomic DNA]
Purify RNase-treated DNA using the High Pure PCR Template Preparation Kit.
TIP: Perform only 4 DNA spin-wash steps instead of 9 recommended steps. Pre-incubate the elution buffer in a heat block set at  .
?. [Extraction of bacterial genomic DNA]
Add  of binding buffer to RNase treated DNA and incubate at  for .
100 µl
70 °C
?. [Extraction of bacterial genomic DNA]
Add  of 2-Propanol and transfer the content to a Roche spin column and spin at  for .
50 µl
?. [Extraction of bacterial genomic DNA]
Discard the flow through and insert the spin column into a new collection tube.
?. [Extraction of bacterial genomic DNA]
Wash by adding  wash buffer and spin at  for .
500 µl
?. [Extraction of bacterial genomic DNA]
Discard the flow through and insert the spin column into a new collection tube.
?. [Extraction of bacterial genomic DNA]
Perform a final spin at  for .
?. [Extraction of bacterial genomic DNA]
Finally, insert the column into a 1.5 ml sterile microfuge tube, add  of pre-heated elution buffer and spin at  for  to elute the purified DNA for next generation sequencing.
50 µl
CRITICAL STEP: For next generation sequencing, contaminant-free, high-molecular weight DNA with an absorbance (260 nm/280 nm) ratio between 1.8 to 2.0 is considered a high-quality template DNA.
?. [Quantification of bacterial genomic DNA]
Dispense  and  of Qubit working solution in standard and sample tubes, respectively.
190 µl
198 µl
?. [Quantification of bacterial genomic DNA]
Add  standards (1 and 2) and  of sample in separate Qubit assay tubes.
10 µl
2 µl
?. [Quantification of bacterial genomic DNA]
Vortex the mixture for  and incubate at  for  before taking the reading.
0 Room temperature
?. [Quantification of bacterial genomic DNA]
Adjust the DNA concentration of each sample to 0.2 ng/μl by diluting with a required volume of distilled water.
CRITICAL STEP: The use of an accurate concentration of DNA is crucial for bacterial DNA genomic library preparation.
?. [Tagmentation and PCR amplification of bacterial genomic DNA]
TIP: For all of the methods below, the recommended 96-well TYC plate can be replaced with 0.2 ml thin wall clear, flat capped PCR tubes. In addition, multichannel pipettes and the high-speed micro plate shaker can be replaced with single channel pipettes and a bench top centrifuge, respectively.
?. [Nextera XT tagment amplicon construction]
In a PCR tube, add  tagmentation DNA buffer and  amplification tagmentation mix to  (0.2 ng/μl) input DNA.
5 µl
2.5 µl
2.5 µl
?. [Nextera XT tagment amplicon construction]
Briefly vortexed the content and transfer to the thermocycler programmed for one step at  for  with heated lid, followed by a hold at  for a volume of .
55 °C
10 °C
10 µl
?. [Neutralization of Nextera XT tagment amplicon]
Immediately after reaching the hold temperature of  in the above step, neutralize NTA by adding  neutralization tagmentation buffer and incubate at  for .
10 °C
2.5 µl
0 Room temperature
?. [PCR amplification]
For amplification, add  Nextera® PCR mastermix and of each index primer, 1 and 2, to a tube containing neutralized NTA.
7.5 µl
2.5 µl
CRITICAL STEP: Primer combinations, S502 with N705 /706 and S503 with N701/702 should be avoided. Avoid any repeated combinations and carefully note the primers used for each sample.
?. [PCR amplification]
Gently pipette the content and perform a quick spin.
?. [PCR amplification]
Proceed to amplification in a thermocycler programmed for a working volume of with the following settings and a heated lid:   ABC1CylesTemperature (°C)Time21723 min319530 s4129510 s5125530 s6127230 s71725 min8110hold
25 µl
ABC1CylesTemperature (°C)Time21723 min319530 s4129510 s5125530 s6127230 s71725 min8110hold
The amplified, tagmented library can be stored at   overnight for PCR clean-up the next day.
?. [Cleaning up the PCR product]
NOTE: Bring AMPure XP beads to room temperature (for ).CRITICAL STEP: Prepare fresh 80% (v/v) ethanol and 0.2 M NaOH.
?. [Cleaning up the PCR product]
To  of PCR product, add  of vortexed () AMPure XP beads and mix by pipetting (10 times).
22.5 µl
11.25 µl
?. [Cleaning up the PCR product]
Incubate at room temperature for .
?. [Cleaning up the PCR product]
Place the tube on a magnetic stand for .
?. [Cleaning up the PCR product]
While leaving the PCR tubes on the magnetic stand, carefully aspirate the supernatant.
CRITICAL STEP: Do not aspirate beads. If aspirated, redo steps 24 and 25.
?. [Cleaning up the PCR product]
Add  of 80% ethanol and leave on the stand in the magnetic stand for .
100 µl
CRITICAL STEP: Do not resuspend the beads.
?. [Cleaning up the PCR product]
Aspirate out the supernatant carefully.
?. [Cleaning up the PCR product]
Add  of 80% ethanol and leave on the stand in the magnetic stand for .
100 µl
?. [Cleaning up the PCR product]
Aspirate out the supernatant carefully.
?. [Cleaning up the PCR product]
Remove the tube from magnetic stand and allow to air dry in a tube stand for approximately .
CRITICAL STEP: Visually check for cracks as over drying the beads will significantly reduce elution efficiency.
?. [Cleaning up the PCR product]
Add  of resuspension buffer and gently pipette 20 times to mix.
26.15 µl
?. [Cleaning up the PCR product]
Incubate the tubes at room temperature for  and then place on a magnetic stand for  (until the supernatant cleared).
?. [Cleaning up the PCR product]
Transfer the supernatant () to a new PCR tube.
25 µl
NOTE: The final supernatant can be stored at −15°C to −20°C for up to 1 week but we recommend proceeding to library normalization immediately.
?. [Library normalization]
Perform the Qubit DNA quantification method as described above to determine the genomic DNA concentration in cleaned up product.
?. [Library normalization]
Pool the genomic DNA from all of the tubes.
NOTE: Sample with the lowest DNA concentration can be used in a volume of   as the reference to prepare a library pool using the formula: Volume required (V2) = Concentration original (S1) x Volume total (V1 = 10 μl) / Concentration required (S2).
?. [Library normalization]
To x μl of library pool, add x μl of freshly prepared 0.2 molar NaOH (final concentration 0.1 molar) and incubate for  at .
0 Room temperature
?. [Library normalization]
To the NaOH treated suspension add an equal volume (2x μl) of LNS1. Label the tube as pooled amplified library (PAL).
TIPS: In this modified step, normalize the library by using LNS1 (Library Normalisation Storage Buffer 1) only.
?. [Library normalization]
Dilute the PAL to 1:1000 by adding  of NaOH-LNS1 treated suspension to  of ultrapure distilled water.
1 µl
999 µl
?. [Library normalization]
Use KAPA library quantification kit (No ROX) to check the concentration of diluted pooled library in a real time PCR system using the following set up:   ABC1CylesTemperature (°C)Time219510 min3409510 s4406030 s
ABC1CylesTemperature (°C)Time219510 min3409510 s4406030 s
NOTE: Include a set of six DNA standards (with concentrations ranging from 20 pM to 0.0002 pM), three sets of negative control (ultrapure distilled water), and three sets of the DNA library in the qPCR run.
?. [Library normalization]
Determine the concentration of DNA in the pooled library by the standard curve method and calculate concentration in picomolar (pM) for each tube.
NOTE: To calculate the original concentration of the pooled library we applied the formula: [Average sample concentration (in pM) . insert size standards (452 bp) . dilution factor (1000)] divisible by the Insert size of pooled library (500 BP) For example, for a qPCR determined concentration of 2.36 pM in a 1:1000 dilution of the pooled library, the library DNA concentration will be: (2.36 pM x 452 bp x 1000) / 500 bp = 2133.44 pM The value obtained from the calculation represents the concentration of DNA in the pooled library.
?. [Library normalization]
To estimate the dilution factor required to achieve a final library concentration of  in a  volume use the formula: Volume required = (Concentration required × Volume total) / Concentration original = (15 pM × 600 μl) / 2133.44 pM = 4.22 μl
600 µl
NOTE: Therefore,   is added to   of HT buffer to produce a final concentration of  , in a final volume of  . The diluted library is then ready to be heat denatured and loaded into the MiSeq reagent cartridge.
?. [Preparing pooled library for loading onto MiSeq]
Thaw the PAL at room temperature and mix by pipetting up and down (5 times) followed by brief centrifugation.
?. [Preparing pooled library for loading onto MiSeq]
Based on the library concentration example above, transfer  of HT buffer to a  diluted amplified library (DAL) tube containing  PAL.
595.78 µl
1.5 ml
4.22 µl
?. [Preparing pooled library for loading onto MiSeq]
Mix using a pipette (5 times).
?. [Preparing pooled library for loading onto MiSeq]
Vortex the DAL tube at top speed, centrifuge briefly , and incubate exactly for  at  ± .
96 °C
2 °C
?. [Preparing pooled library for loading onto MiSeq]
Immediately transfer the DAL tube to ice for at least  or until loading.
CRITICAL STEP: Put the Illumina MiSeq sequencer through a short wash cycle to avoid cross-contamination of the DAL from previous usage.
?. [Preparing pooled library for loading onto MiSeq]
Thaw the MiSeq reagent cartridge at .
0 Room temperature
?. [Preparing pooled library for loading onto MiSeq]
Generate a MiSeq sample sheet using the Illumina Experiment Manager. See step 22 () to identify primer sets for each sample.
?. [Preparing pooled library for loading onto MiSeq]
Use the following configuration to set up the Miseq machine. Generate FASTQ workflow; FASTQ Only application; NexteraXT assay; 151 insert reads; assignment of the samples with a unique identifier and index-pair combination.
CRITICAL STEP: Rinse the flow cell with MilliQ water and remove traces of water using soft tissue paper before inserting into the machine.
?. [Preparing pooled library for loading onto MiSeq]
Transfer the entire  of DAL to the “Load here” well of the MiSeq reagent cartridge.
600 µl
?. [Preparing pooled library for loading onto MiSeq]
Following the setup procedure of the Illumina Experiment Manager, insert the cartridge into MiSeq instrument for sequencing to commence.
TIPS: The raw FastQ sequence reads from whole-genome sequencing can be stored on the local computer as well as on the Illumina BaseSpace server (https://basespace.illumina.com/) for further analysis.
?. [Bioinformatic analyses]
NOTE: The selection of bioinformatics software for the analysis of WGS data will be determined by the objective of the study. Here, we used Geneious 9.1.8 (Biomatters Ltd.), a desktop software to analyse our sequence data. Geneious was used to map the Fastq sequence reads to a publicly available reference genome for each species as follows:
?. [Bioinformatic analyses]
Download Geneious.
?. [Bioinformatic analyses]
Go to File | Import | From File. Import raw-read files (Sample_xx_R1.fastq.gz and Sample_xx_R2.fastq. gz) into Geneious.
?. [Bioinformatic analyses]
Download the Reference Genome from the NCBI database. For example, Enterococcus faecium NC_017960. In the Left panel | Go to NCBI | Nucleotide.
?. [Bioinformatic analyses]
Enter NC_017960 | Click Search.
?. [Bioinformatic analyses]
Once the genome has been found, click Download Full Sequence(s) .
?. [Bioinformatic analyses]
Download the NC_017960 reference genome (The icon changes to a green circular genome when completed).
?. [Bioinformatic analyses]
Drag and drop the NC_017960 reference genome into the working folder.
?. [Bioinformatic analyses]
Mapping the isolate sequence to the reference genome Hold CTRL and select both R1 and R2 raw read files (imported), and the reference genome (NC_017960) (downloaded).
?. [Bioinformatic analyses]
Click Align | Assemble|Map to Reference.
?. [Bioinformatic analyses]
Check the settings
?. [Bioinformatic analyses]
Reference Sequence = NC_017960
?. [Bioinformatic analyses]
Mapper = Bowtie2–fast and accurate read mapper
?. [Bioinformatic analyses]
Trim Before Mapping = Do not trim
?. [Bioinformatic analyses]
Results: Select all options
?. [Bioinformatic analyses]
Results | Save consensus sequences | Options
?. [Bioinformatic analyses]
Threshold = Highest Quality
?. [Bioinformatic analyses]
Threshold for sequences without quality = 95%
?. [Bioinformatic analyses]
No coverage call = ‘–’
?. [Bioinformatic analyses]
When mapping to reference is complete, a new folder will be created containing four files:
?. [Bioinformatic analyses]
Assembly Report
?. [Bioinformatic analyses]
Consensus
?. [Bioinformatic analyses]
Contig
?. [Bioinformatic analyses]
Unused Reads
?. [Bioinformatic analyses]
NOTE: Setting may vary depending on objective of analyses and quality of fastq reads. NOTE: We also used open source databases, for example, TGS-TB, PhyResSe and the Center for Genomic Epidemiology’s [20] ResFinder and VirulenceFinde, to further analyse the whole genome sequence data of our selection of bacterial pathogens. These freely-available databases enable the acquisition of information on bacterial pathogens that included genotype and phylogeny, antibiotic-resistance mutations, and the presence of known virulence genes.