A step-by-step beginner’s protocol for whole genome sequencing of human bacterial pathogens
2Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, Dublin D08 W9RT, Ireland
3School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Victoria 3690, Australia
- Lysozyme (VWR, Australia, Cat.# 0663-10G)
- Ethanol, Pure (Sigma-Aldrich, Australia, Cat. # E7023)
- 2-Propanol (Sigma-Aldrich, Australia, Cat. # I8912)
- Phosphate Buffered Saline (GibcoTM- Thermo Fisher Scientific, UK, Cat. # 10010023)
- Ultrapure™ DNase/RNase Free Distilled Water (Invitrogen, Australia Cat. # 10977-015)
- DNeasy® Blood and Tissue Kit (Qiagen, Germany, Cat. # 69504)
- High Pure PCR Template Preparation Kit (Roche, Germany, Cat. # 11796828001)
- Qubit™ dsDNA HS (High Sensitivity) Assay Kit (Invitrogen, Australia, Cat. # Q32851)
- Nextera® DNA Library Preparation Kit (Illumina, USA, Cat. # FC-121-1030)
- Nextera® XT Library Preparation Kit (Illumina, USA, Cat. # FC-131-1024)
- Nextera® XT Index Kit (Illumina, USA, Cat. # FC-131-1001)
- Miseq Reagent Kit v2 (300 cycles) (Illumina, USA, Cat. # MS-102-2002)
- KAPA Library Quantification Kit (Illumina, USA, Cat. # 07960140001)
- Agencourt® AMPure XP beads (Beckman Coulter, USA, Cat. # A63880)
- Qubit working solution: dilute Qubit dsDNA HS Reagent 1:200 in Qubit dsDNA HS buffer. For n samples, prepare n × 200 µl working solution.
- 80% ethanol: add 2 ml absolute ethanol into 8 ml distilled water.
- 0.2 M NaOH: weigh 0.04 g of NaOH pellet and dissolve it into 5 ml distilled water.
- Qubit™ assay tubes (Life-technologies, USA, Cat. # Q32856)
- PCR tubes (Molecular Bioproducts, USA, Cat. # MBP3412)
- Qubit® 2.0 Fluorometer (Invitrogen, Australia, Cat. # Q32866)
- Agencourt Magnetic stand (Beckman Coulter, USA, Cat. # A32782)
- Applied Biosystems® Veriti 96-Well thermal cycler (Thermo Fisher Scientific, USA)
- Rotor-Gene 6000 real-time thermocycler ( Corbett Research, Australia)
Extraction of bacterial genomic DNA
1.Pellet the liquid culture media (200 µl) by centrifuging at 8000 g for 8 min in a sterile microfuge tube.
2.Resuspend the pellet in 600 µl phosphate-buffered saline (1 ×) until the absorbance at 600 nm (A600) is between 1.0 and 2.0. Lyse the cells by adding 30 µl lysozyme (50 mg/ml), vortex, and incubate at 37°C for 1 h.
3.Follow the DNeasy® Blood and Tissue Kit Quick-start protocol to extract the DNA.
4.Elute the DNA in 100 µl volume and treat it with 2 µl RNase (100 mg/ml) (Qiagen, Hilden, Germany) and incubate at room temperature for 1 h.
5.Purify RNase-treated DNA using the High Pure PCR Template Preparation Kit.
6.Add 100 µl of binding buffer to RNase treated DNA and incubate at 70°C for 10 min.
7.Add 50 µl of 2-Propanol and transfer the content to a Roche spin column and spin at 8000 g for 1 min.
8.Discard the flow through and insert the spin column into a new collection tube.
9.Wash by adding 500 µl wash buffer and spin at 8000 g for 1 min.
10.Discard the flow through and insert the spin column into a new collection tube.
11.Perform a final spin at 8000 g for 1 min.
12.Finally, insert the column into a 1.5 ml sterile microfuge tube, add 50 µl of pre-heated elution buffer and spin at 8000 g for 1 min to elute the purified DNA for next generation sequencing.
Quantification of bacterial genomic DNA
13.Dispense 190 µl and 198 µl of Qubit working solution in standard and sample tubes, respectively.
14.Add 10 µl standards (1 and 2) and 2 µl of sample in separate Qubit assay tubes.
15.Vortex the mixture for 3 s and incubate at room temperature for 2 min before taking the reading.
16.Adjust the DNA concentration of each sample to 0.2 ng/µl by diluting with a required volume of distilled water.
Tagmentation and PCR amplification of bacterial genomic DNA
Nextera XT tagment amplicon construction
17.In a PCR tube, add 5 µl tagmentation DNA buffer and 2.5 µl amplification tagmentation mix to 2.5 µl (0.2 ng/µl) input DNA.
18.Briefly vortexed the content and transfer to the thermocycler programmed for one step at 55°C for 5 min with heated lid, followed by a hold at 10°C for a volume of 10 µl.
Neutralization of Nextera XT tagment amplicon
19.Immediately after reaching the hold temperature of 10°C in the above step, neutralize NTA by adding 2.5 µl neutralization tagmentation buffer and incubate at room temperature for 5 min.
20.For amplification, add 7.5 µl Nextera® PCR mastermix and 2.5 µl of each index primer, 1 and 2, to a tube containing neutralized NTA.
21.Gently pipette the content and perform a quick spin.
22.Proceed to amplification in a thermocycler programmed for a working volume of 25 µl with the following settings: heated lid, initial cycle at 72°C for 3 min followed by 95°C for 30 s and 12 cycles of (95°C for 10 s, 55°C for 30 s and 72°C for 30 s) with a final run at 72°C for 5 min followed by a hold temperature of 10°C. The amplified, tagmented library can be stored at 2 to 4°C overnight for PCR clean-up the next day.
Cleaning up the PCR product
23.To 22.5 µl of PCR product, add 11.25 µl of vortexed (30 s) AMPure XP beads and mix by pipetting (10 times).
24.Incubate at room temperature for 5 min.
25.Place the tube on a magnetic stand for 2 min.
26.While leaving the PCR tubes on the magnetic stand, carefully aspirate the supernatant.
27.Add 100 µl of 80% ethanol and leave on the stand in the magnetic stand for 30 s.
28.Aspirate out the supernatant carefully.
29.Add 100 µl of 80% ethanol and leave on the stand in the magnetic stand for 30 s.
30.Aspirate out the supernatant carefully.
31.Remove the tube from magnetic stand and allow to air dry in a tube stand for approximately 5 min.
32.Add 26.15 µl of resuspension buffer and gently pipette 20 times to mix.
33.Incubate the tubes at room temperature for 2 min and then place on a magnetic stand for 2 min (until the supernatant cleared).
34.Transfer the supernatant (25 µl) to a new PCR tube.
35.Perform the Qubit DNA quantification method as described above to determine the genomic DNA concentration in cleaned up product.
36.Pool the genomic DNA from all of the tubes.
37.To × µl of library pool, add × µl of freshly prepared 0.2 molar NaOH (final concentration 0.1 molar) and incubate for 5 min at room temperature.
38.To the NaOH treated suspension add an equal volume (2× µl) of LNS1. Label the tube as pooled amplified library (PAL).
39.Dilute the PAL to 1:1000 by adding 1 µl of NaOH-LNS1 treated suspension to 999 µl of ultrapure distilled water.
40.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: a hot start run at 95°C for 10 min followed by 40 cycles of (95°C for 10 s and 60°C for 30 s).
41.Determine the concentration of DNA in the pooled library by the standard curve method and calculate concentration in picomolar (pM) for each tube.
42.To estimate the dilution factor required to achieve a final library concentration of 15 pM in a 600 µl volume use the formula:
Preparing pooled library for loading onto MiSeq
43.Thaw the PAL at room temperature and mix by pipetting up and down (5 times) followed by brief centrifugation.
44.Based on the library concentration example above, transfer 595.78 µl of HT buffer to a 1.5 ml diluted amplified library (DAL) tube containing 4.22 µl PAL.
45.Mix using a pipette (5 times).
46.Vortex the DAL tube at top speed, centrifuge briefly, and incubate exactly for 2 min at 96°C ± 2°C.
47.Immediately transfer the DAL tube to ice for at least 5 min or until loading.
48.Thaw the MiSeq reagent cartridge at room temperature .
49.Generate a MiSeq sample sheet using the Illumina Experiment Manager. See step 21 to identify primer sets for each sample.
50.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.
51.Transfer the entire 600 µl of DAL to the “Load here” well of the MiSeq reagent cartridge.
52.Following the setup procedure of the Illumina Experiment Manager, insert the cartridge into MiSeq instrument for sequencing to commence.
53.Download Geneious from https://www.geneious.com/.
54.Go to File | Import | From File. Import raw-read files (Sample_xx_R1.fastq.gz and Sample_xx_R2.fastq.gz) into Geneious.
55.Download the Reference Genome from the NCBI database. For example, Enterococcus faecium NC_017960.
55.1.In the Left panel | Go to NCBI | Nucleotide.
55.2.Enter NC_017960 | Click Search.
55.3.Once the genome has been found, click Download Full Sequence(s).
55.4.Download the NC_017960 reference genome (The icon changes to a green circular genome when completed).
55.5.Drag and drop the NC_017960 reference genome into the working folder.
56.Mapping the isolate sequence to the reference genome
56.1.Hold CTRL and select both R1 and R2 raw read files (imported), and the reference genome (NC_017960) (downloaded).
56.2.Click Align | Assemble|Map to Reference.
56.3.Check the settings
56.3.1.Reference Sequence = NC_017960
56.3.2.Mapper = Bowtie2–fast and accurate read mapper
56.3.3.Trim Before Mapping = Do not trim
56.3.4.Results: Select all options
56.3.5.Results | Save consensus sequences | Options
56.3.6.Threshold = Highest Quality
56.3.7.Threshold for sequences without quality = 95%
56.3.8.No coverage call = ‘–’
56.4.When mapping to reference is complete, a new folder will be created containing four files:
Initial typing analysis
Reference sequence coverage (%)
Mean read depth
|23||Low concentration of AMPure XP bead captured purified products||Bead clean-up affects the quality and quantity of amplified libraries that will be included in downstream sequencing process||Make sure AMPure XP beads are held at room temperature for 20 min before starting the clean-up process. Furthermore, ensure that 80% ethanol is freshly prepared|
|35||Variation in the concentration of amplified library||The concentration of input DNA used for library preparation affects the final yield of genomic data||Measure the concentration of input DNA using Qubit fluorometer rather than a nanodrop and make the appropriate dilution for a DNA concentration of 0.2 ng/μl|
|56||Poor sequencing results||The choice of forward and reverse index primer set affects the sequencing of libraries prepared||Avoid primer combinations S502 with N705 /706, and S503 with N701/702|
|56||Poor sequencing results||Effective denaturation of pooled library not achieved after bead clean-up||Ensure that the NaOH is freshly prepared at the correct concentration|
|56||Poor sequencing results||Repeated thawing and freezing of the pooled library reduces the quality of sequence reads generated||Before preparing the pooled library for loading onto MiSeq, ensure that the machine has already been appropriately cleaned after the previous run and has sufficient storage space (at least 25 GB)|
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