Whole genome sequencing, WGS, can help answer key questions in antimicrobial resistant field through genome interrogation and the identification of molecular mechanisms underlying the antimicrobial resistance phenotype. World genome sequencing improves the exploration of DNA, bacterial DNA at a large scale, facilitating the in depth characterizationof microbes in a single run including closely related pathogenic variants. Next generation sequencing provide rapid identification and characterization of microorganisms and their antimicrobial resistant profile considering a tool for diagnosis, treatment, surveillance and source tracking of pathogens in the clinical context.
Next generation sequencing methodologies can play a determining role in an surveillance and source tracking of pathogens not only in the clinical context but also in the environmental settings and importantly in food safety. Currently, this is not a routine technique. Indeed, a bit of the procedure all will make it easier to understand how successful they carry out the experiment.
Demonstrating the procedure will be Julissa Enciso-Ibarra the genomics technician of our working group. After extracting and quantifying bacterial DNA add 2.5 microliters of tagmentation buffer. Two microliters of input G D N A in a 0.2 milliliter tube and mix by pipetting.
Add one microliter of amplification buffer and pipette up and down to mix. Then spin down the tube at 280 G for one minute. Place the samples in a thermocycler at 55 degrees Celsius for five minutes, followed by holding at 10 degrees Celsius.
Add one microliter of neutralizing buffer to the sample tubes and mix gently by pipetting. Spin down the tube and incubate for five minutes at room temperature. Next, add 1.7 microliters of each index adapter and three microliters of indexing PCR master mix.
After mixing the components, spin down at 280 G for one minute at room temperature. Place the samples in the thermocycler and perform a second PCR reaction as described in the manuscript. Now mix the commercial magnetic bead solution by vortexing.
As per the manufacturer's guidelines. Transfer the tagmented or indexed G D N A sample to a new one five milliliter tube and add 0.6 microliters of magnetic beads for each microliter of the final volume of the G D N A sample. Mix the components gently by pipetting and incubate for five minutes at room temperature.
Place the sample tubes on a magnetic rack for two minutes until the supernatant has cleared. Discard the supernatant carefully without disturbing the beads. Then add 200 microliters of freshly prepared 80%ethanol without mixing.
Incubate for 30 seconds until the eluit clears and carefully remove the supernatant without disturbing the beads. Repeat this procedure and air dry the beads for 10 minutes. Now add 15 microliters of 10 millimolar tris buffer to the beads mixed gently by pipetting and incubate for two minutes at room temperature.
Place the sample tubes on the magnetic rack for two minutes allowing the supernatant to clear. Afterward, carefully transfer 14 microliters of the supernatant from the sample tube to a new 0.2 milliliter tube and get the cleaned up library. Thaw the reagent cartridge following the manufacturer's instructions.
Pull five microliters of the normalized library into a low bind, 1.5 milliliter tube and dilute the pooled library at four nanomolar with the proper volume of RSB. Then add five microliters each of the pooled library and 0.2 normal sodium hydroxide in a new low bind, 1.5 milliliter tube. To denature the pooled library into singles strands, mix the tube by vortexing.
Spin down for one minute and incubate for five minutes at room temperature. Now add 10 microliters of the denatured pooled library 990 microliters of pre chilled hybridization buffer and mix gently by pipetting. Place the tube on ice until the final dilution for sequencer loading is performed.
Pour and prepare a control library at a concentration of four nanomolar by mixing two microliters of the control library and three microliters of nuclease free water. Then add five microliters each of the control library and freshly prepared 0.2 normal sodium hydroxide. Denature the control library into single strands as demonstrated earlier.
Gently mix 10 microliters of denatured control library and 990 microliters of pre chilled hybridization buffer by pipetting, and place it on ice until the final dilution for sequencer loading is done. In a new low bind, 1.5 milliliter tube, combine 594 microliters of the denatured pooled library and six microliters of the denatured control library. Mix properly and dilute the final library mix to a final loading concentration of 1.2 peco molar in a volume of 600 microliters using the pre chilled hybridization buffer.
Before loading the final library mix onto the reagent cartridge perform an additional thermal pretreatment to achieve efficient loading into the flow cell. Incubate the final library mix for two minutes at 96 degrees Celsius. Invert the tube to mix and place the tube on ice for five minutes.
Load 500 microliters of the final library mix into the design reservoir on the reagent cartridge. After initiating the sequencer load the reagent cartridge, the flow cell and set up the sequencing run. For data analysis, connect to the sequencing data server and download the FASTQ files.
Evaluate the initial quality of raw sequence data with third party software. Remove remnant adapter sequences, low quality bases and short reads from the raw sequencing data. Assemble the quality check sequencing data into contig or scaffold level using third party software.
Perform genome annotation by submitting the Phaster file containing the assembled genome to the RAST server. Upload the Phaster file to the Center for Genome Epidemiology and Claremont typing web platforms to identify epidemiological features. ARGS, VAGS and plasmids.
Upload the Phaster file to the Phaster server to identify prophage sequences. Genome annotation revealed 5, 633 encoded features of which 5, 524 are protein coating genes and 109 are RNA related sequences. The predicted Escherichia coli strain belongs to the filo group B one and sequence type 40.
An antimicrobial resistance determinant, the M D F A gene coding for a broad spectrum multi-drug efflux pump was identified. Genes encoding for a heat stable enterotoxin a serum resistance protein and the type three secretion system along with its secreted effector proteins were also identified. The coding sequences are grouped into collections of functionally related proteins called subsystems especially those playing functional roles in carbohydrates, proteins, amino acids and derivative metabolisms and membrane transport.
Good laboratory practice are fundamental through all the protocol. However, a special care must be taken when processing multiple samples during the sectional DNA library preparation to avoid cross-contamination.
