Genome-wide identification of virulence-associated genes in Staphylococcus aureus using Transposon insertion-site deep sequencing
5.7 Mutagenesis in S. aureus
5.7.1 Generation of random Transposon mutant pools in S. aureus
- Pooled mariner transposon mutant libraries were generated as previously described 246.
- Briefly, 6850 transformed with plasmid pBTn was revived on TSA Cm 10 plates.
- An overnight culture was prepared by picking a colony and resuspending in broth with Cm 10 and without Xylose, followed by incubation at 30°C with agitation at 180 rpm.
- The culture was diluted in ratio 1:100 in fresh broth containing 0.5% Xylose, Cm 10 and Erm 5, followed by overnight incubation at 30°C with agitation at
180 rpm.
- This was done in 10 replicates simultaneously. The cultures were again diluted in ratio 1:100 in fresh broth containing 0.5% Xylose and Erm 5, followed by overnight incubation at 42°C with agitation at 180 rpm. This step was repeated two more times.
- All cultures were combined and centrifuged at 3000 xg for 10 minutes.
- The pellets were mixed with fresh broth so that the OD600nm was 10.0.
- Stocks were prepared as previously described (See Section 5.1.2). Successful transposition and subsequent loss of plasmid due to temperature elevation was assessed by enumeration of Erm resistant but Cm sensitive bacteria.
- A trial was made with more than one medium and more than 3 temperatures elevation steps to assess maximum efficiency of transposition (See Section 2.1.1).
- Colonies were randomly chosen from Erm 5 plates and tested for insertion in the genome by Arbitrary PCR (See Section 5.6.1) followed by
5.9 High throughput analysis of nucleic acids
5.9.1 Transposon insertion-site deep sequencing (Tn-seq)
- Chromosomal DNA was isolated from pooled mariner transposon mutant libraries including inocula and output harvested from various conditions, by the method described in Section 5.5. Fragment DNA libraries were prepared and sequenced using the following steps.
5.9.1.1 Fragmentation of S. aureus genomic DNA
- Hydrodynamic shearing of genomic DNA was carried out by application of ultrasonics on a Bioruptorr instrument.
- 1 ml of DNA dissolved in water were taken in 15 ml polystyrene tubes and sonicated for 10 cycles, with the following specifications.
- Ultrasonic wave frequency: 20kHz, wave power: H i.e. 320W, time: 1 minute/cycle (30 seconds ’ON’ and 30 seconds ’OFF’).
- Fragmentation was carried out at 4°C to avoid shearing by the heat generated from the sonication probe.
- This setting consistently resulted in fragments sizes between 100 to 500 nucleotides.
- The size range was analyzed by agarose gel electrophoresis and on Agilent 2100 Bioanalyzer instrument.
5.9.1.2 End-repair and size selection of DNA fragments
- 5 μg of fragmented DNA from each sample were repaired using the commercially available NEBNextr End repair module following manufacturer’s instructions in a volume of 100 μl.
- Fragments within the size range of 200-300 nucleotides were selected using a gel-free double-Solid Phase Reversible Immobilization (dSPRI), method G, as previously described using AMPure XP beads in three steps.
- Step 1: Immediately after end repair, the samples were mixed with 0.9X volume of beads and incubated at room temperature for 20 minutes. The beads were separated using a magnetic rack and the residual solution was transfered to a fresh microfuge tube.
- Step 2: The DNA solution was subsequently mixed with 0.11X volume of beads and incubated at room temperature for 7 minutes. The beads were again separated and the residual solution was disposed.
- Step 3: The beads were washed twice with 70% Ethanol for 30 seconds without removing tubes from the magnetic rack.
- The beads were air-dried for 2-3 minutes and eluted in 42 μl of sterile nuclease-free water.
- If required, the size range was confirmed by agarose gel electrophoresis.
5.9.1.3 dA-tailing of blunt end DNA fragments
- After the correct size of DNA fragments were obtained, NEBNextr dA-Tailing module was used to add non-templated deoxy-Adenosine monophosphate (dAMP) molecules to the 3‘ ends, according to manufacturer’s instructions.
- The DNA fragments were purified using AMPure XP beads and eluted into 30 μl of sterile nuclease-free water.
5.9.1.4 Generation and ligation of adapters to DNA fragments
- For generation of multiplexing dsDNA adapters compatible with Illuminar sequencing platform, two separate oligonucleotides were used.
- MultiPlex-Y-Adapt_f with a 5' phosphorylation and MultiPlex-Y-Adapt_r with a 3' terminal phosphorothioate linkage, were mixed in equimolar concentrations in 1X Oligo-annealing buffer.
- The reaction mixture was heated to 94°C for 5 minutes followed by gradual cooling to room temperature and finally incubated on ice.
- Adaptors were ligated to dA-tailed fragments overnight at 16°C using T4 DNA ligase in the presence of 1X T4 ligase buffer and 50% Polyethylene glycol (PEG), added to the previous reaction from Section 5.9.1.3. Upon completion, DNA fragments were purified with AMPure XP beads and eluted in 25 μl of sterile nuclease-free water.
5.9.1.5 Massively parallel sequencing on Illuminar platform
- Before sequencing transposon ends were enriched by aforementioned PCR steps (See Section 5.6.1), purified with AMPure XP beads and checked for quality (See Section 5.5.7).
- The resulting DNA fragment libraries were sequenced on the Illuminar Hi-Seq 2500 platform obtaining 10-30 million single reads per sample with indices, using the transposon-specific oligonucleotide primer Himar1-Seq.
5.9.1.6 Tn-seq data analysis
- Sequencing files were obtained and was processed and analyzed, as previously described 301,326 .
- In brief, Illuminar adapter sequences were removed using cutadapt version 1.2.1 327.
- The sequence reads were checked for the nucleotide pattern ‘CAACCTGT’ originating from the transposon Inverted Terminal Repeats (ITR). [In my case 'TAAGAGACAG']
- Only reads containing this specific sequence with an allowance of one mismatch or gap and with minimum length of 16 nucleotides were used for further analyses. [In my case 20]
- Further, the reads were mapped to the Staphylococcus aureus 6850 genome (GenBank accession CP006706) using Bowtie 2 algorithm version 2.1.0 328.
- To identify transposon insertion sites (TIS), the aligned start positions of mapped reads were extracted and each position on the genome, covered by at least one alignment start, was annotated as TIS.
- The genomic position was adjusted strand-specifically to account for the 1 nucleotide shift of the reads mapping on the positive or negative strand.
- Statistical analysis of enriched or depleted reads from each TIS, was performed using DESeq2 version 1.6.2 261.
- The HeLa infection experiment was modelled as a time course including a technical replicate with the input libraries as time t0.
- For the mouse lung infection experiment, the 3 output libraries were compared to the input libraries.
- Genes with very low mean normalized read depth (mnrd) <4 were excluded in the HeLa experiment and those with mnrd 8 were excluded from the animal experiments.
- The P-values were corrected for multiple testing and the TIS with adjusted P-value <= 0.05 were considered as significantly increased or decreased.
5.9.2 RNA deep sequencing
- RNA were isolated as previously described (See Section 5.5.5), followed by removal of DNA using DNase I enzyme (See Section 5.5.6).
- Quantity and quality of RNA was determined by spectrophotometric method using Nanodrop and 1.8% agarose gel containing formamide. mRNA enrichment was performed by Ribodepletion Kits followed by library Preparation for Illuminar .
- Depletion of processed transcripts, were performed by using Terminal 5’-phosphate-dependent exonuclease (TEX) as previously described (Sharma 2010).
- Briefly, RNA samples were poly(A)-tailed using poly(A) polymerase.
- 5’-triphosphates were removed by treatment with tobacco acid pyrophosphatase (TAP).
- RNA adapters were ligated to the 5’-phosphate ends and first-strand cDNA were generated using an oligo(dT)-primer and M-MLV reverse transcriptase.
- High fidelity DNA polymerase cDNA was amplified by PCR. cDNA were sequenced on Illuminar HiSeq platform2 , yielding 100 bp paired end reads.
- Adapters removed and trimmed to 70 bp using Trimmomatic (Bolger 2014) and only reads exceeding a mean base quality 5 within all sliding windows of 5bp were mapped to the S. aureus USA300 FPR3757 genome (NCBI accession NC_007793.1), using Bowtie2 (25).
- Only paired and concordant alignments were considered further, yielding at least 12 million uniquely mapped read pairs per replicate. A total of 2693 coding and non-coding transcripts were identified for further analysis.
- Differential transcript abundance analysis was performed using the DESeq2 package v.1.5.9 (12) in R.
[301] Natural mutations in a staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia
and abscess formation. Proceedings of the National Academy of Sciences, 2016.
[326] Transcriptional landscape and essential genes of neisseria gonorrhoeae. Nucleic acids research, 42(16):10579–10595, 2014.
[327] Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. journal, 17(1):
pp–10, 2011.
[328] Fast gapped-read alignment with bowtie 2. Nature methods, 9(4):357–359, 2012.