The second study of the thesis evaluated the suitability of the enzyme ɸ29 for detection and characterisation of low abundant microbial genomes production, for the application of whole genome sequencing. The first question addressed was whether ɸ29 could produce DNA that was suitable for library preparation for next generation sequencing, and how this compared to the DNA produced by current culture based methods. Additionally, the sensitivity of the method was evaluated and the processivity investigated. The quality of the sequencing data produced was assessed and compared to that produced by culture based methods. To establish these properties Escherichia coli K12 substr. MG1655 was used as the test organism. The impact of GC content and cell wall type was also investigated, along with the ability to detect and analyse extra
chromosomal elements using Actinomyces naeslundii (NCTC 10301) and Peptoclostridium difficile 630. To further test the enzyme a mixed bacterial sample was amplified to look for amplification bias, using Haemophilus influenza (NCTC 8143) and Enterococcus faecalis (NCTC 12201).
Additionally, DNA viral genomes from Adenovirus 40 and 41 were amplified to investigate the application to smaller genomes and the extraction of none envel oped viruses. Finally, negative samples were sequenced to monitor contamination, randomly produced DNA and sequencing artefacts.
2.3.1. Development of ɸ29 MDA for whole genome Sequencing
2.3.1.1. Preparation of None Amplification Controls
Overnight cultures of bacteria were extracted using Qiagen QIAamp DNA mini kit as described in 2.2.1.1. The DNA was quantified using Qubit BR kit and 500 ng of DNA taken forward to
sequencing.
2.3.2. Fragmenting DNA for sequencing
The recommended method for fragmenting DNA for sequencing on the 454 Junior was physical shearing for 1 minute using nebulisation. However due to the long lengths and presence of secondary structure in DNA produced by ɸ29 MDA alternative methods were investigated. DNA extracted from cultured bacteria was fragmented using the recommended parameters 30psi for 60 seconds. Three biological replicates of the culture based control were sequenced
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2.3.2.1. Using Nebulisation
500ng of DNA was made up to a final volume of 100 µl with TE buffer. The mixture was added to an assembled nebulisation cup (Roche) to which 500 µl of nebulisation buffer was added (Roche). The sample was then nebulised at 30 psi (pounds per square inch) for 60, 120 or 180 seconds.
2.3.2.2. De-branching of Product
3 µg of ɸ29 MDA DNA was de-branched using S1 nuclease in a 90 µl reaction as follows, 3 µl 10x buffer, 3 µl 0.5M NaCl, 10 µl S1 nuclease (1U/ µl) with water to make the volume to 90µl. The digestion reaction was left at room temperature for 30 minutes and the enzyme deactivated by incubating at 70oC with 6 µl 0.5M EDTA. This was then nebulised at 30psi for 60, 120 or 180 seconds.
2.3.2.3. Using Fragmentase
NEBNext® dsDNA Fragmentase (NEB) generates dsDNA breaks in a time-dependent manner to yield 50–1,000 bp DNA fragments. 1 µg of ɸ29 MDA was added to the following reaction mix, 2µl 10x Fragmentase buffer, 2 µl 10x BSA, and made up to a total volume of 18 µl using the supplied sterile water. The reaction was then incubated on ice for 5 minutes, before the addition of 2 µl dsDNA Fragmentase. After which the reaction was vortexed and incubated at 37oC 10, 15 or 20 minutes. After incubation 5 µl 0.5M EDTA was added to stop the reaction.
2.3.2.4. Combination of S1 Nuclease and Fragmentase
5 µg of DNA was added to the following reaction mix, 10 µl Fragmentase 10x buffer, 10 µl 10x BSA, 24 µl water. This was then incubated on ice for 5 minutes before the addition of the
following, 10 µl dsDNA Fragmentase, 10 µl S1 10x buffer, 10 µl 0.5M NaCl, 30 µl S1 nuclease (1U/ml). Followed by 30-minute incubation at 37oC, after which 2.5 µl 5M EDTA was added to stop the reaction.
2.3.2.5. Sample Purification
The sample was purified using the Qiagen MinElute PCR purification kit, as described in the Roche Rapid Library Preparation method. Briefly, 2.5ml of PB buffer was added to the sample and mixed. 750 µl of this mixture was added to the spin column and centrifuged for 15 seconds and the through flow discarded. This was repeated until the entire sample had been added. The sample was then washed with 750 µl PE buffer and eluted in 20 µl of TE buffer. After which the DNA was quantified using Qubit broad range kit as previously described in 2.2.1.4, 500ng of DNA in a total volume of 16µl in TE buffer was added then taken forward for sequencing.
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2.3.3. Sample sequencing
For sequencing the following Roche 454 Junior protocols were followed:
2.3.3.1. Rapid Library Preparation Method Manual-Match 2012
Briefly, 500ng of fragmented DNA was end repaired and adapters added, the DNA was then size selected using AMPure beads. Library quality was assessed using the Agilent Bioanalyser where successful library preparation consisted of the average read length being between 600 and 900bp. The library was quantified using the QuantiFluor ST Fluorometer (Promega) fluorescence was measured against the standards provided in the library preparation kit. Samples were then diluted to working stocks of 1x107 molecules/ μl, in TE Buffer.
2.3.3.2. EmPCR Amplification Method Manual -Lib-L-March 212
Firstly, an emulsion PCR was set up, aiming at four or two copies per bead. The emulsion was then dived into 100 μl aliquots in a PCR plate. The emulsion PCR used the following parameters in a thermocycler with heated lid turned on. A hold at 94°C for 4 minutes, 50 cycles of 30 seconds at 94°C, 4.5 minutes at 58°C, 30 seconds at 68°C, with a final hold at 10°C. The beads were then harvested and the emulsion broken using isopropanol and ethanol. The beads were then washed using a provided buffer and concentrated into 1 ml. Enrichment primers were then annealed to the libraries and the beads enriched to keep only beads with attached DNA. The recovered beads were then visually quantified before a sequencing primer was attached to the libraries.
2.3.3.3. Sequencing Method Manual – January 2013
Firstly, all frozen sequencing reagents were defrosted in the dark by submersion in water. Then the instruments were primed using the provided pre -wash buffer. Four bead layers were prepared as instructed and added to the Pico Titer Plate (PTP) in the order shown in Table 2-6. The PTP was then loaded onto the sequencer and sequenced usng 200 cycles and full processing for shotgun libraries. Run time was 9 hours and 20 minutes.
Bead layer Bead type
Layer 1 Enzyme beads pre-layer
Layer 2 DNA and packing beads
Layer 3 Enzyme beads post-layer
Layer 4 PPiase beads
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2.3.4. Determining Sensitivity and Processivity of Technique in the
Context of Bacterial Genomes
2.3.4.1. Lowering Reaction Volumes
Reaction volumes were scaled down to total volumes of 25 µl and 12.5 µl with two sets of biological replicates being sequenced for each reaction volume.
2.3.4.2. Preparation of Single Cells for Sequencing
Single colonies from overnight cultures were suspended in 1ml phosphate -buffered saline (PBS) to produce a 10-3 dilution, serial 1:10 dilutions were then prepared until 10-10 was achieved. A blood agar plate was divided into four and 10 µl of the 10-7 – 10-10; dilutions were plated four times on each plate. These were then incubated overnight and a colony count performed. This was then used to calculate the average number of cells in the PBS and the required volume of diluted cells was added to the extraction for ɸ29 MDA amplification. Three biological replicates of estimated single cells were sequenced.
2.3.4.3. Reducing Incubation Time
Incubation times of eight, four, two and one hours were investigated using single cell extracts in 50 µl reactions. Bacteria were extracted as described in 2.2.1.2 , and amplified in the reaction as described in 2.2.1.3 before incubation for the required time at 30oC and inactivation at 65oC for 10 minutes. Two biological replicates were performed per incubation time.
2.3.5. Basic Analysis of Sequencing Data Produced
After 454 junior sequencing run completion run statistics were viewed using the Roche ‘runviewer’ programme, where information on raw reads, reads filter passed and read length were obtained. According to Roche sequencing guidelines, a successful run follo ws the following characteristics,
Raw reads above 200,000 reads
Passed filter wells >100,000 (>50%)
Average read length 400-500 base pairs
After sequencing if these minimum parameters were achieved the sequencing was considered successful and further analysis performed.
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2.3.5.1. Reference Assembly
Initially the SFF files (Standard flowgram format) were mapped against the appropriate reference as listed in Table 2-7. Reference sequences were downloaded in fasta format from the National Centre for Biotechnology Information (NCBI) or Integrated Microbial Genome (IMG) database. The sequencing files were assembled using the standard parameters in Newbler GS Reference Mapper (Roche Diagnostics). This was used for initial run assessment to determine genome coverage and number of mapped reads. The software allows both SFF and FASTQ files to be input and assembled, using Command 2-2. From the output file ‘NewblerMetrics.txt’,
reference assembly information was extracted, including the number of reads which mapped to the reference, the number and size of contigs and the percentage of the reference which was covered. Once the reference assembly was completed, the resulting bam file was sorted and indexed using Sam tools 99. The assembly was then viewed against the reference in Artemis 15.0.0100.