The caeca were removed as a pair. They were then carefully separated at the ileo-caecal-colonic junction and weighed individually. The caeca weights were then combined to give both individual and pair weights per pen.
2.5.17 16S Sequencing study 2 and 4
The V4 hypervariable region of the 16S rRNA gene was amplified from genomic DNA using forward primer 515F: GTGCCAGCMGCCGCGGTAA and reverse primer 806R: GGACTACHVGGGTWTCTAAT (Earth Microbiome, 2015), using the 2X KAPA HiFi Hotstart ReadyMix and primers.
PCR amplification was performed using 25 µl reaction mixtures of 2.5 µl microbial DNA (5ng/ul); 5 ul Amplicon PCR Forward Primer (1uM); 5ul Amplicon PCR Reverse Primer (1 µm) and 12.5 µl 2X KAPA HiFi HotStart ReadyMix (KAPA-Germany). This was added into a 96 well plate fully sealed. PCR was performed in a thermal cycler (Techne, TC-512, UK) using the following program: 95°C for 3 minutes; 25 cycles of :95°C for 30 seconds; 55°C for 30 seconds;72°C for 30 seconds 72°C for 5 minutes. The PCR products were run through 1.5% agarose gel electrophoresis and bands analysed to check the amplicon quality. Gels were run at 100 volts for 45 minutes after loading 2 µl of loading dye. (Cleaver, MP-250v, UK), and 5 µl of amplicon. Gels were viewed under UV light (Syngene, G: Box, USA). The 16S V4 amplicon was prepared using 20 µl of AMPure XP beads and then incubated at room temperature for 5 minutes. The 96 well plate was then placed on a magnetic stand (FastGene MagnaStand, YS, Germany) for 120 seconds or until the supernatant had cleared. The supernatant was then discarded, and the beads washed twice with 80% ethanol. The beads were then allowed to air-dry at room
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temperature for 10 minutes. The amplicon was then suspended in 52.5 µl of 10mM Tris pH 8.5 and gently mixed. The sample was then incubated at room temperature for 2 minutes. 50 µl of the clear supernatant was then transferred onto a fresh 96 well PCR plate.
Index PCR was performed as follows: 5 µl of DNA was moved on to a fresh plate and the index 1 and 2 primers added. The plate containing the re-suspended PCR product was placed in a TruSeq index plate fixture (Illumina, USA). 5 µl of DNA amplicon, 5 µl of Nextera XT Index Primer 1 (N71-12) horizontally, 5 µl Nextera XT Index primer 2 (s51-8) vertically, 25 µl 0f 2x KAPA HiFi HotStart
ReadyMix, and 10 µl PCR grade water were gently mixed then the plate covered with Microseal. This was then centrifuged at 1000 x g at 20°C for 1 minute then PCR performed again on a thermo cycler: 95°C for 3 minutes, 8 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 72°C for 5 minutes, then finally held at 4°C. The index PCR product was then cleaned up as before and 56 µl of AMPure XP beads added to each well. Each well was then mixed by gently pipetting the mixture up and down with a multi-channel pipette. This was incubated at room-temperature for 5 minutes. Once again, the plate was placed on a magnetic stand for 2 minutes or until the supernatant had cleared. The bead was then removed, and the supernatant discarded. Beads were washed with 80% ethanol twice, then the excess ethanol removed, and the beads air-dried. 27.5 µl 0f 10 mM Tris (pH8.5) was added to each well and incubated at room temperature for 2 minutes. 25 µl of the supernatant from each well was then carefully moved to a fresh plate. For validation purposes 1 µl of the library product was run on a tape-station DNA 1000 (Agilent, USA) to verify the size. Quantification, normalization, and pooling were the performed.
DNA concentration was calculated in nM based on the size of DNA amplicons as determined by the Agilent Tape Station 1000. The library sample was then diluted to 4 nM using Tris pH (8.5). 5µl of diluted DNA from each library sample was then pooled. The MiSeq reagent cartridge was removed from -15°C storage and thawed at room temperature. DNA was then denatured by combining 5 µl of 4nM pooled library sample and 5µl 0.2 N NaOH. This was then briefly vortexed and centrifuged at 280
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X g at 20°C for 60 seconds. The sample was then incubated at room temperature for 5 minutes. 990 µl of pre-chilled hybridization buffer HT1 was added to a tube containing 10 µl of denatured DNA. 5 µl of 4 nM phiX library sample and 5 µl of 0.2 n NaOH were combined in a micro centrifuge tube and briefly vortexed before incubating for 5 minutes at room temperature to denature the phiX library sample into individual strands. 990 µl of pre-chilled HT1 (20pM) phiX library was added to the tube containing 10 µl denatured phiX library to result in 20 pM phiX. This was then diluted to the same loading concentration as the amplicon library sample to equal 8mM by mixing 20 pM denatured library and pre-chilled HT1 (360 µl). The amplicon library sample was then set aside on ice until it was time to heat denature the mixture immediately prior to loading it into the MiSeq v3 reagent cartridge. The mixture was once again incubated at 96°C for 2 minutes using a heat block. The tube was then mixed and placed in an ice-water bath. The template allocations of samples were set up in the illumina sheet then the combined library and phiX sample was loaded into the Miseq cartridge then into the illumine machine using version 3 (300x2) chemistry on the Miseq instrument (illumine Inc., USA) according to manufacturer’s instructions.
Results were then analysed through microbiome analyst software that performed comparative diversity analysis and statistical analysis of any differences by either treatment or age. The significance level was set to p=>0.05 to match the rest of the statistical analysis used in this thesis.