Amplification of V3 and V4 segments of the16S rRNA gene and the adjoining of adapters were performed in two separate steps/nested reactions in order to maximize the specificity of the primers.
The latter reaction was not initiated until all samples reached the completion of the first PCR reaction. Positive and negative controls (E.coli genomic DNA and master mix, respectively) were included in all PCR reactions. The water samples were divided and processed in three batches, with duplicates of sample 4-41 in the last batch. The mucosal samples were divided into five batches,
with the last batch containing 6-7 duplicates from each of the four first batches. Comprising as much variation as possible in sample characteristics was considered to be of main importance when choosing the latter duplicates.
In addition to visualizing a subset of amplicons from all PCR reactions on gel, quantitatively and qualitatively measurements were applied, as explained under quality assurance. A subset of water samples from the first PCR reaction were quantitatively measured, while a qualitative measurement was applied on all mucosal samples owing to large variations in the Ct-values from the qPCR. A qualitative measurement was applied on both water samples and mucosal samples after the final PCR reaction. Fluorescence from negative sample was subtracted from the resulting fluorescence to account for excess nucleotides, primer dimers etc. Samples with a fluorescence equal to a non-detectable band were submitted to additional rounds of recovery with the rationale of generating a higher amount of amplicons. The subsequent adjustment of the conditions behind the two nested reactions primarily targeted the number of cycles and the amount of template DNA.
2.3.1 Nested PCR
Template DNA from both reactions was embedded in a 25µl reaction volume of 1x of HotFirePol®
buffer B2, 25mM of MgCl2, (all Solis BioDyne) 200µM dNTP (Solis BioDyne). 1,25U concentration of HotFirePol® and FirePol® DNA polymerase were used in the first and second PCR reaction respectively. 5µl template DNA was used for the amplification of 16S rRNA, with DNA
concentrations in the range of <0,5-1,1 ng/mL and 0,3-25 ng/mL from the water samples and gut biopsies respectively. For the adaptor adjoining 5-10µl template with DNA concentrations of 1-9 ng/mL and 0,1-3,1ng/mL of the respective sample types were used.
0,2 µM of PRK341F (5`-CCTACGGGRBGCASCAG-3`)and PRK806R
(5`-GGACTACYVGGGTATCTAAT-3`) (Invitrogen) was included in the amplification of 16S rRNA, thus allowing for the amplification of V3, V4 and the conservative regions interspersing these variable regions. This reaction will be referred to as PRK PCR. For adjoining of adapters, 0,2 µM of the 16S rRNA forward and reverse indexing primers (Invitrogen) was employed. Indexing primers were added manually on the purified water sample PCR products, and made automatized on the mucosal samples by use of Eppendorf epMotion 5070 machine. (Eppendorf, Hamburg, Germany) 36 forward and 16 reverse primers, each with a unique barcode, were used in each set of samples.
Primers were arranged in a manner giving each sample from each set a unique barcode
combination, allowing for the annotation of sequences to their respective sample after the final sequencing. This reaction will be referred to as indexing PCR.
The following PCR program was implemented for the amplification of 16S rRNA; activation 95oC for 15 min, 25-30 cycles of 95oC for 30s, 50-55oC for 30s, 72oC for 45s, and final elongation at 72oC for 7 min. The adjoining of adapters on the other hand required the following program; 95oC for 5 min, 10 cycles of 95oC for 30s 55oC for 1 min, 72oC for 45s, and final elongation at 72oC for 7min. 2720 Thermal Cycler (Applied Biosystems, Foster city, USA) served as the amplification instrument in both PCR reactions.
2.3.2 PCR product purification
The nested PCR reactions were interspersed by a purification of PCR products with Sera-Mag Magnetic Speed beads in order to remove unincorporated nucleotides, primer dimers, smaller fragments, etc that could pose an impact on the final sequencing process. A 1:1 ratio of PCR product and bead solution were mixed, allowing for DNA fragments over a certain size to bind to the magnetic beads. While on magnet, DNA was washed three times with fresh 80% ethanol, ensued by a release from the beads with nuclease-free water. The purification process was made automatized in Biomek® 3000 Workstation (Beckman Coulter Life Sciences, Indianapolis, USA) in concordance with manufacturers protocols. Bead solution was made from 0,1% carboxyl-modified Sera-Mag Magnetic Speed beads (Fisher Scientific, Thermo ScientificTM) pre-washed with TE, 18%
PEG, 1M NaCl, 10mM Tris-HCl pH 8, and 1mM EDTA pH8. After each purification, DNA was quantitatively measured with Qubit in a subset of samples as described under Quality assurance, to confirm the binding of DNA to the beads. A second post PCR purification process after the adapter adjoining was not considered necessary due to the low amounts of interfering products shown after gel electrophoresis on a subset of samples.
2.3.3 Sequencing preparations.
Ensuing adapter adjoining on all samples, a normalization process was performed in order to achieve that an equal amount of indexing PCR products was transferred to the Illumina sequencing chip, and to avoid an over or under representation of certain samples. This was performed manually on both sample types. The qualitative DNA measurements of the PCR indexing products, were used as a baseline for normalization. The fluorescence from the negative control was subtracted from the value to account for possible primer dimers, excess nucleotides etc. Owing to the even fluorescence
in water samples, an equal volume from each sample was transferred to a common pool. The higher dispersion of fluorescence in the biopsies made it more feasible to divide samples in groups of ten.
An appropriate normalization volume was subsequently assigned to each group, ensuing a fairly equal amount of DNA to be transferred from all samples. Both pools were subsequently subject to a manual purification in order to remove excess nucleotides, primer dimers and non-specific smaller amplicons etc. Pools were mixed with Sera-Mag magnetic speed beads in a 1:0,8 ratio allowing for a removal of DNA under a certain fragment length. While on magnet, the attached DNA was washed twice with 80% fresh ethanol, and eluted with nuclease-free water. Pools from before and after this purification step was checked on 1% agarose gel as described under Quality assurance, in order to confirm the success of the clean-up.
Both cleansed pools, hereby referred to as libraries, were submitted to quantification with Perfecta® NGS Library Quantification Kit for Illumina® Sequencing Platforms, (Quanta BioSciences,
Gaithersburg, USA) following manufacturers instructions and by the use of LightCycler 480 II. A 1:2000 and 1:20000 dilution of the libraries were included, together with five standards ranging from 0,0005pM to 5pM. All samples were run in triplicate reactions to increase the reliability and account for possible deviations. Negative control was also included. Based on the Ct-values from the included standards, an equation from the resulting calibration curve was made, and the corrected concentration in the amplicon libraries was estimated. Libraries were subsequently diluted to 4nM in order to generate a proper cluster density.
2.3.4 Library denaturation and Miseq sequencing
Amplicon libraries consisting of water samples and mucosal samples were sequenced separately.
Before loading onto chip, diluted libraries were prepared and denatured according to Illumina Library Preparation guide and by use of Miseq reagent cartridge (Illumina, San Diego, USA). A denatured control of PhiX was included to serve as a contrast during the reading process and permit error rate calculations. 4nM of PhiX and amplicon library were prepared in a similar manner by first separately combining the samples with equal amounts of 0,5N NaOH, giving samples of 2nM.
Samples were vortexed, centrifuged at 280g at 20oC for 1 min (libraries only), followed by a 5 minute incubation at room temperature in order to separate the strands. Libraries and PhiX were further diluted to 6pM with HT1. Amplicon library of mucosal samples was spiked with15% PhiX according to manufacturers recommendations, while a 30% spike-level was used for the water samples due to recent technical problems with the Miseq machine. Spiked libraries were separately
applied onto the flow cell of an Illumina chip, and sequenced by use of a MiSeq sequencing
platform (Illumina) All library denaturation and MiSeq sequencing steps were performed under the supervision of co-supervisor.