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We demonstrate the utility of this approach by identifying SNVs in eight genes of interest, after performing a pilot experiment in three genomic regions with known SNVs to validate the methodology. Our methodology allows identification of putative variants on the target genes and can be scaled up in the number of genes and individuals to screen large populations.
We conducted three experiments to develop an Ion Torrent-based method for discovery of single nucleotide variants (SNVs) in flax: (i) a pilot experiment with combinations of known SNVs (using an Ion 314 chip); (ii) a proof of concept experiment with a mutagenized population of flax (also using an Ion 314 chip); and (iii) a scale-up experiment using the higher capacity, Ion 316 chip.
Sequence coverage and frequency of variants in gene sections of the pilot experiment. DNA from the cultivar Macbeth was diluted (1:64 or 1:96) in CDC Bethune DNA in several simulated pools as described in methods. Each line in the sequence coverage graphs represents one of 28 pools. The frequencies of the variants are plotted against the position in the respective reference sequence section. Each symbol in the frequency graphs represents a frequency of at least 0.1% for each of the 28 pools in each position. A. S20, B. S900; no graphs for S411 are shown since no variants were detected in that sequence.
Having demonstrated in the pilot experiment that we could detect known SNVs within simulated pools of DNA, we next attempted to discover novel SNVs within pools of DNA obtained from an actual mutagenized population (proof of concept). We used 10 ng of DNA from each of 768 individuals and pooled the DNA as explained in materials and methods. Because of the way our experiment was designed, each one of the 768 individual DNA samples was present in three pools; when a SNV was found in three intersecting pools we could pinpoint the sample of origin. We targeted four genes of the pectin methylesterase (PME) family for discovery of SNVs (LuPME10, LuPME73, LuPME79, LuPME105, Table 2). These genes were selected because they are relevant to ongoing cell wall research in our laboratory . To minimize the amplification of primer dimers, we tested the PME primers (Additional file 1), under a range of annealing temperatures and found that the optimal temperature range for the touch-up first-step PCR was 56-66°C (this was higher than the annealing temperature range 50-60°C in the pilot experiment), and the optimal second-step PCR annealing temperature was 68°C. This highlighted the importance of empirically testing PCR conditions for any new set of primers. Amplicons were analyzed and purified on agarose electrophoretic gels, eluted, and quantified (as in the pilot experiment) before Ion Torrent sequencing.
When comparing to the pilot experiment the percentage of mappable reads increased to over 60% for both replicates (Table 1), but the average read coverage per pool in each of the evaluated genes was proportionally lower than in the pilot since reads in this case were distributed among four genes (Table 2). Furthermore, the proportion of reads mapped was not equally distributed among the four genes in any of the pools. When using the mapped reads in all pools to calculate the coefficient of variation (CV) for each gene and replicate, variability was evident among genes (Additional file 7), but the variation was constant among the two replicates for each gene.
Based on our experience in the pilot experiment, we selected a minimum coverage per position of 500×, with a frequency of at least 0.5% in three intersecting pools, for defining putative mutations. When only two intersecting pools were found with the expected minimum frequency, all individuals from the intersection where sequenced. There was consistency between replicate runs for most SNVs but some of the SNVs were detected by complementary intersecting pools between both replicates. There was no correlation of false positives with the technically consistent SNVs or the ones found by complementarity.
In the pilot experiment, we detected the expected mutations and additional unreported changes in the tested genomic regions (Figure 2 and Additional file 5). Our experiment with PMEs had a larger variability in read depth among pools and genes (Additional file 7), which may have had an influence on the number of false positives. When we increased our read depth by using the larger Ion 316 chip, the number of false positives decreased significantly.
Interestingly while homopolymer errors are the most common error type from this technology [23,36,39], an study with bacteria found that substitutions have the highest variation frequencies, with standard deviations ranging from 26%-56% of the mean substitution rate . This has implications in the detection of rare variants (including false negatives) which may come up at lower frequencies as we detected in our study due to sample dilution in pools. For example, a 0.3% frequency was found for S20 variants in the pilot experiment (Figure 2), and since some random error can reach this frequency, this can lower our detection ability. While there were a few false positives embedded into homopolymeric tracts which constitute the bigger source of error of Ion Torrent technology , no specific position or sequence-specific bias in the SNVs that were not confirmed by sanger could be inferred to make any generalization.
A pilot experiment was performed using known SNVs showing polymorphisms between cultivars CDC Bethune and Macbeth. Since for the pilot experiment we were not trying to discover new mutations we did not have to pool DNA from different individuals as described in the DNA extraction and pooling section; instead Macbeth DNA was diluted 1:64 or 1:96 in Bethune DNA to simulate the presence of an individual with a mutation within the population. Three genomic regions with previously reported SNVs , from three flax scaffolds were chosen to test the methodology and were named S20, S411 and S900 (names were derived from the names of the scaffolds that contained them - see Additional file 1).
For the pilot experiment, first-step amplifications were performed on a template that either included DNA with known SNVs S20, S411 and S900 (Macbeth DNA in 1:64 or 1:96 dilution in CDC Bethune DNA) or homogeneous template without the SNVs (i.e. only CDC Bethune DNA). The PCR products were diluted 1:100 and equal amounts (5 μL) of PCR product from each of the three target regions were mixed in each of 28 pools for barcoding; the changes of Macbeth genic regions SNVs where introduced in all pools for the gene of S20, and in 10 pools for genes S411 and S900. For the second step, a total of 28 PCRs were performed with forward bar-coded primers (Figure 1 and Additional file 2), and a trP1 primer complementary to the tail from the reverse primer of the first-step PCR. The 28 bar codes allowed us to discriminate between the respective DNA pools after sequencing.
Procedures were similar to the pilot experiment, unless stated otherwise. The experimental design was adapted from Tsai et al., 2011 . A total of 28 pools of DNA from distinct individuals (768 lyophilized leaf samples) were created to facilitate detection of mutations as described in DNA extraction and pooling.
Frequency tables were created manually using Microsoft Excel per each gene and per each pool after filtering homopolymeric tracts and indel artifacts created by the sequencing technology. Graphs showing the frequency changes by position for the 28 pools in a specific base change (e.g. G to A) were done to detect outliers visually which were indicative of a rare variant. SNV candidates were chosen if the mutation was present in three intersecting pools or in two intersecting pools with high frequency (>0.3% in pilot experiment and >0.5% in remaining experiments). DNAs from individuals (M3 generation) with potential mutations were re-sequenced using Sanger sequencing to confirm the analysis performed with Ion Torrent.
Two-step PCR from pilot experiment. A. Products of first step PCR from the pilot experiment. Amplifications were performed with 5 or 10 ng of pooled DNA in the different dilutions that simulated the inclusion of the mutated individual. Gel was run in 1.5% agarose in TAE 1X at 90 V for 40 minutes. Size of marker bands is given in bp. Negative control refers to a PCR with no DNA template. B. Products of second step PCR from the pilot experiment. Amplifications were performed with 1:100 dilutions of the first-step PCR mixed products of the three genes (S20, S411, S900). Gel was run in 1.5% agarose in TAE 1X at 90 V for 60 minutes. Size of marker bands is given in bp. Negative control refers to a PCR with no DNA template. 2b1af7f3a8