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Another factor that came into consideration was how to decrease the extent of false positives in information. Whereas it’s has been reported that the extent of false positives in Ion Torrent data is larger than Illumina, it has also been shown that Ion Torrent can detect extra true positives given enough protection . Unfortunately, most second technology NGS technologies nonetheless current excessive error rates . While we tried to regulate for equimolar quantities of DNA when pooling samples, PCR steps result in uncontrollable dilutions of some samples earlier than sequencing which can result within the lack of some SNVs among noise. Improvements to get rid of such technical variability will improve our capability for SNV detection.
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Therefore various factors may have had an influence on the variability in read count that we encountered. When we calculated GC content it was seen that LuPME105 had the lowest GC content (43%) of the four PME sections evaluated. Ion Torrent read protection has been shown to decrease upon high or low GC content or under different ranges of genome complexity . Several variables were optimized throughout our experiment. Since pattern pooling was used, high-quality DNA in equal amounts was needed to extend the likelihood of detecting one mutated individual among the many inhabitants. We standardized a high-throughput CTAB protocol yielding high quality DNA and additional quantified the samples by fluorescence to add equal amounts of DNA from each individual in every pool.
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We used 10 ng of DNA from each of 768 people and pooled the DNA as explained in materials and strategies. Because of the way our experiment was designed, every one of many 768 particular person DNA samples was current in three swimming pools; when a SNV was found in three intersecting pools we could pinpoint the pattern of origin. We focused four genes of the pectin methylesterase family for discovery of SNVs . These genes were chosen because they are related to ongoing cell wall research in our laboratory . This highlighted the significance of empirically testing PCR circumstances for any new set of primers.