Background and aim: Next generation sequencing (NGS) has quickly the tool of choice for genome and exome data generation. The multitude of sequencing platforms as well as the variabilities within each platform need to be assessed. In this paper we used two platforms (ION TORRENT AND ILLUMINA) to assess single nucleotides variants in colorectal cancer (CRC) specimens.
Methods: CRC specimens (n = 13) collected from 6 CRC (cancer and matched normal) patients were used to establish the mutational profile using ION TORRENT AND ILLUMINA sequencing platforms. We analyzed a set of samples from Formalin Fixed Paraffin Embedded and FF (FF) samples on both platforms to assess the effect of sample nature (FFPE vs. FF) on sequencing outcome and to evaluate the similarity/differences of SNVs across the two platforms. In addition, duplicates of FF samples were sequenced on each platform to assess variability within platform.
Results: The comparison of FF replicates to each other gave a concordance of 77% (± 15.3%) in Ion Torrent and 70% (± 3.7%) in Illumina. FFPE vs. FF replicates gave a concordance of 40% (± 32%) in Ion Torrent and 49% (± 19%) in Illumina. For the cross platform concordance were FFPE compared to FF (Average of 75% (± 9.8%) for FFPE samples and 67% (± 32%) for FF and 70% (± 26.8%) overall average).
Conclusion: Our data show a significant variability within and across platforms. Also the number of detected variants depend on the nature of the specimen; FF vs. FFPE. Validation of NGS discovered mutations is a must to rule-out false positive mutants. This validation might either be performed through a second NGS platform or through Sanger sequencing.
Next-generation DNA sequencing has revolutionized genomic studies and is driving the implementation of precision diagnostics. The ability of these technologies to disentangle sequence heterogeneity, however, is limited by their relatively high error rates. A Several single molecule barcoding strategies have been propose to reduce the overall error frequency. A Duplex Sequencing additionally exploits the fact that DNA is double-strand, with one strand reciprocally encoding the sequence information of its complement, and can eliminate nearly all sequencing errors by comparing the sequence of individually tagged amplicons derived from one strand of DNA with that of its complementary strand. This method reduces errors to fewer than one per ten million nucleotides sequenced.